Alveolar macrophages comprising up to 95% of the pulmonary alveoli, are the gate-keepers of homeostasis by ensuring efficient tissue function through metabolizing excessive surfactant and phagocyting inhaled day-to-day and innocuous pathogens and particles, without triggering an immune response. Despite that, they are capable of orchestrating a very well-balanced immune response upon invasion of pathogens. These embryonic-derived cells are capable of self-renewal and therefore maintain themselves in the lungs throughout adult life, with minimal contribution from the circulating monocytes. This self-renewal capacity is attained intrinsically by maintaining low levels of transcription factors MafB and cMaf, and extrinsically through two main cytokines, namely GM-CSF secreted by alveolar epithelial type II cells, and TGFb secreted by AMs themselves in an autocrine manner. However, in inflammatory conditions such as acute respiratory distress syndrome (ARDS), depletion of AM pool and upregulation of MAFB among lung macrophages have been reported. Keeping in mind the role of transcription factors MafB and cMaf in inhibiting proliferative capacity of macrophages; we hypothesized that this depletion is due to upregulation of MafB and hence the suppression of enhancer regions of self-renewal genes in AMs. To investigate the role of MafB and its compensatory partner cMaf in ARDS, we have established a mouse model of ARDS using oropharyngeal instillation of LPS in WT and MafB/cMaf double-knockout (Maf-DKO) mice. Alongside, the molecular mechanisms of the effect of LPS on AMs was investigated ex-vivo. The obtained results have clearly shown that ex-vivo, LPS inhibits proliferation of AMs in a dose dependent manner, and induces apoptosis significantly. Regain of proliferative potential of LPS-stimulated AMs was evident upon TLR4 inhibition, and MyD-88 was shown to be the dominant adaptor downstream of TLR4 (as opposed to TRIF). Both WT and DKO AMs responded to LPS stimulation within 2 hours, by switching from OXPHOS to glycolysis, which accounts for their efficient pro-inflammatory phenotype once activated. Upon activation, MafB and cMaf were upregulated after 48 hours and the inhibition of AM proliferation was shown to be Maf-independent. Similarly, depletion of AM pool was shown to be Maf independent invivo, evident by similar kinetics of AM numbers in WT and DKO at different timepoints upon LPS stimulation. However, several findings indicated potential advantage of Maf-deficiency in tissue regeneration; this includes: 1) higher number of Ly6C+ monocytes and their earlier differentiation into resident AMs, 2) lower degree of tissue damage revealed by H&E staining, 3) higher number of alveolar epithelial type II cells, 4) significantly higher levels of cytotoxicity pointing towards cellular turnover, and 5) significantly higher levels of SP-D and thus its antiinflammatory effects. In a quest for investigating factors which could enhance proliferative potential of AMs and ultimately neutralize the inhibitory effect of LPS, the impact of TGFb and ActivinA was studied. I have shown that TGFb and to a higher extend ActivinA boost the proliferation rate of AMs, ex-vivo. The autocrine effect of these cytokines was validated by blocking signal transduction through inhibition of SMAD2/3, which resulted in a significant increase in doubling time of AMs. Interestingly, Inhba was shown to be significantly upregulated in AMs, as opposed to TGFb. The importance of ActivinA was further demonstrated by its direct inhibition and the resulting reduction in growth rate of AMs. On the contrary to the significant role of these cytokines in enhancing the growth rate of AMs ex-vivo, they could rescue AM proliferation under the effect of LPS. In conclusion, I have demonstrated that LPS inhibits AM proliferation in a dose dependent and Maf-independent manner. Furthermore, neither TGFb nor ActivinA could rescue proliferation of LPS-stimulated AMs. Although Maf-deficiency was not shown to be beneficial during the inflammatory phase of ARDS, due to the fact that both WT and DKO AMs were equally depleted at the peak of inflammation, multiple data indicated potential advantage of Maf deficiency during resolution phase.

Mucopolysaccharidosis VI (MPS VI) is a hereditary lysosomal storage disease caused by the absence of the enzyme arylsulfatase B (ARSB). Craniofacial defects are common in MPS VI patients and manifest as abnormalities of the facial bones, teeth, and temporomandibular joints. Although enzyme replacement therapy (ERT) is the treatment of choice for MPS VI, the effects on the craniofacial and dental structures are still poorly understood. In this study, we used an Arsb-deficient mouse model (Arsb m/m ) that mimics MPS VI to investigate the effects of ERT on dental and craniofacial structures and compared these results with clinical and radiological observations from three MPS VI patients. Using micro-computed tomography, we found that the craniofacial phenotype of the Arsb m/m mice was characterized by bone exostoses at the insertion points of the masseter muscles and an overall increased volume of the jaw bone. An early start of ERT (at 4 weeks of age for 20 weeks) resulted in a moderate improvement of these jaw anomalies, while a late start of ERT (at 12 weeks of age for 12 weeks) showed no effect on the craniofacial skeleton. While teeth typically developed in Arsb m/m mice, we observed a pronounced loss of tooth-bearing alveolar bone. This alveolar bone loss, which has not been described before in MPS VI, was also observed in one of the MPS VI patients. Interestingly, only an early start of ERT led to a complete normalization of the alveolar bone in Arsb m/m mice. The temporomandibular joints in Arsb m/m mice were deformed and had a porous articular surface. Histological analysis revealed a loss of physiological cartilage layering, which was also reflected in an altered proteoglycan content in the cartilage of Arsb m/m mice. These abnormalities could only be partially corrected by an early start of ERT. In conclusion, our results show that an early start of ERT in Arsb m/m mice achieves the best therapeutic effects for tooth, bone, and temporomandibular joint development. As the MPS VI mouse model in this study resembles the clinical findings in MPS VI patients, our results suggest enzyme replacement therapy should be started as early as possible.

Background. The treatment of patients born with cleft lip and palate has been gradually modified over the years as the surgical procedures have developed and improved. Multidisciplinary team care has evolved and provided improved care with enhanced results. Clefts in the alveolus can be reconstructed by alveolar bone grafting or by periosteoplasty. The main goal is to repair and close the alveolar cleft and create a continuous alveolar processes so that the teeth can erupt. Aims. This thesis has several aims: to investigate the impact of dental status and initial cleft width on the outcome of Secondary alveolar bone grafting (SABG) in patients born with unilateral cleft lip and palate (UCLP) at the 10-year follow-up (Studies I and II); to compare the outcomes of primary periosteoplasty (PPP) with those of SABG in patients born with unilateral cleft lip and alveolus (CLA) (Study III); to evaluate clinical and radiographic conditions and identify factors important for the final treatment outcomes after SABG ( Study IV); to evaluate two radiographic methods, i.e. occlusal radiographs and cone beam tomography (CBCT)) for assessing alveolar bone height ( study IV). Results. In UCLP patients, SABG achieved excellent results in terms of bone height; tended to reduce with time, correlated with dental status and dental restoration factors. Occlusal radiographs correspond well with the CBCT, for evaluating alveolar bone height in cleft area. The width of the initial cleft does not seem to affect the success of SABG. Finally, patients with CLA treated with PPP at the time of lip repair have inferior bone formation outcomes in the cleft area compared with patients treated with SABG at the time of mixed dentition. Conclusion.  Poor dental status and malpositioning negatively affect the long-term survival of bone in the alveolar cleft. The initial cleft width affects certain dental status factors. In adults with UCLP, the alveolar bone height in the cleft was correlated to the presence of gingival inflammation and restorations at 20 years follow-up. Specially designed maintenance therapy is beneficial, after complex dental restorations in the cleft area. SABG is preferred to PPP for the reconstruction of alveolar clefts.

Radiographic modalities are used in addition to clinical methods to gain information about the patients. In the examination of the periodontium, which is comprised of the gingiva, alveolar bone, periodontal ligament, and cementum, the radiographic examination plays an integral role for diagnosis of periodontal disease. The diagnosis periodontitis is based on a finding of alveolar bone loss. Changes of the alveolar bone can be assessed by different radiographic modalities, such as intraoral radiography (bitewing and periapical radiography) and panoramic radiography. Analysis of alveolar bone loss (alveolar bone level, detection of vertical bone defect and furcation involvement) in radiographic modalities and a systematic review could be helpful to suggest the more applicable radiographic methods for diagnosis of periodontal disease. The purpose of the present study was to examine diagnostic properties of panoramic radiography for the assessment of alveolar bone loss for the diagnosis of periodontal diseases as compare to posterior bitewing radiography. For 96 patients panoramic and posterior bitewing radiographs were performed and results of analysis of visibility of radiographic images and assessment of alveolar bone loss were compared. Also, the systematic review on diagnostic properties of panoramic radiography in the assessment of alveolar bone loss was performed. Based on the results of this study, recommendations for clinical practice and for research were proposed. / Rentgeniniai tyrimo metodai taikomi, kad būtų papildyti klinikinio tyrimo duomenys. Tiriant periodontą, kurį sudaro dantenos, dantinė atauga, periodonto raiščiai ir cementas, rentgeninis tyrimas svarbus diagnozuojant periodonto ligas, nes jo metu nustatomi dantinės ataugos pokyčiai. Dantinės ataugos pokyčiams vertinti daromos rentgeno nuotraukos taikant vidinių burnos rentgeno nuotraukų darymo metodus, t. y. kandimo ar dantų šaknų rentgeno nuotraukų darymo metodus, ir išorinės burnos rentgeno nuotraukos darymo metodą, t. y. panoraminės rentgeno nuotraukos darymo metodą. Atlikus dantinės ataugos pokyčių (kaulo lygio arba rezorbcijos, kaulo defekto ir tarpšaknio kaulo pažeidimų) analizę, taikant rentgeninius metodus, ir padarius sisteminę literatūros apžvalgą, galima būtų pasiūlyti tinkamiausią rentgeno metodą periodonto ligoms diagnozuoti. Šio darbo tikslas – ištirti ir palyginti panoraminės rentgeno nuotraukos ir kaplių bei krūminių dantų rentgeno nuotraukų darymo metodų diagnostikos ypatybes vertinant dantinės ataugos pokyčius periodonto ligoms diagnozuoti. Kiekvienam, iš 96 pacientų įtrauktų į tyrimą, buvo padaryta panoraminė rentgeno nuotrauka ir kaplių bei krūminių dantų kandimo rentgeno nuotraukos. Atlikta dantinės ataugos rentgeno atvaizdo vizualioji kokybė analizė ir vertinimai skirtingose rentgeno nuotraukose bei palyginti rezultatai. Padaryta sisteminė literatūros apžvalga apie panoraminės rentgeno nuotraukos vertę diagnozuojant periodonto ligas. Pagal šio tyrimo... [toliau žr. visą tekstą]

Rentgeniniai tyrimo metodai taikomi, kad būtų papildyti klinikinio tyrimo duomenys. Tiriant periodontą, kurį sudaro dantenos, dantinė atauga, periodonto raiščiai ir cementas, rentgeninis tyrimas svarbus diagnozuojant periodonto ligas, nes jo metu nustatomi dantinės ataugos pokyčiai. Dantinės ataugos pokyčiams vertinti daromos rentgeno nuotraukos taikant vidinių burnos rentgeno nuotraukų darymo metodus, t. y. kandimo ar dantų šaknų rentgeno nuotraukų darymo metodus, ir išorinės burnos rentgeno nuotraukos darymo metodą, t. y. panoraminės rentgeno nuotraukos darymo metodą. Atlikus dantinės ataugos pokyčių (kaulo lygio arba rezorbcijos, kaulo defekto ir tarpšaknio kaulo pažeidimų) analizę, taikant rentgeninius metodus, ir padarius sisteminę literatūros apžvalgą, galima būtų pasiūlyti tinkamiausią rentgeno metodą periodonto ligoms diagnozuoti. Šio darbo tikslas – ištirti ir palyginti panoraminės rentgeno nuotraukos ir kaplių bei krūminių dantų rentgeno nuotraukų darymo metodų diagnostikos ypatybes vertinant dantinės ataugos pokyčius periodonto ligoms diagnozuoti. Kiekvienam, iš 96 pacientų įtrauktų į tyrimą, buvo padaryta panoraminė rentgeno nuotrauka ir kaplių bei krūminių dantų kandimo rentgeno nuotraukos. Atlikta dantinės ataugos rentgeno atvaizdo vizualioji kokybė analizė ir vertinimai skirtingose rentgeno nuotraukose bei palyginti rezultatai. Padaryta sisteminė literatūros apžvalga apie panoraminės rentgeno nuotraukos vertę diagnozuojant periodonto ligas. Pagal šio tyrimo... [toliau žr. visą tekstą] / Radiographic modalities are used in addition to clinical methods to gain information about the patients. In the examination of the periodontium, which is comprised of the gingiva, alveolar bone, periodontal ligament, and cementum, the radiographic examination plays an integral role for diagnosis of periodontal disease. The diagnosis periodontitis is based on a finding of alveolar bone loss. Changes of the alveolar bone can be assessed by different radiographic modalities, such as intraoral radiography (bitewing and periapical radiography) and panoramic radiography. Analysis of alveolar bone loss (alveolar bone level, detection of vertical bone defect and furcation involvement) in radiographic modalities and a systematic review could be helpful to suggest the more applicable radiographic methods for diagnosis of periodontal disease. The purpose of the present study was to examine diagnostic properties of panoramic radiography for the assessment of alveolar bone loss for the diagnosis of periodontal diseases as compare to posterior bitewing radiography. For 96 patients panoramic and posterior bitewing radiographs were performed and results of analysis of visibility of radiographic images and assessment of alveolar bone loss were compared. Also, the systematic review on diagnostic properties of panoramic radiography in the assessment of alveolar bone loss was performed. Based on the results of this study, recommendations for clinical practice and for research were proposed.

Aims: T1α/(podoplanin) is abundantly expressed in the alveolar epithelial type I cells (ATI) of rodent and human lungs. Caveolin-1 is a classical primary structural protein of plasmalemal invaginations, so-called caveolae, which represent specialized lipid rafts, and which are particularly abundant in ATI cells. The biological functions of T1α in the alveolar epithelium are unknown. Here we report on the characteristics of raft domains in the microplicae/microvillar protrusions of ATI cells, which contain T1α. Methods: Detergent resistant membranes (DRMs) from cell lysates of the mouse epithelial ATI-like cell line E10 were prepared using different detergents followed by flotation in a sucrose gradient and tested by Western and dot blots with raft markers (caveolin-1, GM1) and nonraft markers (transferrin receptor, PDI and β-Cop). Immunocytochemistry was employed for the localization of T1α in E10 cells and in situ in rat lungs. Results: Our biochemical results showed that the solubility or insolubility of T1α and caveolin-1 differs in Triton X-100 and Lubrol WX, two distinct non-ionic detergents. Caveolin-1 was unsoluble in both detergents, whereas T1α was Triton X-100 soluble but Lubrol WX insoluble. Immunofluorescence double stainings revealed that both proteins were colocalized with GM1, while caveolin-1 and T1α were not colocalized in the plasma membrane. Cholesterol depletion modified the segregation of T1α in Lubrol WX DRMs. Cellular processes in ultrathin sections of cultured mouse E10 cells were immunogold positive. Immunoelectron microscopy (postembedding) of rat lung tissue revealed the preferential localization of T1α on apical microvillar protrusions of ATI cells. Conclusion: We conclude that T1α and caveolin-1 are located in distinct plasma membrane microdomains, which differ in their protein-lipid interactions. The raft-associated distribution of T1α may have an impact on a specific, not yet clarified function of this protein in the alveolar epithelium. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Die Inzidenz des Atemnotsyndroms ist bei männlichen Neugeborenen etwa 1,7-mal so hoch wie bei weiblichen. Zur Erforschung der Ursachen dieser Tatsache wurden in der vorliegenden Arbeit geschlechtsabhängige Unterschiede im transepithelialen Natriumtransport an fetalen distalen Lungenepithelzellen von Ratten untersucht. Die zugrunde liegende Versuchsanordnung stellt ein Modell der Typ II Pneumozyten des späten Frühgeborenen dar. In Ussing Kammer Messungen wurde ein höherer Natriumtransport in weiblichen Zellen im Vergleich zu männlichen Zellen nachgewiesen. Des Weiteren zeigten Genexpressionsanalysen eine höhere Expression der am Natriumtransport beteiligten Kanäle und Transporter in weiblichen Zellen. Um mögliche Ursachen der festgestellten Geschlechtsunterschiede zu eruieren, wurde die Genexpression von Hormonrezeptoren untersucht. Die Ergebnisse lassen vermuten, dass die Rezeptoren weiblicher Geschlechtshormone dabei eine wichtige Rolle spielen. Abschließend betrachtet diese Arbeit die absolute Zahl fetaler distaler Lungenepithelzellen in Rattenfeten beider Geschlechter. Hierbei fanden sich ebenfalls Geschlechtsdifferenzen. Zusammenfassend kann die vorliegende Arbeit zu einem besseren Verständnis der Pathogenese und der Inzidenz des Atemnotsyndroms des Frühgeborenen beitragen.

Actinobacillus pleuropneumoniae est l’agent étiologique de la pleuropneumonie porcine. La bactérie se transmet par voies aériennes et contacts directs. Plusieurs facteurs de virulence ont été identifiés, nommément les polysaccharides capsulaires, les lipopolysaccharide, les exotoxines ApxI à IV et de nombreux mécanismes d’acquisition du fer. Aucun vaccin efficace contre tous les sérotypes de la bactérie n’a encore été élaboré. Afin de mieux comprendre de quelle façon A. pleuropneumoniae régule la transcription de ses nombreux facteurs de virulence et de découvrir de nouvelles cibles potentielles pour l’élaboration de vaccins efficaces, le profil transcriptomique de la bactérie a été étudié dans des conditions simulant l’infection ainsi qu’à la suite d’une infection naturelle aiguë chez l’animal. Des biopuces de première et de seconde génération (AppChip1 et AppChip2) comportant respectivement 2025 cadres de lecture ouverts (ORF) de la version préliminaire du génome d’A. pleuropneumoniae sérotype 5b souche L20 et 2033 ORF de la version finale annotée du même génome ont été utilisées. Dans un premier temps, des expériences réalisées dans des conditions de concentration restreinte en fer ont permis d’identifier 210 gènes différentiellement exprimés, dont 92 étaient surexprimés. Plusieurs nouveaux mécanismes d’acquisition du fer ont pu être identifiés, incluant un système homologue au système YfeABCD de Yersinia pestis, impliqué dans l’acquisition du fer chélaté, ainsi que des gènes homologues aux composantes du système HmbR de Neisseria meningitidis impliqué dans l’acquisition du fer à partir de l’hémoglobine. Dans des conditions de culture permettant la formation de biofilms, les gènes tadC et tadD d’un opéron tad (« tight adherence locus ») putatif, les gènes pgaBC impliqués dans la synthèse d’un polysaccharide de la matrice du biofilm ainsi que deux gènes présentant de fortes homologies avec un gène codant pour l’adhésine auto-transporteur Hsf retrouvée chez Haemophilus influenzae ont montré une surexpression significative. Plusieurs de ces gènes ont également été retrouvés lors d’expériences réalisées avec des cellules épithéliales d’origine pulmonaire en culture, qui ont permis d’identifier 170 gènes différentiellement exprimés après la croissance planctonique au-dessus des cellules, et 131 autres suite à l’adhésion à ces cellules. Parmis les gènes surexprimés, les gènes tadB et rcpA de l’opéron tad putatif, les gènes pgaBC ainsi que le gène codant pour l’homologue d’Hsf ont été retrouvés. En présence de liquide de lavage broncho-alvéolaire (BALF), 156 gènes ont montré un profil d’expression modifié, et le gène apxIVA, identifié comme étant surexprimé, a pu être détecté pour la première fois dans des conditions de croissance in vitro. Finalement, des expériences visant à déterminer les gènes utilisés directement chez l’animal en phase aiguë de la pleuropneumonie porcine ont permis d’identifier 150 gènes qui étaient différentiellement exprimés. En plus d’identifier des gènes d’un possible opéron codant pour un fimbriae de type IV, 3 des 72 gènes surexprimés sont conservés chez tous les sérotypes d’A. pleuropneumoniae et codent pour des protéines ou lipoprotéines de surface. Nos expériences ont permis d’identifier plusieurs nouveaux facteurs de virulence potentiels chez A. pleuropneumoniae ainsi que plusieurs nouvelles cibles potentielles pour l’élaboration de vaccins efficaces contre tous les sérotypes. / Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia. Transmission of the disease occurs through direct contact or aerosols. The bacteria possess many virulence factors, namely capsular polysaccharides, lipopolysaccharides, four Apx toxins and iron acquisition mechanisms. To this day, an efficient cross-serotype vaccine has yet to be developed. In order to investigate regulation mechanisms in A. pleuropneumoniae and to identify new potential targets for the synthesis of subunit vaccines, the transcriptomic profile of the bacteria under conditions that simulate the infection and following a natural acute infection in vivo were studied. The experiences relied on first and second generation microarrays (AppChip1 and AppChip2) designed using 2025 ORFs of the draft version of the A. pleuropneumoniae serotype 5b strain L20 genome and 2033 ORFs of the final and annotated version of the same genome respectively. First, experiments were conducted under iron-restricted conditions and 210 genes were deemed differentially expressed, 92 of which were up-regulated. Some new putative iron acquisition mechanisms were identified, including genes homologous to those of the Yersinia pestis YfeABCD chelated-iron acquisition system, as well as other genes homologous to components of the HmbR iron uptake from hemoglobin system of Neisseria meningitidis. When cultured in conditions promoting biofilm production, genes tadC and tadD from a putative tad (« tight adherence locus ») operon, genes pgaABC involved in the biosynthesis of a polysaccharide of the biofilm matrix as well as two ORFs encoding a putative autotransporter adhesins similar to the Haemophilus influenzae Hsf adhesin were all significantly overexpressed. Many of these genes were also overexpressed when lung epithelial cells were infected with A. pleuropneumoniae. While 170 genes were differentially expressed after planktonic growth in the culture medium above the cells, another 131 were identified following direct adhesion to the cells. Genes tadB and rcpA of the tad locus, as well as genes pgaBC and an ORF coding for the Hsf homolog where all found among overexpressed genes. When A. pleuropneumoniae was cultured in contact with broncho-alveolar lavage fluids (BALF), 156 genes were significantly differentially expressed and gene apxIVA, which was up-regulated, was detected for the first time during in in vitro growth conditions. Finally, experiments were conducted in vivo in animals naturally infected with A. pleuropneumoniae in the late stage of the acute phase in order to identify genes that are expressed during the infection of the natural host. While 150 genes were deemed differentially expressed, genes apxIVA as well as two genes from an operon coding for a putative type IV fimbriae were up-regulated. Out of those 72 genes that were overexpressed, 3 encode proteins or lipoproteins of the outer membrane which are conserved among all serotypes of the bacteria. Overall, we were able to identify several new potential virulence factors for A. pleuropneumoniae in the course of our experiments, as well as several new potential targets for the elaboration of an efficient cross-serotype vaccine.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

A quantidade de trabalhos específicos sobre os traumatismos alvéolo-dentários de maior extensão encontrados na literatura é pequena. Este estudo mostra o resultado de 127 casos de traumatismos alvéolo-dentários diagnosticados no Hospital Municipal Arthur Ribeiro de Saboya, em São Paulo, entre junho de 2001 e dezembro de 2002. A análise dos dados permitiu identificar o grupo etário de 0 a 10 anos e o sexo masculino como os mais atingidos, a queda como o fator etiológico mais comum e a fratura alveolar como o principal tipo de lesão encontrada. A maioria dos pacientes apresentou lesões extra e intra orais e 73,2% tardaram até 05 horas para procurar atendimento após o acontecimento do trauma. / There are few specific reports in the literature bearing information on dento-alveolar injuries. A survey was done considering 127 patients recorded from June 2001 to December 2002 showing dento-alveolar injuries treated at the Arthur Ribeiro Saboya Municipal Hospital, Sao Paulo, Brazil. The analysis of data revealed that male patients and the age group 0–10 yr were most prevalently involved in this kind of trauma, the main cause was accidental fall and alveolar fractures were the most frequent type of injury. The majority of the patients suffered some kind of soft tissues injuries and 73,2% of the patients received the first dental assistance during the first 5 hours following the incident.