Next generation risk assessment of biocides (PHMG-p and CMIT/MIT)-induced pulmonary fibrosis using adverse outcome pathway-based transcriptome analysis.

Next-generation risk assessment (NGRA) has emerged as a promising alternative to non-animal studies owing to the increasing demand for the risk assessment of inhaled toxicants. In this study, NGRA was used to assess the inhalation risks of two biocides commonly used as humidifier disinfectants: polyhexamethylene guanidine phosphate (PHMG-p) and chloromethylisothiazolinone/methylisothiazolinone (CMIT/MIT). Human bronchial epithelial cell transcriptomic data were processed based on adverse outcome pathways and used to establish transcriptome-based points of departure (tPODs) for each biocide. tPOD values were 0.00500-0.0510 µg/cm2 and 0.0342-0.0544 µg/cm2 for PHMG-p and CMIT/MIT, respectively. tPODs may provide predictive power comparable to that of traditional animal-based PODs (aPODs). The tPOD-based NGRA determined that both PHMG-p and CMIT/MIT present a high inhalation risk. Moreover, the identified PHMG-p posed a higher risk than CMIT/MIT, and children were identified as more susceptible population compared to adults. This finding is consistent with observations from actual exposure events. Our findings suggest that NGRA with transcriptomics offers a reliable approach for risk assessment of specific humidifier disinfectant biocides, while acknowledging the limitations of current models and in vitro systems, particularly regarding uncertainties in pharmacokinetics (PK) and pharmacodynamics (PD).

Integrated high-throughput drug screening microfluidic system for comprehensive ocular toxicity assessment.

Traditional experimental methodologies suffer from a few limitations in the toxicological evaluation of the preservatives added to eye drops. In this study, we overcame these limitations by using a microfluidic device. We developed a microfluidic system featuring a gradient concentration generator for preservative dosage control with microvalves and micropumps, automatically regulated by a programmable Arduino board. This system facilitated the simultaneous toxicological evaluation of human corneal epithelial cells against eight different concentrations of preservatives, allowing for quadruplicate experiments in a single run. In our study, the IC50 values for healthy eyes and those affected with dry eyes syndrome showed an approximately twofold difference. This variation is likely attributable to the duration for which the preservative remained in contact with corneal cells before being washed off by the medium, suggesting the significance of exposure time in the cytotoxic effect of preservatives. Our microfluidic system, automated by Arduino, simulated healthy and dry eye environments to study benzalkonium chloride toxicity and revealed significant differences in cell viability, with IC50 values of 0.0033% for healthy eyes and 0.0017% for dry eyes. In summary, we implemented the pinch-to-zoom feature of an electronic tablet in our microfluidic system, offering innovative alternatives for eye research.

Bacterial Pathogenesis: Assessment of Intracellular Positioning of Pathogen-Containing Vacuoles During Infection.

Intracellular bacterial pathogens implement a diverse array of strategies to target host cells and establish infection. For vacuolar pathogens, the process of pathogen-containing vacuole movement within host cells, termed intracellular trafficking, is central to both pathogen survival and infection progression. Typically a process mediated by secreted virulence factors that manipulate the host cytoskeletal machinery, internalized pathogen-containing vacuoles traffic to the site of replication to establish a unique replicative niche, and if applicable, traffic back toward the host cell periphery for cell-to-cell spread. As such, the intracellular positioning of pathogen-containing vacuoles represents a fundamental measure of infection progression. Here, we describe a fluorescence microscopy-based method to quantitatively assess bacterial intracellular positioning, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model. This experimental approach can be modified to study infection in diverse host cell types, and with a broad array of pathogens. The system can also be adapted to examine the kinetics of infection, identify secreted virulence factors that mediate host trafficking, investigate host factors that are targeted by the pathogen for trafficking, and assess functional domains within a virulence factor responsible for mediating the phenotype. Collectively, these tools can provide fundamental insight into the pathogenesis of a diverse array of intracellular bacterial pathogens, and new host factors that are hijacked to mediate infection. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture and preparation of host cells Alternate Protocol: Culture and preparation of host cells to assess host factor contribution to bacterial positioning Basic Protocol 2: Infection of epithelial cells with S. Typhimurium Basic Protocol 3: Fluorescence staining for analysis of bacterial positioning Basic Protocol 4: Fluorescence microscopy analysis of bacterial positioning.

Pandemic Risk Assessment for Swine Influenza A Virus in Comparative In Vitro and In Vivo Models.

Swine influenza A viruses pose a public health concern as novel and circulating strains occasionally spill over into human hosts, with the potential to cause disease. Crucial to preempting these events is the use of a threat assessment framework for human populations. However, established guidelines do not specify which animal models or in vitro substrates should be used. We completed an assessment of a contemporary swine influenza isolate, A/swine/GA/A27480/2019 (H1N2), using animal models and human cell substrates. Infection studies in vivo revealed high replicative ability and a pathogenic phenotype in the swine host, with replication corresponding to a complementary study performed in swine primary respiratory epithelial cells. However, replication was limited in human primary cell substrates. This contrasted with our findings in the Calu-3 cell line, which demonstrated a replication profile on par with the 2009 pandemic H1N1 virus. These data suggest that the selection of models is important for meaningful risk assessment.

Assessment of MUC5AC and MUC2 Immunoexpression in Glandular Odontogenic Cysts, Dentigerous Cysts, and Mucoepidermoid Carcinomas.

Glandular odontogenic cysts (GOCs) and dentigerous cysts may show mucous metaplasia. Central mucoepidermoid carcinoma is very rare and mostly associated with dental cysts. It is hypothesized that odontogenic cysts showing mucus differentiation in their lining, have a propensity to transform into MEC. The present study is the first attempt to explore the relationship between odontogenic cysts [GOCs and dentigerous cysts with mucus metaplasia (DCMM)] and MEC by evaluating immunoexpression of MUC5AC and MUC2. Immunoexpression of MUC5AC and MUC2 was evaluated semiquantitatively in GOCs (20 cases), DCMMs (20 cases), and MECs (20 cases). The percentage of positive cells, intensity, and localization of immunoexpression were assessed for each marker in all cases. Of GOCs, DCMMs, and MECs cases, 85%, 70%, and 80%, respectively, were immunopositive for MUC5AC. Strong cytoplasmic immunoreactivity for MUC5AC was noted, particularly in mucous cells present diffusely within MECs. However, the immunoreactivity was limited to the epithelial lining of GOCs and DCMMs. Most of the MECs (60%) showed more than 25% positivity for MUC5AC, followed by GOCs, and the least in DMMCs. Mild cytoplasmic and nuclear positivity of MUC2 was noted only in epithelial lining cells of 70% GOCs and 45% DCMMs. Whereas, 55% of MECs displayed moderate to strong cytoplasmic and membranous immunopositivity for MUC2 exclusively within mucous cells. As MECs showed strong MUC5AC immunoreactivity in mucous cells, immunoexpression of MUC5AC in odontogenic cysts with mucus cells can possibly explain the pathogenesis of MEC from cysts. However, the variable expression of MUC2 did not give any strong evidence regarding its role as a marker.

Characterization and applicability of a novel physiologically relevant 3D-tetraculture bronchial model for in vitro assessment of respiratory sensitization.

Chemical Respiratory Allergy (CRA) is triggered after exposure to Low Molecular Weight (LMW) sensitizers and manifests clinically as asthma and rhinitis. From a risk/toxicity assessment point of view, there are few methods, none of them validated, for evaluating the respiratory sensitization potential of chemicals once the in vivo-based models usually employed for inhalation toxicity addressment do not comprise allergenicity endpoints specifically. Based on that, we developed, characterized, and evaluated the applicability of a 3D-tetraculture airway model reconstructed with bronchial epithelial, fibroblasts, endothelial and monocytic cell lines. Moreover, we exposed the tissue to maleic anhydride (MA) aerosols to challenge the model and subsequently assessed inflammatory and functional aspects of the tissue. The reconstructed tissue presented phenotypic biomarkers compatible with human bronchial epithelium, and MA aerosol exposure triggered an increased IL-8 and IL-6 production, reactive oxygen species (ROS) formation, and apoptosis of epithelial cells. Besides, augmented IL-8 production by monocytic cells was also found, correlating with dendritic cell activation within the co-culture model after MA exposure. Our results demonstrated that the 3D-tetraculture bronchial model presents hallmarks related to human airways' structure and function. Additionally, exposure to a respiratory sensitizer induced inflammatory and functional alterations in the reconstructed tissue, rendering it a valuable tool for exploring the mechanistic framework of chemically induced respiratory sensitization.

Multidimensional assessment of the biological effects of electronic cigarettes on lung bronchial epithelial cells.

Cigarette smoke (CS) exposure is known to cause injury to respiratory tract epithelial cells and is a contributing factor in the development of chronic obstructive pulmonary disease and lung cancer. Electronic cigarettes (e-cigarettes) are gaining popularity as a potential substitute for conventional cigarettes due to their potential for aiding smoking cessation. However, the safety of e-cigarettes remains uncertain, and scientific evidence on this topic is still limited. In this study, we aimed to investigate the effects of CS and e-cigarette smoke (ECS) of different flavors on human lung bronchial epithelial cells. Real-time smoke exposure was carried out using an air-liquid interface system, and cell viability was assessed. RNA-Seq transcriptome analysis was performed to compare the differences between CS and ECS. The transcriptome analysis revealed a significantly higher number of differentially expressed genes in CS than in ECS. Moreover, the impact of mint-flavored e-cigarettes on cells was found to be greater than that of tobacco-flavored e-cigarettes, as evidenced by the greater number of differentially expressed genes. These findings provide a reference for future safety research on traditional cigarettes and e-cigarettes, particularly those of different flavors. The use of omics-scale methodologies has improved our ability to understand the biological effects of CS and ECS on human respiratory tract epithelial cells, which can aid in the development of novel approaches for smoking cessation and lung disease prevention.

Cost-effective 3D lung tissue spheroid as a model for SARS-CoV-2 infection and drug screening.

BACKGROUND: The SARS-CoV-2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus' structure, infection mechanisms, and pathogenesis. Two-dimensional culture systems have been instrumental in shedding light on numerous aspects of COVID-19. However, these in vitro systems lack the physiological complexity to comprehend the infection process and explore treatment options. Three-dimensional (3D) models have been proposed to fill the gap between 2D cultures and in vivo studies. Specifically, spheroids, composed of lung cell types, have been suggested for studying SARS-CoV-2 infection and serving as a drug screening platform. METHODS: 3D lung spheroids were prepared by coculturing human alveolar or bronchial epithelial cells with human lung stromal cells. The morphology, size, and ultrastructure of spheroids before and after SARS-CoV-2 infection were analyzed using optical and electron microscopy. Immunohistochemistry was used to detect spike protein and, thus, the virus presence in the spheroids. Multiplex analysis elucidated the cytokine release after virus infection. RESULTS: The spheroids were stable and kept their size and morphology after SARS-CoV-2 infection despite the presence of multivesicular bodies, endoplasmic reticulum rearrangement, tubular compartment-enclosed vesicles, and the accumulation of viral particles. The spheroid responded to the infection releasing IL-6 and IL-8 cytokines. CONCLUSION: This study demonstrates that coculture spheroids of epithelial and stromal cells can serve as a cost-effective infection model for the SARS-CoV-2 virus. We suggest using this 3D spheroid as a drug screening platform to explore new treatments related to the cytokines released during virus infection, especially for long COVID treatment.

An integrated new approach methodology for inhalation risk assessment of safe and sustainable by design nanomaterials.

The production and use of nanomaterials (NMs) has increased over the last decades posing relevant questions on their risk after release and exposure of the population or sub-populations. In this context, the safe and sustainable by design (SSbD) approach framework requires to assess the potential hazard connected with intrinsic properties of the material along the whole life cycle of the NM and/or of the nano enabled products. Moreover, in the last years, the use of new advanced methodologies (NAMs) has increasingly gained attention for the use of alternative methods in obtaining relevant information on NMs hazard and risk. Considering the SSbD and the NAMs frameworks, within the ASINA H2020 project, we developed new NAMs devoted at improving the hazard and risk definition of different Ag and TiO2 NPs. The NAMs are developed considering two air liquid interface exposure systems, the Vitrocell Cloud-α and the Cultex Compact module and the relevant steps to obtain reproducible exposures are described. The new NAMs build on the integration of environmental monitoring campaigns at nano-coating production sites, allowing the quantification by the multiple-path particle dosimetry (MPPD) model of the expected lung deposited dose in occupational settings. Starting from this information, laboratory exposures to the aerosolized NPs are performed by using air liquid interface exposure equipment and human alveolar cells (epithelial cells and macrophages), replicating the doses of exposure estimated in workers by MPPD. Preliminary results on cell viability and inflammatory responses are reported. The proposed NAMs may represent possible future reference procedures for assessing the NPs inhalation toxicology, supporting risk assessment at real exposure doses.

Human epithelial lung cell toxicity assessment of collected graphite particles from an iron casting industry (in vitro study).

Workers in the iron casting industries are exposed to various chemicals, especially graphite in furnace process. This study aims to investigate the toxic effects of graphite particles on human lung cells. Particle characteristics were confirmed by electron microscope and light scattering. Cell viability and oxidative stress markers were measured. The expression of oxidative repair genes, namely OGG1, MTH1, and ITPA, was evaluated. The average particle size was determined to be 172.1 ± 11.96 nm. The median inhibition concentration (IC50) of graphite particles was 46.75 µg/mL. Notably, 25 and 50 µg/mL concentrations resulted in significant GSH depletion and MDA production. The high concentration of graphite particles (200 µg/mL) led to OGG1 suppression and increased MTH1 expression. Based on these findings, graphite exposure may induce toxicity in human lung cells by increasing oxidative stress. Further research is necessary to fully understand the mechanisms underlying graphite toxicity.

Modulação da expressão gênica no timo: uma perspectiva integrativa da regulação gênica no desenvolvimento de timócitos e involução tímica decorrente de infecção / Gene expression modulation in thymus: an integrative perspective of the gene regulation during thymocyte development and thymic involution due to infection

O timo é um órgão linfoide primário no qual ocorre o desenvolvimento de células T. Distúrbios nesse processo podem levar a doenças diversas inclusive à autoimunidade. [...] Nesse sentido, essa tese aborda os aspectos moleculares envolvidos na linfopoiese T sob a perspectiva das células progenitoras e das TEC. Nosso estudod e meta-análise utilizando microarranjos de timócitos em estágios de desenvolvimento definidos pela expressão de moléculas CD4 e CD8 permitiu a identificação de 14 agrupamentos gênicos,os quais se relacionam intimamente às etapas do desenvolvimento dessas células. A modulaçãodesses genes está vinculada à sinalização via receptor de células T (TCR) e pode sercorrelacionada com a afinidade pelo complexo formado entre peptídeo e o complexo principal dehistocompatibilidade (MHC). Destacam-se ainda, vias de sinalização relacionadas a outrosreceptores de superfície, evidenciando a importância dos estímulos externos no desenvolvimentode células T. Através da integração de dados, nós identificamos as vias e moléculas com maiorimpacto para a diferenciação de timócitos, bem como complementamos a compreensão de comoos eventos envolvidos nesse processo se organizam temporalmente, apresentando umaperspectiva global das ondas de modulação gênica. Já em outra perspectiva, estudamos aregulação gênica em TEC comparando células corticais e medulares, mas também focamos nainvolução tímica, uma resposta natural a diversas fontes de estresse e ao envelhecimento.Nessa tese, esse fenômeno foi estudado em modelos experimentais de fase aguda da doençade Chagas, pois existem evidências de que as alterações no timo durante a fase aguda podemser responsáveis pelo desfecho da doença anos mais tarde...

The thymus is a primary lymphoid organ, where the T cell development takes place. Disturbanceson this process can lead to a diversity of diseases, including autoimmunity. [...] In this sense, this thesis addresses the molecular aspects involved on Tlymphopoiesis under the progenitor cells and TEC perspective. Our meta-analysis study usingmicroarrays of thymocytes sorted based on the developmental stages defined by the CD4 andCD8 expression allowed to identify 14 gene clusters, which are closely related to thedevelopmental stages of these cells. Those genes modulation is linked to the T cell receptor(TCR) signaling pathway and it can be related to the TCR affinity for the complex formed betweenthe peptide and the major histocompatibility complex (MHC). Moreover, other signaling pathwaysrelated to surface receptors stand out, highlighting the importance of external stimuli on the T celldevelopment. Through data integration, we identify pathways and molecules that have morerelevant impact on the thymocyte differentiation, as well as, we improved the comprehension onhow the events are organized temporally, providing a global perspective on the gene modulationwaves. In another perspective, we have studied the gene regulation on TEC by comparingcortical and medullary cells, but also focused on thymic involution, a natural response to varioussources of stress and aging. In this thesis, this phenomenon was studied using Chagas' diseaseacute phase experimental model because there are evidences showing that the thymic alterationsduring the acute phase can be responsible for the disease outcome years later...

Regulação epigenética de FOXN1 no epitélio tímico humano durante a senescência / Epigenetic regulation of foxn1 in human thymic epithelium during senescence

A involução do timo ao longo do envelhecimento se caracteriza por alterações nas células que compõem o seu microambiente, contudo as suas causas ainda não estão completamente esclarecidas. No intuito de contribuir para conhecimento acerca desse fenômeno, esta tese teve por objetivos avaliar a expressão de genes importantes para o desenvolvimento e função linfopoiética do timo como também investigar o mecanismo de regulação da expressão gênica de FOXN1 através de metilação de DNA. Para isso, foram utilizadas amostras de timo humano de diferentes idades provenientes de pacientes submetidos à cirurgia cardíaca. Quanto aos aspectos morfológicos, os timos de doadores entre cinco dias e um ano deidade (grupo pós-natal) apresentaram uma arquitetura microscópica característica do órgão, sem sinais de involução. Nessas amostras, também foi verificada uma deposição normal de citoqueratina, distribuída pelo órgão como uma fina rede de filamentos intracelulares. Já nos timos de doadores acima de 49 anos (grupo adulto idoso),observou-se a perda da arquitetura tímica, presença de uma grande quantidade de tecido adiposo e uma deposição de citoqueratina irregular, quandopresente, com regiões semelhantes a áreas livres de células epiteliais tímicas (TEC).Nos ensaios de PCRq, foi verificado um aumento na expressão dos genes DLL1 eDLL4, e uma diminuição na expressão de FOXN1 em amostras de timo adulto-idosoem comparação com amostras de timo pós-natal. Além disso, não foi possíveldetectar a presença da proteína FOXN1 em cortes congelados de timo adulto-idosoem relação às amostras de timo pós-natal...

Thymic involution during aging is accompanied by alterations on itsmicroenvironment, however little is known about the mechanisms responsible forthese modifications. In this study we aimed to evaluate the expression of genescritical for thymopoiesis and the FOXN1 gene epigenetic regulation by DNAmethylation. We obtained human thymic samples from patients ongoing cardiacsurgery with different ages. In the morphological evaluation, thymus from donors withfive days-old to one year-old (posnatal group) showed a typical thymic architecture,without involution signs, and a normal cytokeratin labelling pattern distributed as athin filament network on the organ. When thymuses from adult-old donors (49 to 78years-old) were analyzed, it was seen a disruption on thymus microenvironment, withlarge adipose tissue infiltration and an irregular cytokeratin pattern revealing someepitelial-free areas. The qPCR assays showed that DLL1, DLL4, FOXN1 e WNT4genes were expressed during aging on thymus, but DLL1 and DLL4 presented highexpression in thymuses from adult-old samples regarding the postnatal group, whilethe FOXN1 expression was lower in adult-old group. By immunofluorescence, wewere not able to detect FOXN1 staining in thymus sections from adult-old sampleswhen compared with samples from postnatal thymus. In human thymic epithelial cellline (THPN), we show that FOXN1 expression is refractory to signals that induceFOXN1 transcription in primary 3D culture conditions and by stimulation of thecanonical WNT signaling pathway. Next, we analyzed the DNA methylation patternon FOXN1 gene by bisulfite sequencing using converted DNA from human thymusand THPN cell line. It was observed differential methylation on intron 1 and exon 2selected regions on the gene sequence. These results demonstrate for the first timethat FOXN1 gene expression in thymus may be regulated through an epigeneticmechanism during aging...

Citogênese e estágios esquizontes em células epiteliais de felinos infectadas pelo Toxoplasma gondii, in vitro / Cystogenesis stages and schizonts in epithelial cells of cats infected with Toxoplasma gondii in vitro

O Toxoplasma gondii é um protozoário parasito intracelular, agente causador da toxoplasmose que é uma das zoonoses mais endêmicas do mundo. O T.gondii possui dois tipos de reprodução : assexuada, que origina dois estágios evolutivos infectivos, os bradizoítos e taquizoítos e a sexuada que ocorre no tecido epitelial intestinal, exclusivamente de felídeos hospedeiros definitivos, resultando na formação de oocistos imaturos que são eliminados nas suas fezes. A disseminação de oocistos no ambiente é o principal fator que explica a distribuição mundial do parasito, associada à formação de cistos teciduais que aumentam a capacidade de transmissão do parasito, através do consumo de carne crua ou mal cozida. Dada a especificidade de o ciclo sexuado ocorrer no tecido epitelial, o principal objetivo do presente trabalho foi investiga, in vitro, a interação do T. gondii com células epiteliais oriundas do intestino de ratos e de rim de felídeos, a fim de estabelecer possíveis correlações entre a origem do tecido (epitelial) e a fonte (rato e felídeos) que possam determinar o destino intracelular do parasito. Assim, culturas de linhagens celulares CRFK e IEC-6 foram infectadas com bradizoítos e taquizoítos de T. gondii com diferentes cargas infectivas. Após os desenhos experimentais as culturas foram processadas como de rotina para microscopia óptica de luz e fluorescência e para microscopia eletrônica de transmissão e varredura. As análises quantitativas mostraram que a linhagem CRFK foi mais suscetível à infecção frente aos dois estágios infectivos do que a linhagem IEC-6 e que essa diferença foi independente da carga parasitária e do estágio infectivo utilizados. As análises qualitativas mostraram que a linhagem CRFK foi mais susceptível à infecção do que a linhagem IEC-6 e esse evento foi independente da carga infectiva utilizada. As análises qualitativas mostraram que a cistogêneses se estabeleceu de forma espontânea na CRFK quando infectada com bradizoítos da cepa ME49 ao se utilizar a carga efetiva de 1:10 (parasito-célula hospedeira). Além disso, esta linhagem celular apontou estágios infectivos morfologicamente distintos de taquizoítos e bradizoítos, similares aos estágios esquizontes de T. gondii. O presente estudo mostrou que existem diferenças no destino intracelular do parasito de acordo com o tipo celular utilizado. O emprego de células epiteliais de felinos como modelo celular da toxoplasmose experimental mostrou que pode contribuir com novos subsídios para o estudo da biologia celular do parasito, assim como contribuir como metodologia alternativa para o melhor entendimento do ciclo enteroepitelial do T. gondii. Este modelo celular abre um novo campo de investigação para aspectos moleculares desta interação que possam contribuir, por exemplo, para introdução de estratégias que venham a interferir em umas das principais vias de disseminação da toxoplasmose. Além disso, a CRFK se mostrou um modelo celular em potencial para a produção de cistos de T. gondii in vitro em larga escala abrindo perspectivas na redução do uso de animais experimentais para isolamento de cistos e inserção na área de inovação e desenvolvimento tecnológico.

Avaliação morfológica de diferentes técnicas de desepitelização da membrana amniótica humana / Morphological assessment of different amniotic membrane epithelial denuding techniques

OBJETIVO: Avaliar as características morfológicas da membrana amniótica desepitelizada por diferentes técnicas. MÉTODOS: A membrana amniótica humana foi coletada no momento do parto, fixada em concentrações crescentes de glicerol (0-50 por cento em DMEM) e preservada a 80°C até a hora de ser usada. O estudo consistiu de 4 grupos: epitélio intacto (controle) e membranas desepitelizadas pela tripsina (2 mg/mL a 1:250), dispase (1,2 U/mL em solução salina balanceada de Hank livre de Mg2+ e Ca2+) e ácido etilenodiaminotetra-acético (EDTA), 0,02 por cento). As amostras foram submetidas à análise por microscopia eletrônica (de varredura e de transmissão). RESULTADOS: A microscopia eletrônica de varredura mostrou epitélio intacto no grupo controle e sua ausência nas membranas amnióticas desepitelizadas pela tripsina e pela dispase. Naquelas tratadas com o ácido etilenodiaminotetra-acético, havia áreas com e sem epitélio. Quando avaliadas pela microscopia eletrônica de transmissão, o epitélio estava intacto e firmemente aderido à membrana basal através de hemidesmossomos nos grupos controle e em parte do ácido etilenodiaminotetra-acético. Havia apenas fibras colágenas nas membranas tratadas com dispase e tripsina. CONCLUSÕES: O tratamento da membrana amniótica com tripsina e dispase pode causar completa retirada do epitélio e da membrana basal, ao passo que o ácido etileno- diaminotetra-acético pode preservar áreas com epitélio intacto e parcialmente destruir a membrana basal em outras.

PURPOSE: To evaluate the morphological features of the amniotic membrane denuded by different techniques. METHODS: Human amniotic membrane was collected at the time of delivery, fixed in increasing concentrations of glycerol (0-50 percent in DMEM) and preserved at -80°C until the time of use. The study consisted of 4 groups: intact epithelium (control) and denuded by trypsin (2 mg/mL at 1:250), dispase (1.2 U/mL in Mg2+ and Ca2+ free Hank's balanced salt solution) or ethylenediaminetetraacetic acid (EDTA), 0.02 percent. Specimens were submitted to electron (scanning and transmission) microscopy analysis. RESULTS: Scanning electron microscopy disclosed intact epithelium in the control group and its absence in the amniotic membranes denuded by trypsin and dispase. In those denuded by ethylenediaminetetraacetic acid there were areas with and without epithelium. When assessed by transmission electron microscopy, the epithelium was intact and firmly adhered to the basement membrane by hemidesmossomes in controls and in parts of ethylenediaminetetraacetic acid group. There were only collagen fibers in the dispase- and trypsin-treated groups. CONCLUSIONS: Trypsin and dispase treatment of the amniotic membrane may cause complete denuding of the epithelium and basement membrane whereas ethylenediaminetetraacetic acid may leave some intact epithelium-areas and partially destroy the basement membrane in others.

A simple and cost-effective protocol for DNA isolation from buccal epithelial cells

Buccal cells provide a convenient source of DNA for epidemiological studies. The goal of this study was to develop a convenient method to obtain buccal cells from mouthwash samples to be used as a source of DNA, and to evaluate the stability of the DNA in mouthwash solution over time. The procedures used in the method described in this paper avoid the use of any organic solvents. This is achieved by salting out the cellular proteins by dehydration and precipitation with a saturated ammonium acetate solution. The protocol described here is fast, simple to perform, sensitive, economical and several samples can be processed at the same time. The analyses provide consistent evidence that DNA extracted by this methodology is sufficient for several PCR amplifications. The total DNA yield ranged from 5 to 93 µg (median 15 µg, mean 20.71 µg). DNA can be extracted and PCR amplified after storage of mouthwash solution at room temperature for periods of up to 30 days.

Células bucais são fontes convenientes de DNA para diagnóstico e estudos epidemiológicos. O objetivo deste trabalho foi desenvolver um método simples e prático para obter células epiteliais, através de bochechos, a fim de serem usadas como fonte de DNA e avaliar a estabilidade do DNA na solução de bochecho no decorrer do tempo. Os procedimentos usados neste estudo evitam o uso de solventes orgânicos permitindo uma pratica laboratorial mais segura. Isto é alcançado pela remoção das proteínas celulares por desidratação e precipitação com uma solução saturada de acetato de amônio. Este protocolo permite a extração de maneira rápida, simples, econômica e garante o processamento de várias amostras ao mesmo tempo, agilizando assim os procedimentos laboratoriais. Nossas análises forneceram evidências consistentes de que o DNA extraído por esta metodologia é suficiente para diversas amplificações por PCR (polymerase chain reaction - reação em cadeia pela polimerase). O produto total de DNA variou de 5 a 93 µg (mediana 15 µg; média 20,71 µg). Além disso, o DNA mostrou-se eficientemente preservado na solução de bochecho, a qual pode ser estocada em temperatura ambiente por até trinta dias.

Valoración de pacientes con atipia escamosa de significado determinado / Valuation of patient with scaly atipia of meaning certain 2005 stage II

El presente estudio tiene como propósito conocer la evolución de pacientes con atipia escamosa de significado indeterminado captados en los centros de salud de la ciudad de León valorados en consulta externa de onco ginecología del Hospital Escuela Oscar Danilo Rosales Arguello (HEODRA), mediante valoración colposcópica y seguimiento posterior ya que estas son pacientes que ameriten intervención o al menos seguimiento estrecho hasta esclarecer un dignóstico. Se valoraron un total de 33 pacientes en la consulta externa el 53 porciento provenian de los centros de salud del casco urbano de León. En cuatno a los grupos de edades, la mayoría de pacientes estuvo entre las edades de 20 a 49 años. El 47 porciento de las pacientes estudiadas planifico con métodos hormonales y de estos el 13 porciento lo hizo por un período mayor de 5 años. El hábito de fumar y el número de compañeros sexuales parece no influir en la presencia de lesiones epiteliales de significado indeterminado, pero por el pequeño tamaño de la muestra estos resultados no son estadísticamente signficativos. En cuanto a la valoración colposcopica de estas pacientes, el 31 porciento tuvo algún grado de patrón colposcópico anormal que incluyó epitelio acetoblanco, punteado + epitelio acetoblanco y vasos atípicos en una zona de transformación anormal; esta valoración fue completada por la toma de biopsias selectivas a como lo ameritó cada caso. El 28 porciento de colposcopías presentaron modificaciones inflamatorias lo cual puede explicar la presencia atipica de origen reactiva en las muestras de Papanicolau. 22 porciento no se observó patrón colposcópico anormal. Existe una buena correlación entre la citología, los hallazgos colposcópicos y los resultados biológicos, ya que el 92 porciento de las biopsias dirigidas por colposcopía presentaron anormalidades que incluyen atipia y lesiones de alto o bajo grado...

Utilidad de la técnica de AgNOR en la interpretación de los derrames de cavidades serosas / Usefulness of AgNOR assay in the assessment of serous effusions

Background: AgNOR technique detects, using silver salts, argyrophylic proteins of the nucleolar organizer region (NOR). The number and size of NOR reflect cell activity, proliferation and transformation and may help to differentiate benign from malignant cells. Aim: To assess the value of AgNOR assay to differentiate reactive mesothelial cells from malignant cells in serous effusions. Material and methods: Thirty one fluids obtained from 16 pleural, 14 peritoneal and one pericardial effusion, were studied. The fluids were processed with Giemsa and Papanicolau stains and with the AgNOR technique. The number of AgNOR dots were counted (only when it was possible to distinguish each individual dot) and the mean value per nucleus was calculated for each smear. Results: Mesothelial cells had a mean of 4,88 ñ 1,5 dots compared with 13,78 ñ 3,88 dots in the malignant cells (p<0,001). Conclusions: AgNOR assay can be useful for the differentiation of benign and malignant cells in serous effusions