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Background: Previous studies have evaluated the implementation of behavioral approaches in individuals with chronic temporomandibular disorders (TMDs)., Objective: To evaluate the benefits of a behavioral approach to craniofacial pain. Second, we assessed the benefits of kinesiophobia, catastrophizing, mouth opening without pain, and forward head posture., Methods: Individuals with chronic TMDs were treated for five weeks. The intervention group ( n  = 17) underwent pain neuroscience education, manual therapy, and therapeutic exercise, whereas the control group ( n  = 17) underwent manual therapy only. Outcomes were evaluated immediately, at seven and 19 weeks follow-up. The assessment tools used were the Craniofacial Pain Disability Inventory, Pain Catastrophizing Scale, Tampa Scale for Kinesiophobia, Mandibular Range of Motion Scale, and Cervical Range of Motion Tool., Results: The interventions did not influence the differences in the improvements between the groups observed for craniofacial pain disability (inter-subject p 0.4). The intervention had a moderate influence on the improvement of kinesiophobia and catastrophizing (Inter-subject p 0.09 and 0.1 respectively) with a clinically significant effect size (Estimated mean (EM) -8.6 standard deviation (SD) ±3.48 p 0.019; and EM -7.6 SD ± 5.11 p 0.15 respectively)., Conclusion: The behavioral approach improved catastrophizing and kinesiophobia outcomes in individuals with chronic TMDs.

Real-time surgical navigation systems (dynamic computer-aided surgery, d-CAIS) and static guided surgery (static computer-aided surgery, s-CAIS) have been shown to enhance the accuracy of zygomatic implant (ZI) placement. The objective of this systematic review was to evaluate and compare the accuracy and risk of complications associated with d-CAIS and s-CAIS in ZI placement. A systematic review of published studies involving more than 4 patients was conducted to assess and compare the accuracy of d-CAIS and s-CAIS in zygomatic implant placement. Only one study included freehand ZI placement as a control. The primary outcomes measured were the accuracy of implant placement relative to preoperative planning, with a secondary focus on evaluating any potential complications. Out of 903 screened studies, 14 met the inclusion criteria. Freehand zygomatic implant placement was used as a control in only 1 study. The results revealed a mean apex deviation of 2.07 mm (95% CI: 2.01 to 2.13; I 2  = 83.14%) for d-CAIS, 1.29 mm (95% CI: 1.15 to 1.43; I 2  = 94.5%) for s-CAIS, and 4.98 mm (95% CI: 3.59 to 6.37; I 2  = not assessable) for freehand placement. Reported complications included mucositis, reversible bilateral sinusitis, oroantral fistula, unspecified reversible postoperative complications, and fracture of the anterior wall of the zygoma. Both CAIS systems demonstrated high accuracy and safety in ZI placement, with a nearly 99% success rate at 6 months of follow-up. These findings suggest that both d-CAIS and s-CAIS are reliable methods for improving the precision and reducing the risks associated with ZI procedures., Competing Interests: Declaration of competing interest The authors declare no conflict of interest related to this article. However, the authors would like to state the following conflicts outside the submitted work: Dr. Rui Figueiredo reports grants, personal fees, and non-financial support from MozoGrau (Valladolid, Spain), Inibsa Dental (Lliçà de Vall, Spain), Dentaid SL (Cerdanyola del Vallés, Spain), non-financial support from Nobel Biocare, personal fees from Geistlich Pharma AG (Wolhusen, Switzerland), BioHorizons Iberica (Madrid, Spain), Araguaney Dental (Barcelona, Spain), Septodont (Saint-Maur-des-fossés, France), and Laboratorios Silanes (Mexico City, Mexico) outside the submitted work. Dr. Figueiredo has also participated as a principal investigator in a randomized clinical trial sponsored by Mundipharma (Cambridge, UK) and in another clinical trial as a sub-investigator for Menarini Richerche (Florence, Italy). Dr. Eduard Valmaseda-Castellón reports grants, personal fees, and non-financial support from MozoGrau (Valladolid, Spain), Inibsa Dental (Lliçà de Vall, Spain) Dentaid SL (Cerdanyola del Vallés, Spain) and personal fees from BioHorizons Iberica (Madrid, Spain) and Laboratorios Silanes (Mexico City, Mexico) outside the submitted work. Dr. Eduard Valmaseda-Castellón has also participated as a principal investigator in a randomized clinical trial sponsored by Geistlich Pharma AG (Wolhusen, Switzerland) and in another clinical trial as a sub-investigator for Mundipharma (Cambridge, UK). The remaining authors report no conflicts of interest. Dr. Octavi Camps-Font reports grants and non-financial support from Inibsa Dental (Lliçà de Vall, Spain) and Dentaid SL (Cerdanyola del Vallés, Spain), non-financial support from Nobel Biocare, and Avinent SLU outside the submitted work., (Copyright © 2024. Published by Elsevier Ltd.)

Objective: To evaluate the diagnostic performance of ChatGPT-4.0 in histopathological diagnoses of oral and maxillofacial lesions and compare its performance with pathologists., Study Design: A retrospective analysis of 102 histopathological descriptions was conducted. Data, including site, age and sex, were anonymized from the General University Hospital's Department of Pathology. ChatGPT-4.0 provided diagnoses, which were categorized as correct, similar, or different compared to pathologists' diagnoses. Descriptive statistics, Chi-squared tests, correlation, and regression analyses were used to assess accuracy and the influence of age and gender., Results: ChatGPT-4.0 correctly diagnosed 61 out of 102 cases, yielding an accuracy of 59.8%. The distribution of diagnostic scores did not significantly deviate from expectations (Chi-squared Statistic: 0.0, P = 1.0). A moderate negative correlation between age and diagnostic scores (r = -0.33) was observed, with age significantly predicting scores (P = .001). No significant difference was found between genders (P = .26). ChatGPT-4.0 performed worst with granuloma and inflammation cases (100% incorrect) and best with mucocele cases (93.3% correct)., Conclusion: ChatGPT-4.0 shows moderate accuracy in histopathological diagnosis of oral and maxillofacial lesions, with performance varying by lesion type. Improvements are needed to enhance its clinical reliability., Competing Interests: DECLARATION OF INTERESTS None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Chronic painful temporomandibular disorders (TMD) are a functional pain syndrome that embodies a spectrum of clinical manifestations and expresses great complexity through the coexistence of multiple comorbidities., Objective: Evaluate the prevalence of pain comorbidities, explore the correlation between comorbidities, kinesiophobia, catastrophizing, and pain disability at baseline, and determine variables of interest for prediction response to physiotherapy interventions at different follow-up times., Methods: This is a secondary analysis of a previously conducted randomized controlled trial (RCT). Outcomes and covariables for this secondary analysis were collected from subjects with chronic TMD (≥3 months), based on the DC/TMD diagnostic criteria, assigned to a multimodal treatment and a second manual therapy control group. The participants underwent follow-up assessment at 7 and 19 weeks. The outcomes (dependent variables) for these analyses were craniofacial pain disability, catastrophizing, and kinesiophobia. However, these outcomes were also used as covariables of interest in addition to comorbidity prevalence and severity at baseline for different analyses. We performed linear regression analyses to determine the association between our outcomes and covariables at baseline and at different follow-ups., Results: Comorbidity severity explained craniofacial pain disability at baseline, showing a strong correlation (Standardized B: 0.40, p-value: 0.017). Higher comorbidity severity, led to higher craniofacial pain score. Comorbidity severity and baseline catastrophizing predicted a better response to treatment for craniofacial pain improvements at short and long term (R 2 .22, p-value : 0.009 and R 2 .19, p-value: 0.02 respectively)., Conclusion: This study provides insight into the prevention and clinical management of chronic pain related to TMD.

The frontal polymerization (FP) of carbon/epoxy (C/Ep) composites is investigated, considering FP as a viable route for the additive manufacturing (AM) of thermoset composites. Neat epoxy (Ep) resin-, short carbon fiber (SCF)-, and continuous carbon fiber (CCF)-reinforced composites are considered in this study. The evolution of the exothermic reaction temperature, polymerization frontal velocity, degree of cure, microstructures, effects of fiber concentration, fracture surface, and thermal and mechanical properties are investigated. The results show that exothermic reaction temperatures range between 110 °C and 153 °C, while the initial excitation temperatures range from 150 °C to 270 °C. It is observed that a higher fiber content increases cure time and decreases average frontal velocity, particularly in low SCF concentrations. This occurs because resin content, which predominantly drives the exothermic reaction, decreases with increased fiber content. The FP velocities of neat Ep resin- and SCF-reinforced composites are seen to be 0.58 and 0.50 mm/s, respectively. The maximum FP velocity (0.64 mm/s) is observed in CCF/Ep composites. The degree of cure (α c ) is observed to be in the range of 70% to 85% in FP-processed composites. Such a range of α c is significantly low in comparison to traditional composites processed through a long cure cycle. The glass transition temperature (T g ) of neat epoxy resin is seen to be approximately 154 °C, and it reduces slightly to a lower value (149 °C) for SCF-reinforced composites. The microstructures show significantly high void contents (12%) and large internal cracks. These internal cracks are initiated due to high thermal residual stress developed during curing for non-uniform temperature distribution. The tensile properties of FP-cured samples are seen to be inferior in comparison to autoclave-processed neat epoxy. This occurs mostly due to the presence of large void contents, internal cracks, and a poor degree of cure. Finally, a highly efficient and controlled FP method is desirable to achieve a defect-free microstructure with improved mechanical and thermal properties.

From over-exploitation of resources to urban pollution, sustaining well-being requires solving social dilemmas of cooperation. Often such dilemmas are studied assuming that individuals occupy fixed positions in a network or lattice. In spatial settings, however, agents can move, and such movements involve costs. Here we investigate how mobility costs impact cooperation dynamics. To this end, we study cooperation dilemmas where individuals are located in a two-dimensional space and can be of two types: cooperators-or cleaners, who pay an individual cost to have a positive impact on their neighbours-and defectors-or polluters, free-riding on others' effort to sustain a clean environment. Importantly, agents can pay a cost to move to a cleaner site. Both analytically and through agent-based simulations we find that, in general, introducing mobility costs increases pollution felt in the limit of fast movement (equivalently slow strategy revision). The effect on cooperation of increasing mobility costs is non-monotonic when mobility co-occurs with strategy revision. In such scenarios, low (yet non-zero) mobility costs minimise cooperation in low density environments; whereas high costs can promote cooperation even when a minority of agents initially defect. Finally, we find that heterogeneity in mobility cost affects the final distribution of strategies, leading to differences in who supports the burden of having a clean environment., (© 2024. The Author(s).)

Background: The 8th edition of the American Joint Committee on Cancer (AJCC) classification has introduced two new parameters: depth of invasion (DOI) and extranodal extension (ENE). The aim of this systematic review was to determine whether this 8th edition referred to oral squamous cell carcinoma (OSCC) offers performance superior to that of the 7th edition in relation to overall survival (OS) and disease-specific survival (DSS)., Material and Methods: The review was carried out following the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-analyses) guidelines. The PubMed (MEDLINE), Scopus and Cochrane Library databases were searched covering the period up until April 7th, 2022., Results: Thirteen retrospective cohort studies were finally included. The introduction of DOI and ENE in the 8th edition of the AJCC classification resulted in improved prognostic performance of the classification., Conclusions: Patients with OSCC can be better classified in relation to OS and DSS, while maintaining the simplicity and ease of use of the classification. This allows more appropriate treatment protocols to be applied and affords a better estimation of the prognosis of each patient.

Background: Demonstration of access to the bile duct through Enlarged Papillary Fistulotomy, a method different from conventional fistulotomy., Aims: Demonstration of the EFP technique with dissection in layers of the papilla for accessing the common bile duct, its efficiency and safety, rescue of cases of failure in cannulation and cases of access failure by EFP in the first attempt, facilitating cannulation in the second attempt., Methods: Cross-sectional study, with retrospective data collection from 2233 ERCP exams with 528 EFP procedures, analysis of success and complications., Results: 528 patients underwent EFP on the first attempt, with success in 465 cases (88.06%) and 63 failures (11.94%). Of these failures, 33 patients (52.38%) returned for a second EFP attempt, with success in 30 cases (90.9%) and failure in 3 cases (9.1%). Deep bile duct cannulation was achieved in 93.75% of EFP procedures, and cannulation failure occurred in 33 cases (6.25%)., Conclusion: EFP showed efficiency in CBD cannulation, did not induce post-ERCP pancreatitis, no cases of perforation or false tract, but resulted in higher rates of minor bleeding, rescued cases of access failure by EFP, facilitated the posterior approach on the second attempt, it is safe, effective, low risk and associated with few comorbidities., (© 2023. The Author(s).)

Background: There is a sex-related difference in strength and endurance in trunk muscles: males have more strength while females have more endurance., Objective: Investigate sex-related differences in motor control strategies in back muscles during isometric contractions (IC) in the Sorensen test posture., Methods: Thirty-six healthy and young volunteers performed different tasks: three maximal voluntary contraction (MVC) tests recorded with a bio-feedback force sensor, and followed by five loaded IC (LIC) tests using bodyweight and loads of 0 to 8 kg with a 2-kg step. Surface electromyography was used to measure the activity of bilateral lumbar paravertebral (LP) and quadratus lumborum (QL) muscles., Results: Sex-related and load-related differences in EMG amplitudes were highlighted during LIC tests. Females showed significantly (p-value = 0.02) greater EMG amplitude for the highest load (8 kg) than males. Besides, significant differences between low (2 and 4 kg) and high (6 and 8 kg) loads for both LP and QL muscles and for both sexes were observed. Finally, for MVC tests, males produced significantly (p-value = 1.02e-4) greater strength during MVC tests (4.25 ± 1.37 N/kg vs 2.60 ± 0.78 N/kg)., Conclusion: The results were clinically relevant to bring attention to load strategy during rehabilitation, particularly in females who seemed to recruit the QL muscle more.

Background: The primary objective of this study was to determine the position and course of the greater palatine artery using color doppler ultrasound. The secondary objective was to determine the thickness of the palatine fibromucosa., Material and Methods: A pilot case series study was performed in a private clinic during February 2020. The scans were performed with a Mindray® M9 ultrasound machine (Mindray North America, NJ, USA) coupled to an L16-4Hs® hockey-type angled probe. For each participant, the arterial path and thickness of the palatal fibromucosa were determined at 5 different points., Results: A total of 6 volunteers (3 males and 3 females) with a mean age of 39.2 (±16) years were included. While the thickness of the fibromucosa decreased along the anterior area, the distance from the cementoenamel junction to the position of the artery was generally maintained up to the canine position, where it was found to be closed to teeth., Conclusions: Color doppler ultrasound allows accurate localization of the artery as well as measurement of the thickness of the palatine fibromucosa. It would help to select the best area for graft harvesting in order to avoid bleeding complications due to vascular sectioning. Key words: Hard palate, doppler ultrasonography, diagnosis, connective tissue graft., Competing Interests: Conflicts of interest The authors Raúl Sampietro-Martínez, Javier Pérez-Monreal and Javier Bara-Casaus declare that they have no conflict of interest. Dr. Sánchez-Torres reports personal fees from Unither Pharmaceuticals (Paris, France) and from Mundipharma Research (Cambridge, United Kingdom), both outside the submitted work. Prof. Dr. Gay-Escoda reports personal fees from Unither Pharmaceuticals (Paris, France), from Mundipharma Research (Cambridge, United Kingdom) and Menarini Research (Florence, Italy), outside the submitted work., (Copyright: © 2022 Medicina Oral S.L.)

Alkaptonuria is a rare autosomal recessive metabolic disorder with wide systemic involvement including pigment deposition. We present an unusual case diagnosed by an image obtained via telemedicine showing pigment deposition in the earlobe. We highlight how this clue may allow prompt diagnosis of alkaptonuria and prevent disease progression. Click https://www.wileyhealthlearning.com/#/online-courses/5da3bb51-40d1-4d42-9c4b-610d68106e25 for the corresponding questions to this CME article., (© 2022 British Association of Dermatologists.)

Background Surgical management of headache due to anomalies in the cervical spine is uncommon, as most cases improve with drugs and/or physical therapy. Case We report two instances of a very uncommon congenital upper cervical spine anomaly due to the presence of a pseudoarthrosis between a unilateral paracondylar process in the base of the skull and an epitransverse process arising from the transverse apophysis (PCP/ETA). The first one corresponds to a male on whom an endoscopic guided puncture was performed, and the second to an adult male from the Neolithic period who showed two cranial trepanations together with the presence of morphine metabolites in both bones and dental calculus. Discussion We draw a parallel between the treatment of two individuals separated by a gap of more than 4800 years: contemporary direct vision of the false joint through a small endoscope, which provides an accurate puncture, and ancient double trepanation with clear signs of bone eburnation.

Objective: Dengue virus consists of four antigenically distinct serotypes (DENV 1-4) that are transmitted to humans by Aedes aegypti and Aedes albopictus mosquitoes. In many dengue-endemic regions, co-circulation of two or more DENV serotypes is fairly common increasing the likelihood for exposure of the two vectors to multiple serotypes. We used a model system of DENV-2 and DENV-4 to investigate how prior exposure of Aedes aegypti to one DENV serotype affects its susceptibility to another serotype., Methods: Aedes aegypti mosquitoes were sequentially infected with DENV-2 and DENV-4 and the infection and dissemination rates for each virus determined., Results: We found that prior infection of Ae. aegypti mosquitoes with DENV-4 rendered them significantly less susceptible to secondary infection with DENV-2. Although the results were not statistically significant, mosquitoes infected with DENV-2 were also less susceptible to secondary infection with DENV-4. The midgut dissemination and population dissemination rates for DENV-2 were significantly higher than those of DENV-4 when either virus was administered 7 days after administration of either a non-infectious blood meal or a blood meal containing a heterologous dengue serotype., Conclusion: These results demonstrate that superinfection interference between DENV serotypes is possible within Ae. aegypti mosquitoes, but its effect on DENV epidemiology may be dependent on the fitness of interacting serotypes., (© 2017 John Wiley & Sons Ltd.)

Background: Mosquitoes host diverse microbial communities that influence many aspects of their biology including reproduction, digestion, and ability to transmit pathogens. Unraveling the composition, structure, and function of these microbiota can provide new opportunities for exploiting microbial function for mosquito-borne disease control., Methods: MiSeq® sequencing of 16S rRNA gene amplicons was used to characterize the microbiota of adult females of Culex pipiens L. and Cx. restuans Theobald collected from nine study sites in central Illinois., Results: Out of 195 bacterial OTUs that were identified, 86 were shared between the two mosquito species while 16 and 93 OTUs were unique to Cx. pipiens and Cx. restuans, respectively. The composition and structure of microbial communities differed significantly between the two mosquito species with Cx. restuans hosting a more diverse bacterial community compared to Cx. pipiens. Wolbachia (OTU836919) was the dominant bacterial species in Cx. pipiens accounting for 91% of total microbiota while Sphingomonas (OTU817982) was the dominant bacterial species in Cx. restuans accounting for 31% of total microbiota. Only 3 and 6 OTUs occurred in over 60% of individuals in Cx. pipiens and Cx. restuans, respectively. There was little effect of study site on bacterial community structure of either mosquito species., Conclusion: These results suggest that the two mosquito species support distinct microbial communities that are sparsely distributed between individuals. These findings will allow investigations of the role of identified microbiota on the spatial and temporal heterogeneity in WNV transmission and their potential application in disease control.

Despite the growing awareness that larval competition can influence adult mosquito life history traits including susceptibility to pathogens, the net effect of larval competition on human risk of exposure to mosquito-borne pathogens remains poorly understood. We examined how intraspecific larval competition affects dengue-2 virus (DENV-2) extrinsic incubation period and vectorial capacity of its natural vector Aedes albopictus. Adult Ae. albopictus from low and high-larval density conditions were orally challenged with DENV-2 and then assayed for virus infection and dissemination rates following a 6, 9, or 12-day incubation period using real-time quantitative reverse transcription PCR. We then modeled the effect of larval competition on vectorial capacity using parameter estimates obtained from peer-reviewed field and laboratory studies. Larval competition resulted in significantly longer development times, lower emergence rates, and smaller adults, but did not significantly affect the extrinsic incubation period of DENV-2 in Ae. albopictus. Our vectorial capacity models suggest that the effect of larval competition on adult mosquito longevity likely has a greater influence on vectorial capacity relative to any competition-induced changes in vector competence. Furthermore, we found that large increases in the viral dissemination rate may be necessary to compensate for small competition-induced reductions in daily survivorship. Our results indicate that mosquito populations that experience stress from larval competition are likely to have a reduced vectorial capacity, even when susceptibility to pathogens is enhanced.

Regenerative medicine approaches to cartilage tissue repair have mainly been concerned with the implantation of a scaffold material containing monolayer expanded cells into the defect, with the aim to differentiate the cells into chondrocytes. While this may be a valid approach, the secretome of the implanted cells and its effects on the endogenous resident cells, is gaining in interest. This review aims to summarize the knowledge on the secretome of mesenchymal stem cells, including knowledge from other tissues, in order to indicate how these mechanisms may be of value in repairing articular cartilage defects. Potential therapies and their effects on the repair of articular cartilage defects will be discussed, with a focus on the transition from classical cell therapy to the implantation of cell free matrices releasing specific cytokines., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Mosquitoes transmit a number of arboviruses associated with disease outbreaks in humans and other animals. The majority of medically important arboviruses belong to three families: Togaviridae, Flaviviridae and Bunyaviridae. Several members of these families have overlapping distributions and share common vectors, increasing the potential for arboviral coinfections. This study examined how two model viruses: Sindbis virus (SINV, Togaviridae: Alphavirus) and dengue-4 virus (DENV-4, Flaviviridae: Flavivirus) may interact in C6/36 Aedes albopictus cells and in the mosquito vector Ae. albopictus., Methods: C6/36 cells were coinfected, superinfected, or singly infected with SINV and DENV-4 and the two viruses quantified at different time points. Four to seven day old adult females of Ae. albopictus were also fed blood containing one or both viruses and viral infection and dissemination rates determined., Results: Sindbis virus suppressed replication of DENV-4 in C6/36 Ae. albopictus cells with greater inhibition occurring when the two arboviruses were inoculated simultaneously compared to sequentially. In addition, Ae. albopictus simultaneously exposed to both arboviruses had significantly lower DENV-4 infection and population dissemination rates compared to those exposed to DENV-4 alone., Conclusion: These results suggest that certain Alphaviruses may interfere with DENV-4 transmission by suppressing its replication and increasing vector refractoriness. The findings provide important insights into the potential contribution of mixed arboviral infections to DENV transmission dynamics.

Objectives: Biomarker detection in pancreatic cyst fluids is of importance to improve the diagnosis of mucinous cystadenoma, a precancerous lesion. However, assay protocols are generally established for serum testing., Methods: Immunoradiometric assay of gastric M1/MUC5AC mucin was performed on pancreatic cyst fluids with well-characterized monoclonal antibodies., Results: Among 1466 pancreatic cyst fluids tested, about 10% to 15% of samples presented abnormal behaviors: (i) radioactivity measured after immunoradiometric assay much lower than the blank of the assay and (ii) increasing dilution of the fluids leading to apparent increase of M1/MUC5AC concentration. In contrast, none of the 109 hepatic cyst fluids tested presented interference.We demonstrate that some (n = 54) interfering fluids cause mucin degradation as well as antibody degradation. Western blot analysis showed that the C-terminal part of the M1/MUC5AC apomucin is most sensitive to degradation., Conclusions: The presence of proteases that degrade antibodies as well as mucin may explain the pitfalls observed in 3.6% of the samples. To detect this interference, each fluid has to be systematically tested at 1:100 dilution in the presence of a saturating concentration of M1/MUC5AC mucin standard and in the absence of antiprotease reagents. Detection of interference could prevent false results caused by mucin degradation in situ.

Aims: During colonic carcinogenesis, mucin-type glycoproteins are known to undergo quantitative and qualitative alterations. The aim of this study was to determine the value of infrared (IR) spectral histology for the histopathological recognition of colonic adenocarcinomas based on mucin-associated IR spectral markers., Methods and Results: Paraffin-embedded tissue sections of normal human colon and adenocarcinomas were analysed directly by IR-microspectroscopy (IR-MSP), without prior chemical dewaxing. IR-MSP imaging combined with multivariate analysis permitted the construction of IR colour-coded images of the tissue sections providing spatially resolved biochemical information. This allowed localization of mucin-rich areas and provided label-free spectral-based staining of secreted mucus related to the biochemical heterogeneity of its mucin content. IR images of secreted mucus display the same spectral clusters in both normal and adenocarcinomatous colonic tissues, but with significant differences in surface percentages. Such differences allow a distinction between these two tissue types. Spectral variations associated with changes of mucin secondary structure were the most accurate mucus spectral marker for discriminating between normal colon and adenocarcinomas in the sample set., Conclusions: IR-MSP imaging provides a new type of histology, independent of visual morphology, presenting tremendous possibilities for discovery and clinical monitoring of cancer markers.

Unlabelled: Mucins are related to infectious and non-infectious diseases in Veterinary and Human Medicine. MUC1 mucin is a transmembrane glycoprotein expressed on the apical surface of human epithelia while MUC5AC is the predominant secreted mucin expressed in human gastric epithelium and goblet cells of lung and eyes. MUC5AC C-terminus cysteine rich regions and the cytoplasmic tail of MUC1 domains are conserved among several mammalian species., Objective: to compare the expression of MUC1 and MUC5AC mucins in mammalian epithelia. CT33 anti-MUC1 cytoplasmic tail (MUC1CT) polyclonal antibody and 45M1 anti-MUC5AC monoclonal antibody were employed. By immunohistochemistry, MUC1CT was expressed in most tissues while MUC5AC was restricted to gastric surface epithelium and goblet cells from trachea and lung. By western blot, MUC1CT showed a band at approximately 35 kDa in most tissues; MUC5AC revealed bands at >180 kDa in stomach and lung secretions from rat, cat, pig and cow. When rat MUC5AC was immunoprecipitated, a band at about 180 kDa was obtained.

Mucins are large glycoproteins protecting mucosal surfaces throughout the body. Their expressions are tissue-specific, but in disease states such as cystic fibrosis, inflammation and cancer, this specificity can be disturbed. MUC5AC is normally expressed in the mucous cells of the epithelia lining the stomach and the trachea, where it constitutes a major component of the gastric and respiratory mucus. A number of mAbs have been raised against the gastric M1 antigen, an early marker for colonic carcinogenesis. Several of these mAbs recognize epitopes present on MUC5AC, suggesting that MUC5AC is the antigen. However, some of the mAbs raised against the gastric M1 antigen are widely used as antibodies against MUC5AC, despite the fact that their specificity for MUC5AC has not been clearly shown. In this study, we have tested the reactivity of the latter antibodies against a recombinantly expressed C-terminal cysteine-rich part of human MUC5AC. We demonstrate for the first time that the widely used mAb 45M1, as well as 2-12M1 and 166M1, are true antibodies against MUC5AC, with epitopes located in the C-terminal cysteine-rich part of the mucin.

The abnormal expression of gastric M1/MUC5AC mucin in precancerous lesions and colon cancer evidenced by immunohistochemistry led us to check for its presence in the mucus obtained directly from patients undergoing surgery for cancerous (adenocarcinoma) or inflammatory (diverticulitis or ulcerative colitis) diseases. In parallel, the authors quantified aberrant crypt foci (ACF) and their immunolabelling by M1/MUC5AC in mucosae of cancer and diverticulitis patients. Immuno-Radio-Metric Assay of M1/MUC5AC mucin developed by the authors was used to detect M1/MUC5AC mucin in the colonic mucus scraped from surgical specimens. M1/MUC5AC mucin was detected in the mucus of 51/69 (74%) patients with colon adenocarcinoma, versus 7/27 (26%) patients with diverticulitis (threshold: 30 units of M1 mucin per mg protein, area under ROC curve: 0.80). M1/MUC5AC was present in significantly (p < 0.001) larger amounts in the mucus of cancer versus diverticulitis patients. All (10/10) patients with ulcerative colitis tested showed levels above the threshold and their mucosae were strongly labelled with the anti-M1/MUC5AC antibody by immunohistochemistry. Patients with cancer exhibited 3 fold more ACF than those with diverticulitis, but no significant difference was observed in the mean size and M1/MUC5AC expression pattern of ACF between these two groups. The expression of M1/MUC5AC was in correlation with their size. In macroscopically normal mucosa, ACF were the most important source of M1/MUC5AC mucin. Testing of M1/MUC5AC can enhance the detection of precancerous lesions and colon cancer.

Aberrant crypt foci (ACF) are microscopic lesions which have been postulated to precede the development of adenomas, precursors of colon cancer. The gastric M1/MUC5AC mucin has also been described as an early marker of colon carcinogenesis in the human and in the rat. To study changes in mucin expression associated with the genesis of tumors, Wistar rats were treated by intrarectal instillations of MNNG, twice a week for 2 weeks, and were sacrificed 10 (n = 20), 14 (n = 20), 22 (n = 20), 30 (n = 10) and 66 (n = 16) weeks after the beginning of the treatment. In the treated rats, the MUC5AC mucin was mainly expressed in ACF compared with the histologically normal mucosae, which showed few isolated MUC5AC-positive normal crypts. During carcinogenesis, the percentage of large ACF [> or =10 aberrant crypts] increased and the number of MUC5AC-positive (NCs) decreased. At Week 30, small tumors were observed arising from large ACF, both types of lesions expressing MUC5AC. At Week 66, large tumors showed remnants of MUC5AC-positive ACF in their adjacent mucosae. This observation suggests that the expression of MUC5AC is associated with the ACF/adenoma sequence and supports the notion of large ACF as precursors of adenomas/adenocarcinomas. Moreover, the expression of MUC5AC in the transitional mucosa adjacent to both rat and human colon tumors suggests that some human tumors could arise from large ACF, and reinforces the concept of the premalignant potential of these lesions.

Our study was performed to sequentially analyze the expression of the intestinal mucin MUC2 and of the gastric mucin MUC5AC as indicators during progression of preneoplastic biomarkers in rat colon. F344 rats were sacrificed 2, 4, 8, 12, 24 and 36 weeks after injection of 1,2-dimethylhydrazine (DMH, 200 mg/kg, i.p.). The expression of MUC2 and of MUC5AC was studied by immunohistochemistry in preneoplastic lesions classified in two categories: histologically altered foci (HAF) and beta-catenin accumulated crypts (BCAC). HAF appeared 4 weeks after DMH injection. Their crypt multiplicity stagnated with time (3-4 crypts/foci) but gastric MUC5AC mucin was always observed in some goblet cells of the lesions of this category. In contrast, MUC2-immunostaining was not modified compared to the adjacent crypts. Double-immunofluorescence revealed that goblet cells which produced MUC5AC continued to express MUC2. In BCAC, crypt multiplicity and mucin expression strongly evolved with time. These lesions were observed only 8 weeks after DMH-injection. At this stage, 20% of BCAC showed a decreased MUC2 expression and 33% were MUC5AC immunopositive. At the 36-week point, 43% of BCAC had a reduced MUC2 staining and 90% were positive for MUC5AC. This immunopositivity was often observed in all the cells of these lesions. Seldom, some BCAC were depleted at the same time in MUC2 and in MUC5AC. Similar alterations in mucin expression were observed in human colonic pre-neoplastic lesions. These findings suggest that a decrease in MUC2 expression and staining of MUC5AC in non-goblet-like cells predicts histological progression of preneoplastic lesions.

Trefoil factor family (TFF) of proteins are involved in mucosal protection and healing and are induced in inflammatory diseases and neoplastic progression. The purpose of this investigation was to determine if expression of the trefoil factor family (TFF) proteins is altered in human pterygium compared to in normal conjunctiva. Fourteen pterygia (P) and 21 biopsies from normal human conjunctiva (NC) were studied. TFF1, TFF2 and TFF3 mRNA levels were measured by semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR), and TFF1 mRNA levels in addition by real-time PCR. The cellular expression of TFF1 (pS2), TFF3 (intestinal trefoil factor) and M1/MUC5AC mucin in ten pterygia and ten normal human conjunctiva specimens was analyzed by immunohistochemistry using specific monoclonal antibodies. TFF1 mRNA levels were higher in P than in NC (p=0.02). Accordingly, intensity of TFF1 and mucin MUC5AC immunostaining was higher in P than in NC. Mucus-secreting goblet cells (GC) were more densely packed in P than in NC. In both cases, TFF1 protein was detected in GC only, but was not systematically expressed in all GC. In addition, TFF3 mRNA levels were similar (p=0.89) in NC and P, while TFF2 (spasmolytic polypeptide) mRNA were not detected. Both TFF3 and MUC5AC proteins were clearly detected in all GC identified in NC and P. Increased expression of TFF1 mRNA and protein is observed in pterygium GC, suggesting that this trefoil protein might exert protective and beneficial roles during the pathogenesis of this benign and inflammatory conjunctival tumor.

Purpose: We sought to assess dark lumen magnetic resonance (MR) colonography for the detection of colon polyps in a rodent model with histology as the gold standard., Material and Methods: Fourteen male Wistar rats were subjected to carcinogenic N-methyl-N'-nitro-N-nitrosoguanidine at the age of 4 months to induce colon neoplasms. MR imaging was performed after a time interval of 1 year. Preparation and data acquisition was performed with the animals under full anesthesia. After a body-warm saline enema images were acquired on a clinical 1.5-T whole-body MR system using a standard extremity coil. Plain and contrast-enhanced (0.3 mmol/kg; Gd-DOTA; Dotarem, Guerbet, France) 3-dimensional T1-weighted gradient recall echo images were acquired. Two radiologists analyzed the MR data sets in consensus for lesion depiction. Contrast uptake in colonic wall and polyps was quantitatively assessed by signal-to-noise ratio and contrast-to-noise ratio measurements and compared using a Wilcoxon-Mann-Whitney U test with statistical significance at a P value < 0.05. Finally, all animals were killed, and the MR imaging results were compared with pathologic findings. Sensitivity and specificity were calculated., Results: By pathology, a total of 15 polyps were found in 9 of 14 rats. MR colonography detected 13 of 15 polyps measuring between 4 and 11 mm (mean 7 +/- 0.6 mm) in 8 of 9 animals, resulting in a sensitivity and specificity of 0.87 and 1.0, respectively. Compared with the precontrast data, all polyps showed a statistically significant increase in signal-to-noise ratio (78.2 +/- 6.3 to 167.4 +/- 17.7) and contrast-to-noise ratio (45.4 +/- 5.2 to 124.6 +/- 11.2)., Conclusion: MR colonography with a dark colon lumen and a bright, contrast-enhanced colon wall appears well suited for the detection of colonic lesions in a rodent model.

Aim: To study the MUC5AC and the blood group related antigen expression in ocular cicatricial pemphigoid (OCP) according to the distribution of Lewis and secretor phenotypes in OCP patients compared to normal subjects., Methods: Immunostaining was performed on conjunctival biopsy specimens from 22 consecutive patients suffering from OCP, using monoclonal antibodies (Mabs) directed against the peptidic core MUC5AC mucin (anti-M1/MUC5AC Mabs) and against the saccharide moieties (anti-blood group related antigens). These latter included anti-Le(a), anti-Le(b), anti-sialyl Le(a), and H type 2 Mabs, which immunoreact with Lewis positive and non-secretor (Le(a)), Lewis positive and secretor (Le(b)), Lewis positive (sialyl Le(a)), and secretor (H type 2) phenotypes respectively. Serological tests were also performed to confirm the phenotype of each patient. The immunohistopathological patterns and the distribution of Lewis and secretor phenotypes were compared with the results of a previous study in normal individuals., Results: (1) In OCP patients compared to the normal population, anti-M1 immunoreactivity of goblet cells was unchanged, whereas anti-Le(a), anti-Le(b), and anti-sialyl Le(a) immunoreactivities of epithelial and/or goblet cells were markedly decreased. (2) 41% of OCP patients had a non-secretor phenotype, which is statistically significantly more than the estimated incidence of the same phenotype in the French population (20%) (p approximately 0.04)., Conclusions: Mucins in OCP patients showed a decreased expression of blood group related antigens whereas the MUC5AC peptidic core detected by anti-M1 Mab remained unchanged. These results also seem to indicate that OCP may be associated with a non-secretor phenotype.

The expression of mucin genes was evaluated in rat intestinal cell lines in order to establish an in vitro model for investigating the regulation of intestinal mucin expression in this species. Two rat intestinal cancer cell lines (DHE, LGA) and three nontumoral rat intestinal cell lines (IEC6, IEC17, IEC18) were screened. The mRNA expression of rMuc1, rMuc2, rMuc3, rMuc4, and rMuc5AC mucin genes was studied by semiquantitative RT-PCR, real-time RT-PCR and Northern-blot analysis. Results were correlated with immunohistochemical expression of rat gastric and intestinal mucin proteins, and secretion of glycoconjugates was examined by enzyme-linked lectin assay. We showed that mRNA of rMucl and rMuc2 were constitutively expressed in all IEC cell populations but periodic acid Schiff staining of these cells did not reveal the presence of glycoproteins. DHE cells expressed rMuc1-5AC mRNA and LGA expressed the same mucins but the level of rMuc4 was much lower. Mucin mRNA expression also differed in relation with the length of cultivation. Immunocytochemical studies revealed the presence of gastric and intestinal mucins in the two tumoral cell lines. Functional experiments showed that bethanechol, A23187 and PMA stimulated release of glycoconjugates in DHE but not in LGA cells. Treatment of DHE cells with dexamethasone (10(-7) mol/l) enhanced rMuc2 mRNA but decreased rMuc1 and rMuc5AC mRNA. Real-time RT-PCR showed that the expression of rMuc1 and rMuc5AC genes was reduced by more than tenfold after 24 h. The increased expression of rMuc2 gene was confirmed by Northern blot analysis. In conclusion, DHE cells provide a valuable cellular model for research on rat mucin secretion and expression.

We have developed 11 monoclonal antibodies (MAbs) against human gastric mucin, (1-13M1, 2-11M1, 2-12M1, 9-13M1, 58M1, 19M1, 21M1, 45M1, 463M, 589M, 62M1), which specifically stained by immunohistochemisty both the human gastric surface mucosa and colon adenoma. Among them, five (19M1, 21M1, 463M, 589M, 62M1) immunoreacted with the peptide encoded by the 3' region of the MUC5AC gene (Nollet et al: Int J Cancer 2002;99:336-343). In this study, we identified in the 5' region of this gene the nucleotide fragments encoding peptides immunoreacting with three other anti-M1 MAbs (1-13M1, 2-11M1 and 9-13M1), as well as the SOMU1 MAb (Sotozono et al: J Immunol Methods 1996;192:187-196). 1-13M1 MAb immunoreacts with peptides, including the Cys 2 and Cys 4 domains. The SOMU1 MAb recognized the Cys 5 domain, and the MAbs 2-11M1 and 9-13M1 the globular D1/D2 and D3 domains, respectively. Using serial sections of the mucosae adjacent to colon adenocarcinomas and colon adenomas, we observed that the anti-M1 and anti-SOMU1 MAbs displayed the same immunostaining patterns. The three anti-M1 MAbs (2-12M1, 58M1, and 45M1) did not react with the products of the MUC5AC gene tested until now. The MUC5AC apomucin is now well characterized by MAbs immunoreacting against seven different epitopes belonging to the different main cystein globular domains of this macromolecule. Such antibodies are useful tools for studying the biosynthesis, polymerization, and degradation of mucin.

Many theories mention hypersensitive, promiscuous, outlaw or bypass signalling pathways to explain the acquisition of hormone independence in prostate cancer. Hormonal escape of prostate tumours is marked by many biological changes, including mucinous and neuroendocrine differentiation. Since expression of several mucins has been linked to carcinoma tumour progression, we have characterised the expression of mucins at both RNA and protein levels in an in vivo model of prostate cancer in hormonal escape. Using PAC120, a xenograft of a human hormone-dependent prostate tumour, and its hormone-independent variants, we analysed the expression of mucins (MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC6) by immunohistochemistry or reverse transcriptase (RT)-PCR. While the parental PAC120 tumour was a compact poorly-differentiated tumour of Gleason score 9 (5+4), hormone-independent variants displayed mucinous, neuroendocrine-like or mixed histological changes; these changes were stable through serial transplantations or after testosterone supply. MUC1 mRNA was expressed in both PAC120 and the hormone-independent variants, although at variable levels. All tumours displayed a high and constant expression of MUC2 and no expression of MUC4 mRNA. While MUC1 was expressed in all xenografts whatever their hormone dependence status, MUC2, MUC5B and MUC6 were preferentially expressed in hormone-independent variants. The loss of hormone dependence in this prostate cancer xenograft model is therefore marked by irreversible histological alterations, mucinous or neuro-endocrine, associated with an expression of secretory MUC2, MUC5B and MUC6, independent of the histological differentiation subtype. These data point to mucinous differentiation as an important step in the acquisition of hormone independence in this cancer, and suggest that secretory mucins might participate in an unknown pathway of hormonal escape in prostate cancer.

Purpose: Trefoil factor family (TFF) peptides (formerly P-domain peptides; trefoil factor) are small (7-12 kDa) protease-resistant secreted peptides designated pS2 (or TFF1), SP (TFF2) and ITF (TFF3). Human conjunctival goblet cells (GCs) are known to synthesize TFF, but TFF expression by these cells has not been studied in pathological conditions. We quantified trefoil factor family (TFF) gene transcripts in pterygium, and we immunolocalized TFF protein., Methods: Eleven pterygium specimens were studied, together with 19 biopsy specimens of normal human conjunctiva as controls. TFF1 (pS2), TFF2 (spasmolytic peptide) and TFF3 (intestinal trefoil factor) mRNA expression was semiquantified by means of reverse-transcription polymerase chain reaction amplification (RT-PCR). TFF1, TFF2 and TFF3 mRNA levels were determined individually, relative to beta2 microglobulin housekeeping gene mRNA (internal standard), by coamplification of the target fragments and beta2 microglobulin in the same tube. Five pterygia and five normal human conjunctival biopsy specimens were also analyzed for TFF1 and mucin (MUC5AC) protein expression by immunostaining with monoclonal antibodies. Anti-PS2 (Zymed Laboratories, San Francisco), a mouse monoclonal antibody (MAb) against the 30 C-terminal amino acids of human TFF1, and P2802 (provided by Doctor Marie-Christine Rio, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM, Strasbourg, France), a mouse MAb directed against a synthetic peptide corresponding to the last 28 amino acids of TFF1, were used at 1/20 dilution. A mouse monoclonal antibody directed against the peptidic core of gastric M1 mucin was used as previously described. M1 immunoreactivity is encoded by the MUC5AC gene., Results: TFF1 and TFF3 mRNA was expressed in all normal conjunctival and pterygium specimens. TFF2 mRNA was not expressed by either sample type, but was expressed by the positive control (human stomach cDNA). TFF1 mRNA expression was stronger in pterygium than in controls (p=0.02). TFF3 mRNA expression was similar in the two sample types (p=0.89). TFF are coexpressed and act in concert with mucins to protect mucous epithelia and trigger wound-healing responses. Inflammation and ulceration of the gastrointestinal tract are associated with increased TFF expression. Conjunctival GCs secrete TFF in both pigs and humans. We found that TFF1 mRNA was overexpressed in pterygium relative to healthy conjunctiva, whereas the TFF1 immunostaining patterns were similar. TFF1 protein expression was confined to goblet cells. However, whereas all GCs were positive for MUC5AC, not all GC were labeled by anti-TFF1 mAbs in either normal conjunctiva or pterygium. The observed TFF1 mRNA overexpression in pterygium was not associated with abnormal TFF1 peptide localization. Increased MUC5AC protein expression would be expected in pterygium, because of increased GC density. Indeed, in conjunctival diseases such as dry-eye syndrome in which GC density is decreased, mucin secretion is also decreased. This could explain the increased expression of TFF1 mRNA in pterygium, although not all GCs expressed TFF1 protein. TFF proteins are copackaged within mucous cell granules; TFF1 preferentially colocalizes with MUC5AC, and TFF3 with MUC2. However, we found some cell granules containing MUC5AC but not TFF1. The proportion of TFF1-negative GCs was similar in pterygium and normal conjunctiva. The normal TFF3 mRNA expression in pterygium was unexpected and suggests that only GCs involved in TFF1 secretion are overrepresented in this pathological tissue. TFF2 mRNA was undetectable in both normal conjunctiva and pterygium, possibly because of its copackaging in mucous cell granules and its preferential cosecretion with MUC6, which is not expressed in the conjunctiva., Conclusion: As in normal conjunctiva, the TFF1 and TFF3 genes are expressed by conjunctival goblet cells in pterygium, contrary to the TFF2 gene. Only TFF1 gene expression was elevated in pterygium compared to normal conjunctiva.

Background and Objective: Sentinel node (SN) biopsy represents an alternative to full lymph node dissection in the surgical treatment of several malignant tumors., Patients and Method: Prospective study of 32 consecutive patients with clinically node-negative oral cancer comparing SN biopsy results with standard neck dissection., Results: An effective SN localization was achieved in 31 patients (97%) and a complete agreement with neck dissection was observed: 16 were true negative and 15 were true positive. In 11 out of the 15 positive cases, the SN was the only node containing metastasis (73%)., Conclusion: SN biopsy predicts the subclinical lymph node status in oral cancer patients.

Colorectal cancer is a major cause of death in Europe and the USA, and much effort is therefore devoted to improve its early detection. In this article, we report the abnormal expression of gastric mucin in aberrant crypt foci (ACF) that appear in the colon mucosae removed from colorectal cancer patients and rats treated with methyl-N'-nitro-N-nitroso-guanidine (MNNG). We performed the immunoperoxidase test using monoclonal antibodies raised against gastric M1 mucin encoded by the MUC5AC gene and against rat gastric mucins (MAb 660), respectively. In both human and rat colon, these anti-gastric mucin MAbs stained specifically goblet cells within ACF. In humans, the M1/MUC5AC mucin was expressed in the upper part of the glands in hyperplastic ACF and in the typical ACF. In addition, the anti-gastric mucin MAbs stained some rare, scattered, histologically normal glands in the human and rat colon mucosae. These glands may be regarded as precursors of ACF. The abnormal expression of the MUC5AC gene constitutes a novel change in addition to genetic modifications already observed in ACF, and supports our previous findings demonstrating the potential of this gastric mucin as an early marker of human and rat colon carcinogenesis., (Copyright 2003 S. Karger AG, Basel)

The secreted thiol-activated cytolysin listeriolysin O (LLO) was responsible for L. monocytogenes-induced high-molecular glycoproteins (HMGs) exocytosis in cultured human mucosecreting HT29-MTX cells. By biochemical analysis we demonstrate that the majority of secreted HMGs in LLO-stimulated cells are of mucin origin. In parallel, analysis of the expression of MUCs genes showed that the transcription of the MUC3, MUC4 and MUC12 genes encoding for membrane-bound mucins was increased in LLO-stimulated cells. Upregulation of the MUC3 gene correlates with an increased expression of the membrane-bound MUC3 mucin. In contrast, increase in secretion of the gel-forming MUC5AC mucin develops without upregulation of the MUC5AC gene. Finally, results showed that NF-kappaB and AP-1 transcription factors were not involved in LLO-induced upregulation of MUCs genes in HT29-MTX cells, whereas L. monocytogenes infection was able to promote the degradation of IkappaB proteins in the cells.

Three hybridomas secreting monoclonal antibodies (MAbs) against human (62M MAb) or rat (463M and 589M MAbs) gastric mucins were isolated. These MAbs immunoreacted against a human recombinant protein encoded by the 3' region of the MUC5AC gene. We have mapped 2 new gastric mucin epitopes and the M1-f epitope previously characterized by the 19/21M1 MAbs on MUC5AC-encoded apomucin. The M1-f, 463/589M and 62M epitopes are located in the MUC11p15/von Willebrand factor (vWF)-A3uD4 domain, in the D4-(vWF)-like domain and in the C- and CK-vWF-like domains of MUC5AC, respectively. The 463/589M and 62M MAbs stained the surface epithelium of human gastric mucosae, but not the normal colon mucosae (except 463/589M MAbs, which immunoreacted with 5 of 49 cases). All hyperplastic polyps are stained strongly with the 463/589M MAbs and faintly with the 62M MAb. In addition, 463/589M epitope was detected in 64% of the adenomas and in 93% of the mucosae adjacent to adenocarcinomas; in contrast, only 9% of the adenomas and 29% of the mucosae adjacent to adenocarcinomas expressed the 62M epitope. The expression pattern of the 463/589M epitope in colonic carcinogenesis is different from that of the 19/21M1 epitope, although the 2 epitopes are encoded by MUC5AC gene., (Copyright 2002 Wiley-Liss, Inc.)

Purpose: To evaluate subclinical inflammation and mucus production of the conjunctiva in asymptomatic contact lens (CL) wearers, and to obtain an estimation of the chronologic variations in each group., Methods: Eighteen eyes fitted with rigid CL (RCL) and 28 eyes with soft CL (SCL) worn daily were compared with 10 eyes from five healthy non-CL wearers. Impression cytology (IC) specimens were collected after clinical examination and were analyzed by flow cytometry using antibodies directed to HLA DR and intercellular adhesion molecule type 1 (ICAM-1) (CD 54), as inflammatory markers, and to the peptidic core of the conjunctival mucin (M1/MUC5AC) for mucus and goblet cell detection. The percentage of positive cells was calculated, and levels of fluorescence expression were quantified and compared between each group., Results: A significant increase of HLA DR and ICAM-1 was observed in the SCL group in comparison with the control group. The two inflammatory markers were highly positively correlated with each other. Mucin detection with M1/MUC5AC did not find a significant difference between each group in terms of percentage of positive cells, but analyses of mean levels of fluorescence showed a significant decrease in the two CL groups. Evolution in time was different for each group, with a regular low level of inflammation in the RCL group in the first 10 years in comparison with the SCL group. In the SCL group, inflammation seemed to be higher before 2 years and after 10 years of wear. Mucin expression was variable in time, but without significant difference at any time., Conclusion: This study confirms difference in expression of subclinical conjunctival inflammation in asymptomatic CL wearers, with lower levels for RCL than SCL wearers with daily or extended wear. The mucin system is also modified by this low but chronic aggression of the ocular surface, with a tendency to decrease with time in the RCL and SCL groups.

Mucin staining can be used to evaluate secretory activity of human airways. However, mucin epitopes may be masked by physicochemical properties of the secretions. The aim of this investigation was to examine the effects of the calcium chelator, ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) on the detection of M1/MUC5AC mucin in isolated human bronchial preparations. Immunohistochemical investigation and immunoradiometric assays with anti-M1 monoclonal antibodies (Mabs) were used to detect M1/MUC5AC mucin derived from bronchial preparations with an intact surface epithelium, or in tissues where the epithelium had been removed (rubbed preparations). The Mabs labelled both epithelial goblet cells and submucosal glandular cells in EGTA (4 mM)-exposed bronchial preparations, while only goblet cells were stained in EGTA (0.4 mM)-exposed tissues. The quantities of M1/MUC5AC mucin detected in either the bronchial fluids derived from EGTA (4 mM)-exposed intact and rubbed preparations or in bronchial fluids treated with EGTA (4 mM) were significantly increased by two-fold when compared with untreated control values (p<0.001). In addition, lactate dehydrogenase (LDH) activity and protein measurements were unaltered during exposure of human airways to EGTA (4 mM) suggesting that this treatment did not affect tissue viability. These results provide evidence that ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (4 mM) facilitates the detection of M1/MUC5AC mucin by altering the physicochemical properties of respiratory mucin, thereby exposing epitopes with which anti-M1 monoclonal antibodies are reactive. This will allow more accurate measurement of secretory activity in human airways in vitro.

Background: Increased secretion of mucus is a hallmark of many respiratory diseases and contributes significantly to the airflow limitation experienced by many patients. While the current pharmacological approach to reducing mucus and sputum production in patients is limited, clinical studies have suggested that drugs which inhibit the cyclooxygenase and/or 5-lipoxygenase enzymatic pathways may reduce secretory activity in patients with airway disease., Aim: This study was performed to investigate the effects of indomethacin (cyclooxygenase inhibitor) and Bay x 1005 (5-lipoxygenase inhibitor) on MUC5AC release from human airways in vitro., Methods: An immunoradiometric assay was used to determine the quantities of MUC5AC present in the biological fluids derived from human airways in vitro. The measurements were made with a mixture of eight monoclonal antibodies (MAbs; PM8) of which the 21 M1 MAb recognized a recombinant M1 mucin partially encoded by the MUC5AC gene., Results: The quantities of MUC5AC detected in the biological fluids derived from human bronchial preparations were not modified after treatment with indomethacin (cyclooxygenase inhibitor) and/or an inhibitor of the 5-lipoxygenase metabolic pathway (BAY x 1005)., Conclusion: These results suggest that the cyclooxygenase and 5-lipoxygenase metabolic pathways play little or no role in the release of MUC5AC from human airways.

Erythrocyte polyagglutination antigens T and Tn are truncated O-glycan chains that are also carcinoma-associated antigens. We investigated whether Tk polyagglutination antigen could similarly be a carcinoma-associated marker and a target of immunotherapy. Monoclonal antibody LM389 was raised against Tk erythrocytes and tested by immunohistochemistry. LM389 strongly reacted with 48% human colorectal carcinomas. Labeling of normal tissues was visible on epithelial cells, mainly digestive, but was confined at a supranuclear level. Expression of the antigen on cloned human carcinoma cells correlated with sialosyl-Tn expression. O-Sialoglycoprotein endopeptidase treatment revealed that on carcinomas and cell lines, the epitope was present on O-glycans. Antibody specificity was determined using synthetic carbohydrates. Direct binding and inhibition studies indicated that LM389 best ligands were terminated by two branched N-acetylglucosamine units. Screening of murine cellular cell lines with LM389 allowed development of an experimental model with Tk-positive and -negative cells in syngeneic BDIX rats. Vaccination of rats with Tk erythrocytes provided a protection against growth of rat Tk-positive, but not of Tk-negative, tumor cells in association with the development of antibodies. Taken together, the results indicate that Tk polyagglutination antigen is a new colorectal carcinoma-associated antigen, absent from the normal cell surface, resulting from alteration of O-glycans biosynthesis and with potential as a target of immunotherapy.

Purpose: To investigate by flow cytometry and impression cytology (IC) specimens the inflammatory status of the conjunctival epithelium and goblet cell density in two series of patients with rosacea and dry eye syndrome compared with a population of healthy subjects., Design: Nonrandomized, prospective, comparative case series., Participants: Twenty-six eyes of 13 patients with rosacea, 26 eyes of 13 patients with dry eye syndrome, and 24 eyes of 12 control subjects were included in this study., Methods: IC specimens were collected after clinical examination of the ocular surface and analyzed by flow cytometry, using antibodies directed to human lymphocyte antigen-DR (HLA-DR), intercellular adhesion molecule-1 (ICAM-1) (CD 54), and the peptidic core of the conjunctival mucin (M1/MUC5AC). The percentage of positive cells was calculated and levels of fluorescence expression quantified and compared with those obtained in a series of 12 healthy subjects., Main Outcome Measures: Tear break-up time (TBUT), Schirmer test, fluorescein and lissamin green stainings, and IC were realized in this study., Results: A significant increase of HLA-DR and ICAM-1 expressions by epithelial cells was consistently found in the two pathologic groups compared with levels calculated in normal eyes. The two markers were well correlated with each other and inversely with TBUT and Schirmer test. The percentage of goblet cells was significantly decreased in rosacea patients and in dry eye patients compared with the normal group with a significant negative correlation with both HLA DR and ICAM-1 markers., Conclusions: Ocular rosacea and keratoconjunctivitis sicca were associated with severe ocular surface changes, such as an overexpression of inflammatory markers and a significant decrease in the number of goblet cells.

In an attempt to study the functional aspects of respiratory mucin secretion and the effects of mediators of inflammation on the release of M1/MUC5AC mucins in airways diseases, an ex vivo human bronchial model of mucin secretion was developed. Anti-M1 mucin monoclonal antibodies raised against the peptidic core of ovarian cyst M1 mucins were used. PAS and Alcian blue stainings of sections of bronchial rings revealed the presence of mucins in epithelial goblet cells as well as in glandular mucous cells. Immunohistochemical labelling of these sections with anti-M1 monoclonal antibodies revealed a preferential localization of M1/MUC5AC mucins in epithelial goblet cells. Functional studies were performed on this bronchial model using various secretagogues (methacholine, leukotrienes D4 and anti-human immunoglobulin E antibodies). No statistical difference of M1/MUC5AC mucin secretion was observed after a one-hour stimulation of bronchial rings with these agents. The development of an ex vivo functional human bronchial model of mucin secretion and the use of specific anti-M1 antibodies are essential tools in studying the regulation of the M1/MUC5AC mucin release from human airways.

Background: Chronic airway diseases are often associated with marked mucus production, however, little is known about the regulation of secretory activity by locally released endogenous mediators., Aim: This investigation was performed to determine the release of MUC5AC mucin from human bronchial preparations using the purinergic agonists adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP)., Methods: Immunohistochemical and immunoradiometric assays (IRMA) were used to detect the MUC5AC mucin. Immunohistochemical analysis were performed using individual 1-13 M1 and 21 M1 MAbs recognizing a recombinant M1 mucin partially encoded by the MUC5AC gene. IRMA measurments were performed using a mixture of eight anti-M1 mucin MAbs (PM8), which included both 1-13 M1 and 21 M1 MAbs. Lysozyme and protein were also measured in the biological fluids derived from human bronchial preparations obtained from patients who had undergone surgery for lung carcinoma., Results: The anti-M1 monoclonal antibodies labelled epithelial goblet cells. After challenge of human bronchial preparations with ATP, the goblet cells exhibited less staining. In contrast, UTP did not alter the immunolabelling of goblet cells. MUC5AC mucin in the bronchial fluids derived from ATP-challenged preparations was increased while UTP had no effect on release. ATP did not alter either the quantities of lysozyme or protein detected in the biological fluids., Conclusion: These results suggest that ATP may regulate epithelial goblet cell secretion of MUC5AC mucin from human airways in vitro.

A series of monoclonal antibodies which bind to a mucin known as M1 (anti-M1 MAbs) have also been shown to detect the product of the human gene MUC5AC. The aim of this investigation was to determine the concentration of the M1 mucin in the surface epithelium of human bronchial preparations by means of immunohistochemistry and in the bronchial fluid derived from human airways by means of an immunoradiometric assay. Human bronchial ring preparations from the resection material of 20 patients were challenged with methacholine, leukotriene D4, or anti-immunoglobulin E. Experiments were performed in preparations with an intact epithelium as well as in tissues in which the epithelium had been mechanically removed. The anti-M1 MAbs stained the goblet cells in the epithelium intensely and there was also light and less uniform staining in the submucosa. The M1/MUC5AC mucin in the fluids secreted by the bronchial preparations was not modified during either the experimental protocol or stimulation with the different secretagogues. However, in preparations in which the epithelium had been removed, there was a significant reduction in the amount of M1/MUC5AC mucin detected. These data suggest that the M1/MUC5AC mucin detected in the biological fluids produced by human airways in vitro may be released constantly, and principally from the goblet cells in the epithelial layer.

Purpose: Mucin-related antigens are abundantly expressed by the cells of the normal human conjunctiva. The pattern of these antigens in pterygium, and especially the role of Galbeta1-3GlcNAc alpha2,3-sialyltransferase (ST3Gal III), sialyltransferase necessary to build the sialyl-Le(a) (Lewis(a)) antigen, were studied., Methods: Immunoperoxidase staining was performed on 28 pterygia using different monoclonal antibodies: anti-M1 (against the peptidic core of gastric mucins encoded by MUC 5AC gene), anti-Le(a)(7LE), anti-sialyl Le(a)(NS 19-9), and anti-Le(b)(2-25LE). A serologic Lewis determination was done in 18 patients. ST3Gal III sialyltransferase expression was also studied in 10 healthy conjunctiva and 10 pterygia by reverse transcriptase-polymerase chain reaction (RT-PCR). Glyceraldehyde-3-phosphate-dehydrogenase was used as an endogenous internal control., Results: First, Le(a), sialyl Le(a), and Le(b) immunoreactivities either decreased or were no longer detectable in pterygium goblet cells as opposed to normal conjunctiva. Second, unlike in pterygium, the Lewis immunoreactivity, which is mainly located in the surface epithelial cells in the normal conjunctiva, was occasionally restricted to the epithelial cells of the deep layers. However, M1 mucins did show an identical pattern expression in a normal conjunctiva and pterygium. ST3Gal III expression was significantly lower in pterygium (0.20+/-0.02 AU [arbitrary units]) than in normal conjunctiva (0.95+/-0.12 AU)., Conclusions: ST3Gal III gene is less expressed in pterygium than in normal conjunctiva. This observation could explain the decrease of sialyl Le(a) expression observed in pterygium by immunohistology.

This study examines the canine model of keratoconjunctivitis sicca (KCS, 'dry eye') in order to establish the biochemical basis of altered ocular mucin secretion in this condition. It follows a previous examination of ocular mucins in the normal dog. Mucus was collected by suction from the ocular surface of dogs with KCS, and dispersed in guanidine hydrochloride containing a cocktail of protease inhibitors. Caesium chloride density gradient centrifugation was used to separate floating 'rafts' of cell membranes from gradients containing secreted mucins. Gradient fractions were collected into pools on the basis of differential staining by Periodic Acid Schiff, Wheat Germ Agglutinin, and antibodies to MUC5AC peptide. High molecular weight glycoproteins were purified from the pooled material by gel filtration chromatography. Membrane-associated glycoproteins were also derived from the membrane rafts using octyl glucoside extraction and/or reduction and alkylation. Secreted mucins and membrane extracts from KCS samples were compared to equivalent material obtained from normal eyes. Density gradient staining profiles for normal and KCS mucus were similar over the buoyant density range typical for secreted mucins, enabling the collection of identical pools of gradient fractions for direct comparison. The following differences were observed in KCS secreted mucins compared to normal samples: an increase in the proportion of mucin with low buoyant density; a decrease in mannose content detected with Concanavalin A lectin; an increase in N-acetylglucosamine structures detected with Lycopersicon esculentum lectin; increased migration and lack of evidence for distinct subunit structure on agarose gels. In membrane extracts, the main difference was the presence of T antigen (Gal beta 1-3GalNAc) in KCS. These results demonstrate alterations in the subunit linkage of mucins in KCS, and suggest that glycosylation, core protein expression and/or post-synthetic modification of ocular surface mucins may also be changed.

CAM 17.1 is an antimucin monoclonal antibody which has recently been proven valuable as a reagent for serological diagnosis of pancreatic cancer. A series of studies have been performed to characterise its epitope. First it was screened immunohistochemically against a wide range of formalin-fixed normal and neoplastic human tissues and showed widespread binding to mucin throughout the gastro-intestinal tract, in both normal and malignant tissues. In pancreas, strong intracellular staining of acinar and ductal cells was found in normal tissue and in carcinoma cells in tumours. Normal stomach showed only weak staining (n = 6), but gastritis with metaplasia showed strong staining (n = 4). Staining of colonic mucosa from patients of known Lewis phenotype showed Le(a+b-) (7/8) and Le(a-b+) (4/6) samples to be positive, but not Le(a-b-) (0/3) samples. CAM 17. 1 agglutinated all donor erythrocytes tested at 4 degreesC regardless of blood group, whereas cord blood red cells were not agglutinated. Since I antigen is the only antigen known to be present on all adult red blood cells but absent from cord blood, this suggests probable involvement of this antigen in the binding site. The agglutination was abolished by sialidase treatment of the red cells and immunoblotting with slot-blotted mucin showed that binding was both acid and sialidase sensitive indicating the involvement of sialic acid in the binding site. These studies show that CAM 17.1 binds to a sialic-acid-containing determinant of mucin, probably sialyl-I, which epitope shows wide distribution throughout the gastro-intestinal tract.

Gastric M1 mucin and the MUC5AC gene show a similar oncofetal expression in the colon. Our aim was to determine whether M1 mucin is the product of the MUC5AC gene. A recombinant baculovirus encoding the C-terminal portion of the MUC5AC gene as a fusion protein was isolated and the immunoreactivity of the recombinant mucin (rM) toward M1 antibodies studied. Chicken antibodies also were raised against purified rM. Besides its reactivity with L56/C, a serum recognizing the bacterially expressed MUC5AC gene product, rM was endowed with M1 immunoreactivity: (i) rM-expressing cells were stained specifically with anti-M1 serum and with the monoclonal antibody (MAb) 21M1, defining the M1-f epitope; (ii) both L56/C and anti-M1 antibodies recognized the same bands in immunoblots of rM-containing cell extracts; (iii) the 21M1 antibody reacted with rM in an immunoradiometric assay. Among the 7 M1 epitopes, M1-f was the only one encoded by the 3' portion of the MUC5AC gene. It was the only epitope detected in a native mucin M1-derived 170 kDa bromelain proteolytic fragment. Furthermore, the staining patterns of human tissues obtained with either anti-rM chicken antibodies or anti-M1 antibodies were identical. We conclude that M1 immunoreactivity is encoded at least in part by the MUC5AC gene.

Aims: This study investigated mucus changes in the tears in various eye conditions using impression cytology. The quantity of mucins was measured by enzyme linked immunosorbent assay (ELISA) using the tumour marker CA 19-9. This assay quantifies the sialylated Lewis(a) structure mainly associated with ocular mucins., Methods: Impression cytology was performed using a cellulose nitrate membrane, on 53 healthy patients, 50 glaucoma patients treated with beta blockers, 24 patients suffering from dry eye syndrome, and 45 contact lens wearers. The tear film glycoproteins were eluted and CA 19-9 was measured., Results: CA 19-9 content expressed as kilo units (kU) per microgram of tears was significantly decreased in dry eye syndrome (25.8 kU (SD 17.3)/microgram) (p < 0.05), glaucoma patients over 60 years (28.9 (19.5) kU/microgram) (p < 0.05), and contact lens wearers (28.4 kU (18)/microgram) (p < 0.05), when compared with healthy individuals (39.4 kU (22.2)/microgram)., Conclusion: Impression cytology can be regarded as a valuable method for obtaining samples of glycoconjugates of mucin. The decrease of sialylated chains observed with this method confirms the hypothesis that some quantitative changes in the tear film may be encountered in ocular surface disorders.