Objective: The rectal mucosa is highly susceptible to HIV infection. Mucosal fluid contains soluble immune proteins that influence HIV infection, and previous studies have shown unique mucosal protein expression in HIV-exposed seronegative (HESN) populations, which may contribute to reduced HIV susceptibility. However, the key correlates of susceptibility at the rectal mucosa have not been well defined, which is a critical knowledge gap for our understanding of HIV pathogenesis. Methods: Rectal lavage from low risk men was screened for HIV-neutralizing activity in a TZM-bl reporter cell line against an R5-tropic HIV virus. Label-free tandem mass spectrometry was used to characterize soluble proteins within rectal lavage samples from a low-risk cohort of men (n=15), and HESN men who have sex with men (MSM; n=25). Protein expression between populations was compared using adjusted t tests (p<0.05), and was interpreted using hierarchical clustering and DAVID biofunctional analysis. Protein expression was further analyzed using survey data on sexual behaviours. Proteins associated with the HESN population were screened for antiviral activity in TZM-bl and PBMC culture against an R5- and X4-tropic virus. Major Results: Rectal mucosal fluid was able to inhibit HIV infection in vitro by 40% (p<0.05). Mass spectrometry identified 30/341 (9%) proteins deferentially expressed (DE) in HESN MSM. DE proteins held functions in immunity (p=6.68x10-6, p=0.001) and epithelial barrier development (p=1.81x10-4; p=0.01); notably, specific antiproteases were elevated in HESN secretions, two of which were screened for antiviral activity. Serpin B4 (+2.52 L2FD; p=1.09x10-5), showed significant inhibition of HIV in TZM-bl (45% BaL, 34% IIIB; p<0.05) and PBMC culture (37% BaL, 49% IIIB; p<0.05); cystatin A (+1.52 L2FD; p=1.40x10-3) showed no inhibitory effects. Serpin B4 expression was not associated with frequency of oral intercourse (p=0.32), partner viral load (r=0.16; p=0.29) or presence of HIV neutralizing IgA in secretions (p=0.52). Conclusions: This thesis reports the use of proteomics to understand HIV-susceptibility at the rectal mucosa, and identified serpin B4 as a novel antiviral immune correlate in a population of HESN MSM. These results may help guide future studies of prevention technologies, such as microbicides or vaccines, which would ultimately help limit the spread of HIV. / February 2016

The development of mucosal dendritic cells (DCs) in cattle is poorly understood and an analysis of myeloid cells in the bovine small intestine is required to increase our knowledge in this area. The phenotype, frequency and distribution of mucosal myeloid and lymphoid lamina propria leukocytes (LPL) and intraepithelial leukocytes (IEL) in the ileum and jejunum of newborn calves (3-5 weeks old) were analyzed using flow cytometry and immunohistochemistry (IHC). LPL and IEL were isolated through the use of chemical and enzymatic incubations. Costaining with a CD45-specific monoclonal antibody allowed us to exclude all non-leukocytic cells from our analysis of IEL and LPL. The morphology of CD45+CD11c+MHC Class II+ cells isolated from the lamina propria (LP) of ileum and jejunum showed myeloid characteristics, validating the use of CD11c and MHC Class II co-expression to identify myeloid cells. Regional differences in the frequency and number of leukocytes isolated from the IEL and LP compartments of the ileum and jejunum were analyzed in newborn calves. The CD11cHiCD14+ and CD335+ NK cell populations were significantly more abundant in the ileum than the jejunum. IHC was then used to identify the distribution of myeloid cells within the intestine. This analysis confirmed the presence of a variety of myeloid cell populations within the LP. Furthermore, CD11c+ cells were uniquely distributed within the jejunal, but not the ileal IEL compartment. In contrast, CD11b+ cells were present in the ileal, but absent from the jejunal, IEL compartment. A comparison of myeloid cell populations isolated from jejunum and blood dentified distinct mucosal DC populations, such as CD11c+CD13+ cells, which were present in he jejunum but absent from blood. The phenotype, frequency and distribution of IEL and LPL in the ileum and jejunum of weaned calves (6 months old) were then investigated. Significant regional differences were observed when comparing mucosal T cell populations with CD8+ and &#947;&#948; T cells more abundant in the ileum and CD4+ T cells more abundant in the jejunum. Proportionally, there were no significant differences between the frequency and number of myeloid populations in the two regions. IHC was, once again, used to confirm these unique distributions of cells within each region. CD11b+ cells were present in the LP of both the ileum and jejunum, although a small number of CD11b+ cells were found in the ileal epithelium. CD4+ T cells were restricted to the LP, while CD8+ and &#947;&#948; T cells were restricted to the IEL compartment. Significant age-related changes were observed when comparing mucosal leukocyte populations in the ileum and jejunum of newborn and 6 month old calves. In the ileum there was an age-related enrichment of CD8+ and &#947;&#948; T cells, while in the jejunum there was enrichment in CD4+ and CD8+ T cells. In contrast, total myeloid (CD11c+MHC Class II+) cells number remained unchanged but there was a significant age-related enrichment of DC subpopulations (CD13, CD26, CD205). In conclusion, the ileum and jejunum of the newborn calf was populated by diverse myeloid subpopulations, some of which were distinct from myeloid subpopualtions identified in blood. Furthermore, the total number of CD11cHiMHC Class II+ myeloid cells isolated from a 10 cm segment of intestine did not change with age. If neonatal DCs are functionally equivalent to DCs present in weaned calves then the neonatal mucosal immune system appears to have an equivalent capacity to acquire and present antigens acquired from diet, commensal microflora, or pathogens. The one limitation to this conclusion may be the marked difference in the distribution of intraepithelial DC and macrophage distribution when comparing newborn and weaned calves.

CD8+ T cells responses are critical in the immune defense against human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection. A major challenge for vaccine development is that HIV/SIV can rapidly mutate to escape containment by the CD8+ T cell response. Therefore, optimal virus control by a vaccine will likely require clonally diverse CD8+ T cells capable of recognizing mutant viruses. Mucosal tissues play a fundamental role in early HIV/SIV pathogenesis by serving as the site for viral entry, CD4+ T cell depletion, and a reservoir for viral replication. Vaccine strategies that induce effective mucosal immunity will likely be critical for protection against HIV/SIV. We examined the SIV Gag p11C-specific CD8+ T cell responses in peripheral blood, gastrointestinal (GI) mucosal and lung mucosal tissues of rhesus monkeys expressing the MHC class I molecule Mamu-A*01. We first investigated the clonal composition of this cell population during the acute and chronic phases of SIVmac infection. We showed that there is a narrowing of the clonal repertoire from acute to chronic infection and the same clonal populations of virus-specific CD8+ T cells are present in the systemic and mucosal compartments of chronically SIV-infected animals. These data indicated that virus-specific CD8+ T cells establish broadly distributed immune responses. Next, we examined the clonal diversity of systemic and mucosal p11C-specific CD8+ T cells induced by prime-boost vaccination. We found that systemic prime-boost vaccination induced clonally diverse p11C-specific populations in mucosal tissues. There were high levels of clonal sharing between systemic and mucosal compartments soon after vaccination. However, later following vaccination there was decreased clonal sharing between the GI mucosa and the systemic circulation. We showed that this was due to limited trafficking of p11C-specific CD8+ T cells to the GI mucosa following vaccination. Overall, these studies indicate that following SIV infection and systemic vaccination the same p11C-specific clones are present in mucosal and systemic compartments. Moreover, the apparent immune compartmentalization is a consequence of differences in cell trafficking between systemic and mucosal CD8+ T cells. These observations have important implications for the design of HIV vaccines that generate effective mucosal immunity.

Η παρούσα διδακτορική διατριβή επιχειρεί να διερευνήσει την επίδραση της πειραματικής ηπατεκτομής στην δομή του εντερικού βλεννογόνου μελετώντας την απόπτωση και τον πολλαπλασιασμό των επιθηλιακών κυττάρων στις κρύπτες, τα επίπεδα ενδοτοξίνης στην πυλαία φλέβα και την αορτή, καθώς επίσης και τα επίπεδα του οξει-δωτικού stress. Μελετά επίσης τα επίπεδα του οξειδωτικού stress στο εναπομείναν ήπαρ. Επιπλέον, σε μια προσπάθεια θεραπευτικής παρέμ-βασης, διερευνήθηκε ο ρόλος των ρυθμιστικών εντερικών πεπτιδίων BBS και NT στις ανωτέρω παραμέτρους . Προηγούμενες μελέτες έχουν δείξει ότι τα πεπτίδια αυτά εξα-σκούν ένα ευρύ φάσμα δράσεων στον εντεροηπατικό άξονα και βελτι-ώνουν την ακεραιότητα του γαστρεντερικού βλεννογόνου έπειτα από την επίδραση διαφόρων βλαπτικών παραγόντων. Η απουσία χολής ενδοαυλικά, αποστερεί τον εντερικό βλεννο-γόνο, από τις βακτηριοστατικές, αντιενδοτοξινικές και τροφικές της ιδιότητες, οδηγώντας σε αύξηση των βακτηριδίων και της ενδοτοξίνης ενδοαυλικά και σε εντερική ατροφία. Οι μεταβολές αυτές προάγουν τη μετακίνηση βακτηρίων και ενδοτοξινών στην πυλαία φλέβα και ακο-λούθως, μέσω μιας κατασταλμένης εκκαθαριστικής ικανότητας των κυττάρων Kupffer εξαιτίας της μειωμένης μάζας τους, στη συστη-ματική κυκλοφορία. Η συστηματική ενδοτοξιναιμία ενεργοποιεί τη συστηματική φλεγμονώδη απάντηση, η οποία σχετίζεται με τη δυσλει-τουργία που αναπτύσσεται σε απομακρυσμένα όργανα, ενώ συνεισφέ-ρει και στην περαιτέρω επιδείνωση της λειτουργίας του εντερικού φραγμού και της ηπατικής βλάβης. Τα αποτελέσματα της παρούσας μελέτης επιβεβαίωσαν την παρουσία πυλαίας και συστηματικής ενδο-τοξιναιμίας σε πειραματική ηπατεκτομή. Η προκαλούμενη από την πειραματική ηπατεκτομή ατροφία του εντερικού βλεννογόνου τεκμη-ριώθηκε με μορφομετρική ανάλυση και με μετρήσεις του DNA και της πρωτεΐνης. ΄Ενας πιθανός μηχανισμός προαγωγής της ατροφίας του εντερικού βλεννογόνου είναι η διαταραχή της ισορροπίας μεταξύ κυτταρικού πολλαπλασιασμού και κυτταρικού θανάτου στις κρύπτες, με αύξηση της απόπτωσης και μείωση της μιτωτικής δραστηριότητας. Επιπλέον η πειραματική ηπατεκτομή οδήγησε τις εντερικές λειτουργί-ες σε μια κατάσταση χαμηλού οξειδωτικού stress όπως φαίνεται καθαρά από τη μείωση της υπεροξείδωσης των λιπιδίων και της οξει-δωμένης γλουταθειόνης (GSSG) καθώς και από την αύξηση της ανηγ-μένης γλουταθειόνης (GSG). Είναι άξιο παρατήρησης ότι το οξειδωτι-κό stress δεν φαίνεται να είναι το πρωταρχικό αίτιο της πρόκλησης κυτταρικής απόπτωσης και ατροφίας λαχνών που παρατηρείται μετά από μερική ηπατεκτομή, αν και αυτή η επέμβαση μειώνει το οξειδωτι-κό stress κάτω από το control. Από το άλλο μέρος αυτές οι επιδράσεις εξαλείφθηκαν υπό συνθήκες ακόμη πιο μειωμένου οξειδωτικού stress που προκλήθηκαν μετά την χορήγηση των ρυθμιστικών πεπτιδίων BBS και NT Τα ρυθμιστικά εντερικά πεπτίδια, BBS και NT, δρώντας είτε άμεσα, μέσω ειδικών υποδοχέων των επιθηλιακών κυττάρων του εντέ-ρου, είτε έμμεσα, βελτιώνοντας τη μικροκυκλοφορία του εντέρου, μείωσαν σημαντικά την απόπτωση και ανέστρεψαν την εντερική ατρο-φία. Η πρόληψη, από τα ρυθμιστικά πεπτίδια, των επαγόμενων από την ηπατεκτομή κυτταρικών και βιοχημικών μεταβολών του εντερικού βλεννογόνου, οδήγησε σε σημαντική μείωση της πυλαίας και συστη-ματικής ενδοτοξιναιμίας. Επιπλέον, η BBS και η NT, εξασκώντας αντιοξειδωτική δράση και στο ήπαρ, προστάτεψαν το εναπομείναν ήπαρ από δυο μείζονες παράγοντες ηπατικής βλάβης, που είναι το οξειδωτικό stress και η ενδοτοξιναιμία. Συμπερασματικά, τα αποτελέσματα της παρούσας μελέτης δείχ-νουν ότι η δυσλειτουργία του εντερικού φραγμού στην ηπατεκτομή σχετίζεται με την επαγωγή κυτταρικών και βιοχημικών μεταβολών στον εντερικό βλεννογόνο, οι οποίες χαρακτηρίζονται από πρόκληση οξειδωτικού stress και επαγωγή της απόπτωσης. Τα ρυθμιστικά εντε-ρικά πεπτίδια BBS και NT προλαμβάνοντας τις μεταβολές αυτές του εντερικού βλεννογόνου μειώνουν σημαντικά την πυλαία και συστημα-τική ενδοτοξιναιμία. Επίσης, εξασκούν προστατευτική δράση εναντίον του οξειδωτικού stress στο ήπαρ. Η συνδυασμένη ευεργετική επίδραση των ρυθμιστικών πεπτιδίων, τόσο στη δυσλειτουργία του εντερικού φραγμού και την ενδοτοξιναιμία, που ευθύνονται για την ανάπτυξη σηπτικών επιπλοκών και βλάβης απομακρυσμένων οργάνων, όσο και στο οξειδωτικό stress, εισηγούνται μια νέα θεραπευτική προσέγγιση στην ηπατεκτομή. / This doctoral thesis explores the effect of experimental hepatectomy on the structure of the intestinal mucosa by studying apoptosis and proliferation of epithelial cells in crypts, endotoxin levels in portal vein and aorta, as well as levels of oxidative stress. It also investigates the level of oxidative stress on the remaining liver tissue. In addition, in an attempt of therapeutical approach, the role of the regulatory intestinal peptides BBS and NT in the above parameters has also been evaluated. Previous studies have demonstrate that the above peptides present a wide range of actions on the intestinal-liver axis, thus improving integrity of the gastrointestinal mucosa after it has been affected by various harmful factors. Intestinal mucosa in the absence of bile in the lumen lacks its bacteriostatic, anti-endotoxin and trophic effects, leading in a raise of bacteria and endotoxin in the lumen as well as intestinal atrophy. These changes are promoting bacteria and endotoxin translocation to the portal vein and from there, to the systematic circulation, a procedure augmented by the supression of the cleaning ability of Kuppfer cells due to their reduced mass in hepatectomy. Systemic endotoxinaemia activates the systematic inflammatory response (SIR) related to the dysfunction of distal organs, and contributing to further damage to the mucosal barrier and the liver. This study confirmed the presence of portal and systemic endotoxinaimia in experimental hepatectomy. The subsequent atrophy of the intestinal mucosa has been documented with morphologic analyses as well as DNA and protein measurements. A possible explanation of the observed atrophy might be disturbance in balance between cellular proliferation and cellular death in the crypts by increased apoptosis and decreased mitotic activity. In addition, experimental hepatectomy led the intestinal functions in a state of low oxidative stress, as it is clearly shown by the decrease of lipid peroxidation and oxidized glutathione (GSSG), and the increase of glutathione (GSH). Interestingly enough, oxidative stress does not seem to be the primary cause of cellular apoptosis and decreased cell proliferation in the crypts (leading to mucosa atrophy), which are observed after partial hepatectomy, although this operation decreased oxidative stress below the control levels. On the other hand, these effects are prevented at more decreased levels of oxidative sress induced by the regulatory intestinal peptides BBS and NT. BBS and NT may act either directly, through specific receptors of the intestinal epithelial cells, or indirectly, by improving the intestinal microcirculation, leading to reversal of intestinal atrophy. Prevention, by these peptides, of the hepatectomy induced cellular and biochemical changes in the intestinal mucosa led to significantly reduced portal and systematic endotoxinaemia. In addition, BBS and NT, through antioxidative action to the liver, protected the remaining liver tissue from two major factors of liver lesions: oxidative stress and endotoxinaimia. Concluding, the results of this study show that dysfunction of the intestinal mucosa barrier in the hepatectomy is associated with the induction of cellular and biochemical changes in the intestinal mucosa, which are characterized by the promotion of oxidative stress and apoptosis. The regulatory intestinal peptides BBS and NT preventing these changes in the intestinal mucosa are significantly reducing portal and systematic endotoxinaimia. They also act by protecting against oxidative stress in the liver. This combined beneficial effect of these peptides, both in the dysfunction of the intestinal mucosa barrier and endotoxinaimia, which are responsible for the development of septic complications and distal organ lesions as well as in oxidative stress, might suggests a new therapeutical approach in hepatectomy.

Apart from motor and sensory function loss, an injury to the spinal cord can cause sympathetic dysfunction, which has been shown to affect immune responses. In this thesis, data from five experimental studies have been collected to compare physiological and psychophysiological exercise responses between wheelchair athlete subgroups with different disabilities (tetraplegic, paraplegic, and non-spinal cord-injured). In two preparatory studies, physiological exercise responses to exhaustive (Chapter 4) and submaximal exercise (Chapter 5) were investigated in all three disability subgroups. Whilst reliability measures for peak oxygen uptake (VO2peak) were in a range observed previously in able-bodied athletes, the variation in tetraplegic athletes was larger when expressed relative to their VO2peak, questioning the use of this variable to track small changes in aerobic capacity in athletic populations. Submaximal physiological and psychophysiological exercise responses were found to be similar between disability subgroups when expressed as a percentage of VO2peak, justifying the protocol used in the laboratory study on mucosal immune function, which was based on the same percentages of VO2peak for all disability subgroups. The most extensive study of this thesis, detailed in Chapter 6, showed that single laboratory-controlled 60-min exercise sessions increase both salivary secretory immunoglobulin A (sIgA), a marker of mucosal immunity, and α-amylase, a marker of sympathetic activation in all three disability subgroups. However, the impaired sympathetic nervous system in tetraplegic athletes seemed to influence the fine-tuning of their sIgA response when compared with paraplegic and non-spinal cord-injured athletes, resulting in a larger exercise-induced increase of sIgA secretion rate when compared to paraplegic and non-spinal cord-injured athletes. Based on these results, the study detailed in Chapter 7 investigated sIgA responses in tetraplegic athletes during wheelchair rugby court training. Despite their disability, these athletes showed responses thought to be governed by the sympathetic nervous system, such as reductions of saliva flow rate as a result of strenuous exercise. Similarly, the responses observed in Chapter 8 imply a comparable trend of chronic sIgA exercise responses in tetraplegic athletes as found in the able-bodied population, namely a decrease in sIgA secretion rate during periods of heavy training. These are the first studies in wheelchair athlete populations to investigate mucosal immune responses. Interestingly, despite the disruption of their sympathetic nervous system, some responses in tetraplegic athletes are comparable with findings in able-bodied populations. It is possible that due to their highly trained nature, these tetraplegic individuals are able to compensate for their loss of central sympathetic innervation. This may be by way of adapted spinal reflex or parasympathetic nervous system activity, or increased sensitivity of receptors involved in autonomic pathways. Therefore, sympathetic nervous function in tetraplegic athletes may be qualitatively altered, but in parts still be functional.

Inflammatory Bowel Diseases, Crohn's Disease and Ulcerative colitis, are idiopathic chronic conditions with multifactorial determinants. In general, terms, intestinal inflammation results from abnormal host-microbe interactions. Alterations in homeostasis involve host genetic factors, environmental cues and unique luminal microbial niches. We have examined the coordinated expressions of several molecular targets relevant to the mucosal immune system and identified signature biomarkers of IBD. Qualitative and quantitative changes in the composition of microbiota can be related to unique immuno-phenotypes. This in turn can have more systemic effects that involve energy metabolism. Adiponectin, an adipose tissue derived adipokine, can restore cellular ATP levels and fulfills innate immune functions. We have concluded that IBD might represent a state of adiponectin resistance relating to chronic inflammation and obesity status. Lastly we hypothesized that activation of xenobiotic pathway (AHR-aryl hydrocarbon receptor) can further modulate host immune and metabolic responses, and thus contribute to IBD phenotypes. We found that IBD is associated with robust mucosal, aryl hydrocarbon receptor pathway and related to proinflammatory cytokine secretion. We conclude that IBD heterogeneity is reflected through distinct immunophenotypes. Furthermore, environmental cues that involve the AhR receptor and adipose tissue derived adiponectin are important regulators of the inflammatory process in IBD.

Mucoadhesive drug delivery systems which are being used from 1980’s to avoid first pass metabolism of drugs, commercially exist for only systemic drug delivery with fast erosion times (15-60 min), that may not be appropriate for local oral disorders. The goal of this research was to develop and characterize mucoadhesive films with flexibility of carrying different drugs and proteins and provide sustained release for local treatment of oral disorders. Mucoadhesive films composed of polyvinylpyrrolidone (PVP) and carboxymethlycellulose (CMC) were formulated with imiquimod, an immune response modifier. Problems such as solubilization of imiquimod to increase drug loading, uniformity in films and total amount of drug released into supernatants were addressed by use of acetate buffer after investigating multiple methods. Subsequently, other relevant properties of mucoadhesive systems, such as adhesion (shear, pull-off), tensile properties, swelling profiles, transport kinetics, and subsequent changes in release profiles as a function of film composition were characterized. The potential of the system for local retention of imiquimod, determined in oral mucosa of hamsters showed time dependent decrease in imiquimod amount through 12 hours, with no traces of drug in blood. Further testing in humans revealed that the residence time of the mucoadhesive films depended on the application site, increasing in the order of tongue < cheek < gingiva. In parallel, mucoadhesive films loaded with epidermal growth factor (EGF) were developed to promote treatment of oral mucosal wounds. Bioactivity was tested in vitro on buccal tissues by creating a wound followed by application of films. Although EGF-loaded films did not accelerate wound healing, but rather elicited a hyperparakeratotic response. In vitro buccal tissues may not be appropriate for testing the effects of EGF in wound healing without incorporation of other biochemical factors. Overall, a mucoadhesive system capable of delivering bioactive small molecules and proteins in sustained manner was developed in this work. A thorough understanding of the system properties was achieved to further tune for future applications. In vitro studies and in vivo studies in hamsters and humans clearly showed the potential and usefulness of the system to translate in to clinic for treatment of oral precancerous lesions.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Thesis (Ph. D.)--West Virginia University, 2005 / Title from document title page. Document formatted into pages; contains ix, 155 p. : ill. Vita. Includes abstract. Includes bibliographical references.