Introdução: Gengivite descamativa (GD) se refere a uma manifestação clínica associada com diversas doenças mucocutâneas. Suas causas mais comuns são penfigóide das membranas mucosas (PMM), pênfigo vulgar (PV) e líquen plano oral (LP). A diagnose específica é melhor estabelecida através de avaliação histopatológica e de imunofluorescência. Objetivos: Examinar casos de gengivite descamativa utilizando microscopia confocal a laser e comparar os achados com aqueles encontrados na gengiva normal. Além disso, comparar os achados de microscopia confocal da gengivite descamativa com os da histopatologia convencional das lesões biopsiadas a fim de estabelecer critérios para este método diagnóstico não invasivo. Método: Doentes com manifestações clínicas de gengivite descamativa foram incluídos, totalizando quarenta e três casos. A microscopia confocal foi realizada na gengiva de um indivíduo saudável e nas lesões gengivais. Todas as lesões sem exame histopatológico prévio foram biopsiadas a fim de permitir uma correlação entre a microscopia confocal e a histopatologia. Resultados: O exame de microscopia confocal das lesões suspeitas de penfigóide das membranas mucosas revelou uma separação ao nível da junção dermo-epidérmica, preenchida por pequenas estruturas brilhantes, interpretadas como hemáceas. Os aspectos histopatológicos e de imunofluorescência confirmaram o diagnose. Para os casos de pênfigo vulgar, os achados da microscopia confocal foram de fenda intraepitelial com células arredondadas interpretadas como queratinócitos acantolíticos. Hiperqueratose e espongiose, associadas com infiltrado inflamatório, caracterizado por células pequenas e brilhantes permeando a estrutura intraepitelial de queratinócitos conhecida como favo de mel foram vistos no líquen plano. Estruturas arredondadas pouco brilhantes, interpretadas como queratinócitos necróticos, e estruturas estelares também pouco brilhantes, interpretadas como melanófagos, foram encontrados na derme. Conclusões: Propõe-se o uso da microscopia confocal como uma ferramenta adicional no diagnose e avaliação da gengivite descamativa / Background: Desquamative gingivitis refers to a clinical manifestation associated with several mucocutaneous disorders. The most common are mucous membrane pemphigoid, pemphigus vulgaris and lichen planus. Their specific diagnosis is better established by histopathological and immunofluorescence evaluation. Objective: To examine cases of desquamative gingivitis using reflectance confocal microscopy and compare the findings with those of normal gingiva. Moreover, confocal microscopy findings in desquamative gingivitis were compared to conventional histopathology of the biopsied lesions, in order to establish criteria for this non-invasive diagnostic technique. Methods: Patients with clinical manifestations of desquamative gingivitis were included, totalizing forty-three cases. Reflectance confocal microscopy was performed the gingival of a healthy person and on gingival lesions. All lesions were biopsied in order to perform a reflectance confocal microscopy- histopathologic correlation. Results: Reflectance confocal microscopy exam of the gingival lesions suspected of mucous membrane pemphigoid revealed a separation at the level of dermal-epidermal junction, filled with small bright structures interpreted as blood cells. Histopathological and immunofluorescence aspects confirmed the diagnosis. For pemphigus vulgaris, reflectance confocal microscopy aspects were of intraepithelial cleft with round detached cells interpreted as acantholytic keratinocytes, similar to the histopathological features. Hyperkeratosis and spongiosis associated with infiltration of inflammatory cells, recognized as small bright cells intermingling the honeycomb keratinocyte epithelial structure, were seen in lichen planus. Mild bright round structures interpreted as necrotic keratinocytes and mild bright stellate structures, interpreted as melanophages in the dermis were also seen. These features were present in histopathology, confirming the diagnosis of lichen planus. Conclusion: We propose the use of reflectance confocal microscopy as an additional tool in diagnosis and evaluation of desquamative gingivitis

Se han desarrollado y optimizado dos nanoemulsiones que contienen Nistatina (N1 y N2) para su aplicación en piel y mucosa oral, respectivamente. Estas formulaciones tuvieron un tamaño de gota nanométrico y resultaron ser estables a lo largo del tiempo. Adicionalmente se determinó el comportamiento reológico de ambos sistemas resultando ser fluidos newtonianos. Se evaluó la actividad antifúngica in vitro, observado que la potencia de la nistatina aumentó al ser introducida en las nanoemulsiones, cuando se comparaba con la nistatina libre. El mecanismo de liberación de la nistatina de las nanoemulsiones siguió una cinética de orden uno para ambas formulaciones. Trás el ensayo de permeación ex vivo en piel humana con la nanoemulsion N1 se observó que la cantidad de fármaco permeada fue muy baja, pudiendo descartarse la posible aparición de efectos adversos a nivel sistémico. De la misma manera se realizó el ensayo de permeación ex vivo en mucosa oral porcina para la formulación N2 llegando a la misma conclusión que en el caso de la permeación en piel. En ambos casos la cantidad de fármaco retenido en el tejido (tanto en piel o mucosa) es suficiente para observar un efecto fungistático, fungicida y un prolongado efecto post-antifungico (PAFE). Con el fin de determinar el efecto de la nanoemulsion N2 sobre la mucosa oral se visualizó la ultra-estructura del tejido y no observándose variaciones significativas cuando se comparaban con el control (mucosa no tratada con la nanoemulsion). Las nanoemulsiones N1 y N2 son formulaciones prometedoras para el potencial tratamiento clínico de candidiasis muco-cutáneas. / Muco-cutaneous candidosis is a common opportunistic infection must be treated to prevent other tissue invasion. Nystatin is one of the most prescribed drugs to treat this pathology, but due to its physicochemical properties its pharmaceutical-technological requirements make it a challenge. The purpose of this work was the development and characterization of an optimal nystatin delivery system for the potential treatment of oral candidosis avoiding undesirable side effects and toxicity of potential systemic absorption. Two nanoemulsion (N1 and N2) was developed, evaluated and characterized. It has been formulated successfully as a stable nanoemulsion with a droplet size of 75 and 138 nm, respectively. First order release parameters were estimated using different mathematical approaches and ex vivo permeation of nystatin through human skin and porcine buccal mucosa were found no systemic effects would happen. Microbiologic studies performed revealed an enhanced antifungal effect of the nystatin loaded nanoemulsion. Also the evaluation of buccal mucosa ultrastructure by transmission electron microscopy methodology showing a harmless effect in the mucosa microstructure. We can infer that the selected nystatin nanoemulsion could be potentially used on candidosis infection under mucositis conditions.

[Truncated abstract] Atopic asthma pathogenesis is driven by the combined effects of airway inflammation generated during responses to viral infections and aeroallergens, and both of these pathways are regulated by dendritic cells (DC) that differentiate locally from monocytic precursors. These DC normally exhibit a sentinel phenotype characterised by active antigen sampling but attenuated presentation capability, which limits the intensity of local expression of adaptive immunity. How this tight control of airway DC functions is normally maintained and why it breaks down in some atopics leading to immunopathological changes in airway tissues, is unknown. In the airway mucosa, DC are intimately associated with airway epithelial cells (AEC), which are a source of a range of both pro- and anti-inflammatory mediators. A few studies have previously examined the effects of AEC-derived surface-expressed and soluble mediators upon the function of pre-differentiated DC, although there is a dearth of information as to the extent of AEC-conditioning of DC during their generation from incoming monocytic precursors within the airways. Therefore, this study was designed to test the hypothesis that signals from adjacent AEC contribute to regulation of local differentiation of airway mucosal DC, especially in the context of allergic airway disease. A direct co-culture model was developed containing the AEC line 16HBE 14o- as a surrogate for primary AEC, and purified peripheral blood monocytes derived from atopic patients in a GM-CSF/IL-4-enriched cytokine milieu. Cells were cultured for 5 days, at which time the phenotype and functional attributes of the monocyte-derived DC (MDDC) generated in the presence of AEC (AEC-MDDC) were compared to the control MDDC population generated without AEC contact (Ctrl- MDDC). ... In parallel, an attenuation of mRNA boosting for 7 out of 12 selected Th2-asscociated genes as well as IL-13 protein, was observed in AEC-MDDC supplemented cultures compared to ctrl-MDDC supplemented cultures. The data collected in the initial characterisation of the AEC-MDDC in Chapter 3 and further analysis of their gene expression profiles by microarray suggest a number of DC-associated factors could be involved in directing a potential bias against Th2 immunity within the T-cell recall response. These include increased expression of IL- 12 subunit mRNA and the enhanced levels of surface MHC Class II, CD80, ICAM-1 and SLAM. Further to Th1/Th2 modulation, a number of T-regulatory (Treg) genes were differentially expressed in the AEC-MDDC-re-activated CD4+ T-cells, and members of the chemokine and metallothionein families were elevated in the same population. Collectively the results of this study suggest that in the context of the atopic airway microenvironment where there is an abundance of Th2-related mediators, healthy AEC arm locally maturing DC with an arsenal of anti-microbial defences that can be rapidly employed in response to encounter with inhaled pathogens, in particular viruses. In this way, the DC are maintained in an ideal functional phenotype to efficiently mobilise both innate and Th1-polarised adaptive immune defences against infection, whilst achieving tight control of potentially-damaging Th2 immunity to aeroallergens, thus contributing to the maintenance of immunological homeostasis within the respiratory tract.

The present study was undertaken to evaluate the rat as a model for middle ear re­search. The rat was chosen primarily because the gross structure of its middle ear shows several similarities to that of man. It was considered of great importance to make a thorough structural study of the rat middle ear and to compare the results with those reported for the human middle ear. The thesis therefore includes indepen­dent studies on various aspects of rat middle ear structure and function as well as a review of the literature. The most pertinent findings in the experimental part of this study were the following. The rat Eustachian tube consists of a nasopharyngeal, and a cartilaginous and bony portion. The orifice of the nasopharyngeal portion is composed of two soft tissue lips, which appear to be opened mainly by the action of the salpingopharyngeal mus­cle, but also by the levator and tensor veli palatini muscles. The cartilaginous por­tion appears to be opened solely by the tensor veli palatini muscle. The tensor tympani muscle seems to have no effect on the tube. A ciliated and secretory epithelium lines the inferomedial walls of the tube throughout its length. In the tympanic cavity these thelial cell types extend as two tracts - one anterior and the other inferoposterior to  the promontory - which communicate with the epitympanic/attic compartments. The remaining parts of the tube and the tympanic cavity are covered by a squamous/cuboidal, non-ciliated epithelium. The subepithelial loose connective tissue contains vessels, nerves, and connective tissue cells, among these mast cells. The mast cells are confined to areas covered by the ciliated epithelium, and in the floor of the bulla, in the pars flaccida, and along the manubrial vessels. Glands are restricted to the Eustachian tube. In the clearance/transport of serum-like material, from the epitympanum towards the tube, hydrostatic forces appear to be important. The tympanic membrane is vascularized from meatal and tympanal vessels. Meatal ves­sels branch in the pars flaccida and along the handle of the malleus, where they are localized directly beneath the outer, keratinizing, stratified, squamous epithelium. Furthermore, meatal vessels form a vascular network at the junction between the fi­brocartilaginous annulus and the tympanic sulcus. Tympanal vessels send branches to the periphery of the pars tensa, where they run immediately beneath the tympanal, simple, squamous epithelium. In the major portion of the pars tensa, no blood vessels were found. The rat stapedial artery is a thin-walled vessel with a wide lumen. Without branch­ing, it runs through the tympanic cavity to the extratympanal regions it supplies. In contrast to the corresponding artery in man, the rat stapedial artery persists throughout life. The artery does not seem to be affected by the fluid produced during experimentally induced otitis media with effusion. The middle ear structure in the rat and in man show both similarities and differ­ences. If the differences are kept in mind and considered, it would seem that the rat is indeed a suitable model for experimental middle ear research. / digitalisering@umu

In 2008 the Nobel Prize in Physiology or Medicine was awarded to the co-discoverers of the Human Immunodeficiency Virus Type 1 (HIV-1), the causative agent of Acquired Immunodeficiency Syndrome (AIDS). This award acknowledged the enormous worldwide impact of the HIV-1/AIDS pandemic and the importance of research aimed at halting its spread. Since the syndrome was first recognized, 25 million people have succumbed to AIDS and over 33 million are currently infected with HIV-1 (www.unaids.org). The most effective strategy for ending the pandemic is the creation of a prophylactic vaccine. Yet, to date, all efforts at HIV-1 vaccine design have met with very limited success. The consistent failures of vaccine candidates stem in large part from the unprecedented diversity of HIV-1. Among the novel theories of vaccine design put forward to address this diversity is the targeted vaccine approach. This proposal is based on the finding that mucosal transmission of HIV-1, the most prevalent form, occurs across a selective bottleneck such that typically only a single (or a few) variants of the viral swarm present in a donor are passed to the recipient. While the mechanisms controlling the selection are largely unknown, the targeted vaccine approach postulates that once they are identified, we can utilize this understanding to design vaccines specifically targeted to the characteristics shared by the rare, mucosally transmissible HIV-1 variants. The studies described in this work were conducted to improve our understanding of the factors influencing viral variant selection during mother-to-child-transmission of HIV-1, a route of mucosal transmission which has globally become the leading cause of child infection. A unique panel was generated, consisting of nearly 300 HIV-1 envelope genes cloned from infected mother-infant pairs. Extensive characterization of the genotypes, phenotypes and phylogeny of these clones was then done to identify attributes differentiating early infant from maternal variants. Low genetic diversity of HIV-1 envelope variants was detected in early infant samples, suggesting a bottleneck and active selection of variants for transmission. Transmitted variants did not differ from non-transmitted variants in CD4 and CCR5 use. Infant isolates replicated poorly in macrophages; a cell subtype hypothesized to be important in the establishment of infection. The sensitivity of infant envelope variants to neutralization by a panel of monoclonal antibodies, heterologous and autologous plasmas and HIV-1 entry inhibitors varied. Most intriguingly, envelopes cloned from infants infected during delivery exhibited a faster entry phenotype than maternal isolates. Together, these findings provide further insight into viral variant selection during mother-to-child transmission. Identification of properties shared by mucosally transmitted viral variants may allow them to be selectively targeted, resulting in improved methods for preventing HIV-1 transmission.

Syfte: Är att ta reda på om man med hjälp av immunhistokemi (IHC) med infärgning avantikroppar mot laminin-5 och C3d kan särskilja mellan slemhinnepemfigoid (MMP) ochorala lichenoida reaktioner (OLR) med epitelsläpp. Vidare undersöks graden inflammation för MMP och OLR för att fastställa om det går att se ett samband mellan grad av inflammation och antikroppsinfärgning.Material och metod: En pilotstudie utfördes på 10 prover med diagnosen MMP respektive 9 prover med OLR, som hämtades från Malmö universitets biobank. Proverna genomgickrutinfärgning respektive antikroppsinfärgning mot laminin-5 och C3d. Granskning av prover skedde i digitalmikroskop. Efteråt delades proverna in i grupper efter var infärgningen sågs. Sammanställning gjordes i Excel med stapeldiagram.Resultat: Ingen tendens till särskiljning ses mellan MMP och OLR avseende infärgning mot laminin-5 och C3d. Positiva utslag för infärgning mot Laminin-5 ses enhetligt på enbart en sida om epitelsläppet hos både snitten för MMP och OLR. Vidare kan det inte ses att snitt med diagnosen OLR har slumpartad infärgning mot laminin-5 på båda sidor om släppet. Det finns en tendens till att MMP-snitt får mer positiva utslag för infärgning mot C3d än för OLR snitt. Graden inflammation var högre i OLR snitt än för MMP snitt.Slutsats: Enligt studien går det inte att, med IHC, med infärgning av antikroppar mot laminin5 och C3d, kunna särskilja MMP och OLR med epitelsläpp. Större urval krävs för definitiva slutsatser. Vidare studier behövs för att utreda om det går att använda IHC för att särskilja MMP och OLR.Nyckelord: C3d, immunhistokemi (IHC), laminin-5, orala lichenoida reaktioner (OLR),slemhinnepemfigoid (MMP) / Aim: To investigate if it is possible to differentiate between the diagnoses mucous membrane pemphigoid (MMP) and oral lichenoid reactions (OLR) with epithelial detachment using immunohistochemistry (IHC) with antibodies against laminin-5 and C3d. Furthermore, the extent of inflammation was examined for MMP and OLR to determine if a correlation between the inflammation and immunostaining is evident.Material and method: A pilot study is conducted using 10 samples diagnosed with MMP and 9 samples diagnosed with OLR, collected from Malmö University’s biobank. H&E staining and immunostaining against laminin-5 and C3d is performed on the samples. Analysis is conducted using a microscope. Samples are then divided into groups depending on the staining. Excel is used to compile the results.Result: No differentiating tendencies are observed between MMP and OLR regardingimmunostaining against laminin-5 and C3d. Immunostaining against laminin-5 is positive for MMP and OLR and is seen continuously on only one side of the epithelial detachment.Furthermore, staining with laminin-5 is not seen as random staining on both sides of theepithelial detachment. Samples with MMP have a higher tendency to stain against C3dcompared to OLR samples. Inflammation is higher in OLR samples than those for MMP.Conclusion: According to this study it is not possible to differentiate between the diagnoses MMP and OLR with epithelial detachment using immunostaining against laminin-5 and C3d. A larger sample size is needed for a definitive conclusion. Additionally, further studies are required to conclude if IHC can be used to differentiate between MMP and OLR.Keywords: C3d, immunohistochemistry (IHC), laminin-5, mucous membrane pemphigoid(MMP), oral lichenoid reactions (OLR)

O objetivo foi evidenciar a presença do espaço intercelular dilatado do epitélio esofágico após a infusão de ácido clorídrico (HCl) à 0,1N comparando com a infusão de soro fisiológico (SF) em pacientes sem sintomas típicos da DRGE com mucosa esofágica normal e compará-los com os de sintomas típicos e esofagite erosiva. Foram entrevistados e realizaram o exame de endoscopia digestiva alta 60 pacientes destes, 29 foram incluídos no estudo sendo 18 com esôfago normal (9 foram infundidos SF e 9 HCl) e 11 com esofagite erosiva (6 foram infundidos SF e 5 HCl) e foram realizados 4 biópsias da mucosa esofágica (2 antes e 2 depois das infusões). Não foi encontrado diferença estatisticamente significante no espaço intercelular da mucosa esofágica dos pacientes com e sem esofagite erosiva com ácido clorídrico ou soro fisiológico não sendo um marcador da DRGE / The purpose was to prove the presence of extended intercellular space of the esophagic epithelium after chloridric acid infusion (HCI) to 0,1N comparing to the physiologic serum infusion (PS) in patients without typical symptoms of DGER with normal esophagic mucous membrane and compare them to ones with typical symptoms and erosive esophagitis. 60 patients were interviewed and took the high digestive endoscopy; 29 were included in the research, among them 18 with normal esophagus (9 were infused PS, and 9 HCI) and 11 with erosive esophagitis (6 were infused PS and 5 HCI); 4 biopsies of esophagic mucous membrane were made (2 before and 2 after infusions). It was not found any statistically meaningful difference in intercellular space of esophagic mucous membrane in patients with or without erosive esophagitis with chloridric acid or physiologic serum, and thus it is not a DGER

O objetivo foi evidenciar a presença do espaço intercelular dilatado do epitélio esofágico após a infusão de ácido clorídrico (HCl) à 0,1N comparando com a infusão de soro fisiológico (SF) em pacientes sem sintomas típicos da DRGE com mucosa esofágica normal e compará-los com os de sintomas típicos e esofagite erosiva. Foram entrevistados e realizaram o exame de endoscopia digestiva alta 60 pacientes destes, 29 foram incluídos no estudo sendo 18 com esôfago normal (9 foram infundidos SF e 9 HCl) e 11 com esofagite erosiva (6 foram infundidos SF e 5 HCl) e foram realizados 4 biópsias da mucosa esofágica (2 antes e 2 depois das infusões). Não foi encontrado diferença estatisticamente significante no espaço intercelular da mucosa esofágica dos pacientes com e sem esofagite erosiva com ácido clorídrico ou soro fisiológico não sendo um marcador da DRGE / The purpose was to prove the presence of extended intercellular space of the esophagic epithelium after chloridric acid infusion (HCI) to 0,1N comparing to the physiologic serum infusion (PS) in patients without typical symptoms of DGER with normal esophagic mucous membrane and compare them to ones with typical symptoms and erosive esophagitis. 60 patients were interviewed and took the high digestive endoscopy; 29 were included in the research, among them 18 with normal esophagus (9 were infused PS, and 9 HCI) and 11 with erosive esophagitis (6 were infused PS and 5 HCI); 4 biopsies of esophagic mucous membrane were made (2 before and 2 after infusions). It was not found any statistically meaningful difference in intercellular space of esophagic mucous membrane in patients with or without erosive esophagitis with chloridric acid or physiologic serum, and thus it is not a DGER