As the use of probiotics and postbiotics is increasingly gaining ground in the past decade, the possibility of using bacterial strains with postbiotic activity to restore homeostasis in pathologic conditions such as IBD is extensively debated. However, clinical data as far as induction of remission is concerned has not been encouraging so far, while researchers observe that only a small number of treatments which seem promising on in vitro or mouse models translate to significant clinical benefit. This could mean that the models used so far to test potential treatments are not an accurate representation of the human intestine's rather complex micro-environment, and thus there is a need for the development of more relevant models. In this thesis, a novel organ culture model for polarized stimulation of intestinal mucosa is described. In the intestine, apical and basolateral challenge of the mucosal layer can elicit completely different results, and this is one of the issues addressed in this work. First of all, we show that it is possible to keep human intestinal mucosa in polarized culture for at least 24 hours, provided the explants are cultured in an atmosphere that is rich in oxygen. Polarized challenge is achieved by mechanical means, namely by attaching a cave plastic cylinder on the apical side of the mucosal layer, in order to confine the stimuli. We examine the impact of the cylinder and the surgical glue used to attach it on tissue morphology and survival, and show that there is no negative impact. Once optimized, this experimental set-up is used to challenge explants with pathogenic and probiotic bacteria alike and evaluate the immune response. On this model we are able to mimic a classical pathogen infection using a highly invasive Salmonella enterica serovar typhimurium strain, FB62. The response of the tissue to the pathogen was monitored by assessing the phenotype of explants after culture, while cytoine secretion profiles of the tissue were also studied. Salmonella challenge led to a damaged explant phenotype, upregulation of TNF-α and downregulation of IL-10. This response was abolished in the presence of an antiinflammatory postbiotic component, namely culture supernatant of the probiotic Lactobacillus paracasei. Moreover, as far as challenge with pathogens is concerned, we use this novel system to examine the mechanism of Shigella induced apoptosis of epithelial cells. Importantly, no animal models are available for the study of Shigella infection, as these 18 bacteria are poorly virulent in rodents. Thus, in an effort to study the involvement of Gadd45a in the apoptotic route triggered by Shigella on intestinal epitelial cells, HeLa cells, which are however a poor model for an intestinal pathogen were used. Gadd45a participates in the responses to a variety of DNA damaging agents and interacts with proteins such as Cdc2, PCNA and p21. In this work, the authors showed that after infection of HeLa cells with Shigella, Gadd45a is involved in the induction of the apoptotic process. The data was confirmed in a more relevant setting, by applying Shigella on human intestinal mucosa. Finally, we use the novel organ culture platform to test three different strains thought to exert probiotic actions. Surprisingly, we show that this is not the case, and the three strains can exert different activities even on healthy tissue. More specifically, Lactobacillus paracasei and Lactobacillus rhamnosus GG did not significantly alter the phenotype or cytokine secretion profiles of healthy explants, but challenge with Lactobacillus plantarum resulted in a detrimental effect. Of note, all three strains were detrimental for IBD explants when administered as live bacteria, even though one of the strains (L. paracasei) had previously been found to exert a prophylactic effect in a mouse model of colitis. On the contrary, we show that a potent postbiotic (L. paracasei culture supernatant) is able to ameliorate overt inflammation on both ulcerative colitis and Crohn's disease tissues as attested by cytokine secretion profiles of challenged explants. In conclusion, this work introduces a valid alternative system on which to study the interaction of various components (bacterial, pharmacological, and others) with the human intestinal mucosa.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

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Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Autor no autoriza el acceso a texto completo de su documento / El objetivo de este estudio fue determinar las patologías de la mucosa oral más frecuentes entre los pacientes que acuden al Servicio de Diagnóstico de la Facultad de Odontología de la Universidad de Chile, y su asociación con factores demográficos. Se llevó a cabo un estudio descriptivo retrospectivo donde se analizaron las fichas clínicas y registros de 14.479 pacientes que acudieron al servicio entre Enero de 2001 y Diciembre de 2008, registrándose, edad, sexo, diagnóstico, localización anatómica y expresión clínica de las lesiones. El 3.40% (n=493) de los pacientes presentaron patologías de la mucosa oral, de ellos 65.11% mujeres y 34.88% hombres. Las lesiones más frecuentes fueron las reaccionales (23.32%), la localización anatómica más frecuente fue la cara interna de la mejilla (19.91%) y lengua (19.49%). La expresión clínica más frecuente fue el tumor (34.92%). Las diez lesiones de mucosa oral más frecuentes fueron el pseudofibroma (15.41%), Liquen plano (8.52%), UROs (8.11%), Candidiasis (7.10%), Granuloma telangectásico (6.50%), CEC (6.90%), Penfigoide mucoso (5.07%), Papiloma (4.26%), Leucoplasia (3.66%) y Queilitis Actínica (2.84%). Los resultados del presente estudio coinciden en gran medida con los reportados por la literatura internacional.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master in Pharmacy. Johannesburg, South Africa, 2006 / The aqueous extract of rooibos has been used for more than three hundred years since its discovery by the indigenous people. Currently, rooibos is gaining popularity in the cosmetic industry and incorporation of rooibos extracts in topical cosmetic formulations has become a fashionable trend. Both topical and intestinal absorption of rooibos tea were investigated. The transport of aspalathin in the unfennented (green) rooibos aqueous extracts and aqueous solution of pure aspalathin were studied. / IT2018

90% of Human Immunodeficiency Virus (HIV) infections are acquired across the genital or gastrointestinal mucosa, and infection leads to profound depletion of CD4+ lymphocytes. Antiretroviral therapy can restore blood CD4+ T cells. However, immune dysfunction and defects in mucosal antimicrobial defence persist. Some CD4+ T-subsets, particularly antimicrobial Th17 cells, show enhanced susceptibility to HIV infection and are also preferentially depleted in the course of HIV infection; the latter may allow microbial translocation into the bloodstream. Genital infections have been shown to have direct mucosal immune effects and to increase susceptibility to HIV; however, the effect of systemic infections, such as Malaria (which is holo-endemic in some HIV prevalent regions) is unknown. Understanding the relationship between HIV, highly susceptible immune cells, immune activation and malaria infection on mucosal tissues has been the main focus of my thesis. In HIV-infected individuals, I explored whether HIV antiretroviral therapy restores gut Th17 populations and improves gut antimicrobial defences. Therapy restored gut Th17 populations in some, but not all individuals, but antimicrobial defence remained impaired. I then piloted a novel mucosal-optimized PCR assay to measure cervical immune gene responses, as standard mucosal assays are inadequate. I succeeded in measuring mitogen-induced, but not HIV-specific, cervical immune responses in HIV-infected individuals. Next, using this PCR platform I examined mitogen-induced cervical immune responses in individuals demonstrating reduced susceptibility to HIV, and found that they had reduced production of both Th17-associated and pro-inflammatory cytokines from cervical cells. Finally, in a murine model I found that malaria caused genital and gastrointestinal mucosal immune activation, and increased both the expression of mucosal HIV susceptibility immune markers, and mucosal T cell immune activation. In summary, insufficient gastrointestinal Th17 cells restoration does not underlie persistent mucosal immune activation and microbial translocation in HIV-infected people on therapy. A reduced frequency of highly susceptible Th17 cells in the cervix of HIV-exposed but uninfected individuals was identified as a correlate of reduced HIV susceptibility. Malaria, a common systemic infection in HIV-endemic countries, may enhance susceptibility to HIV through increasing putative immune markers of HIV susceptibility and immune activation in potential mucosal sites of HIV exposure.

Thesis--M. Phil., University of Hong Kong.