L’infection par le VHB est endémique en Thaïlande. Malgré l’introduction des programmes de vaccination contre le VHB, la transmission périnatale reste une cause majeure d’infection chronique. Les objectifs de ce travail étaient d’identifier les mutants du VHB pouvant être associés à des échecs de vaccination, de diagnostic et de thérapeutique. Le travail présenté ici est divisé en trois parties. Dans une première partie, nous avons analysé la prévalence de la transmission périnatale du VHB dans une cohorte issue d’un protocole thérapeutique de prévention de la transmission materno-fœtale du VIH. Nous avons cherché à caractériser les mutants d’échappement à la vaccination contre le VHB. Parmi 3349 femmes enceintes séropositives pour le VIH, l’antigène (Ag) HBs était positif dans 7% des cas. L’Ag HBs était détectable à l’âge de 2 et 18 mois chez 11 enfants nés de mères porteuses chroniques. Les variants du VHB présents au sein de 9 de ces paires mère-enfant ont pu être étudiés après séquençage et clonage. Trois types de transmission du VHB ont pu être décrites ; i) transmission de variants non mutés par les mères présentant une charge virale VHB élevée ii) transmission d’un virus mutant minoritaire isolé chez la mère, et iii) transmission de mutants déjà présents à plus de 20% chez la mère. La capacité in vitro de ces mutants à échapper à la réponse neutralisante anti-HBs sera étudiée en utilisant un modèle de pseudo-particules portant les mutations identifiées. / Thailand is an endemic area for chronic HBV infection. Despite implementation of HBV vaccination, perinatal HBV transmission remains a major cause of chronic infection. This study aimed at identifying HBV mutants that may be associated with vaccine failure, misdiagnosis of chronic HBV infection and antiviral treatment failure. The dissertation is divided in three parts. In the first part, we analyzed the prevalence of perinatal HBV transmission in a large HIV prevention cohort in Thailand and characterized the HBV vaccine escape mutants. Among 3,349 HIV-infected pregnant women, 7% were found HBsAg positive. Eleven children born to HBsAg-positive mother were found HBsAg-positive at 2–18 months of age. Complete series of samples were available for 9 mother-child pairs. Based on direct sequencing and cloning analysis, 3 patterns of transmission were observed : i) transmission of wild-type variants from mothers with high HBV DNA level, ii) transmission of maternal minor variant and iii) transmission of variants already present in maternal blood samples. The capacity of HBV variants to escape from anti-HBs neutralization in vitro will be further studied using HBV-pseudoviral particles harboring the characterized mutations.

Salmonella typhimurium (ST) infection not only causes salmonellosis in humans, but also can result in great economic losses in the typically narrow-margin, high-volume broiler business due to reduced growth rates and mortalities. Over the last decade, the use of antibiotics and attenuated vaccines to restrain or prevent bacterial infections in domestic animals has been criticised because of the possible development of antibiotic resistance and the potential dangers of residual antibiotics and vaccines in animal-derived food products for human consumption. For these reasons, many countries have begun phasing out growth promoting antibiotics in broiler diets. It is therefore essential for the poultry production industry to develop feed additives and processing techniques as alternatives for sub-therapeutic dietary supplementation of antibiotics. However, innovative research is needed to evaluate the efficacy of new and existing alternative products. The general aim of this trial was to determine the effects of Salmonella typhimurium colonisation of the gastrointestinal tract of broiler chicks on gastrointestinal health and production performance. The effect of Zinc-Bacitracin (Zn-BC), a commonly used antibiotic growth promoter in the poultry industry, on Salmonella colonisation was also measured. A pilot trial was first conducted to determine the level of Salmonella typhimurium required to infect broiler chicks, and the necessity of administering an immunosuppressive agent in order to obtain infection. The main trial followed to determine the effects of Salmonella typhimurium on gastrointestinal health and function. The ultimate aim of the study was to obtain baseline values of various parameters that could be used in future trials for the evaluation of antibiotic alternative products. The results obtained from the pilot trial showed that it was not necessary to administer cyclophosphamide as the Salmonella typhimurium proved to be highly virulent. The cloacal swabs taken in the second trial showed that the use of Zn-BC as an antibiotic did not inhibit Salmonella colonisation in the challenged birds. The inclusion of Zn-BC in this trial inhibited the growth of the gut microflora allowing the Salmonella to proliferate in the body of the chicken, which lead to the conclusion that the routine inclusion of Zn-BC at sub-clinical levels as a growth promoter may be detrimental when the bird gets exposed to Gram(-) bacteria, such as Salmonella. In both of the trials, Salmonella challenge resulted in enlargement of the organs with a consequent increase in the organ weights. In the pilot trial there was a significant difference (P<0.0033) of the control weights for the duodenum, ileum, caeca and liver and those of the Salmonella infected birds. Control birds that did not receive CY had duodenum weights of 1.00 (±0.236) while the birds infected with 1 x 108 CFU/mL had weights of 1.99 (±0.310), while the control birds that did receive CY had duodenum weights of 0.98 (±0.244) with the Salmonella infected birds having weights of between 1.79 (±0.299) and 2.13 (±0.006). Significant results (P<0.016) in the main trial were found to occur predominantly at 7 days of age for the duodenum, jejenum, ileum and caeca weights. Control birds in the group that did receive antibiotics had 7 day duodenum weights of 1.80 (±0.301) compared to the Salmonella infected bird which had weights of between 2.33 (±0.376) and 2.51 (±0.424). In general Salmonella did not affect the growth and performance of the challenged birds. Birds challenged with Salmonella showed a tendency to have enlarged livers, possibly due to hepatic damage. In the main trial there was a significant difference (P<0.016) in liver weights at 28 days of age between the control and Salmonella infected groups regardless of whether the birds received antibiotics or not. The control birds that received antibiotics had liver weights of 3.24 (±0.234) while the birds infected with the higher level of Salmonella had weights of 3.86 (±0.542). This finding, together with the noticeable, although mainly insignificant, trend of decreased serum albumin levels and increased serum globulin and total serum protein levels noted in infected birds can be used in conjunction to measure the effect of ST on liver damage. Salmonella colonisation resulted in an increase in the severity of lesions seen in the gastrointestinal tract (P<0.0016). Histopathology results proved to be inconsistent and did not provide any conclusive evidence on the effect of Salmonella on the organs. Villi measurements taken in the second trial showed that Salmonella significantly (P<0.016) shortened the length of the villi in the duodenum and jejenum of challenged birds when measured at 28 days of age. Control birds had duodenum villi length of 662.5 (±56.79) while those birds infected with Salmonella had lengths of between 558.9 (±77.74) and 537.0 (±51.66). There was a significant difference in the duodenum villi length regardless of antibiotic inclusion into the diet. In the birds that did receive antibiotics, there was a significant difference (P<0.016) in the jejenum villi length with the control birds having the longest villi 725.7 (±90.92) while the birds infected with the higher level of Salmonella having the shortest villi 557.2 (±124.5). It would appear that using all of the information and results obtained for liver weights, broiler performance, serum biochemical level, lesion scoring, histopathology and villous morphological measurements should be used in conjunction with one another to measure the effect of Salmonella on the broiler chicken. The results obtained in this trial clearly show just how significant a problem Salmonella infection can be in the poultry industry due to seemingly healthy adult birds displaying little or no systemic disease being non-symptomatic carriers. Many of the Para-typhoid salmonellae do not always produce clinical signs in chicks, and their presence in the poultry industry may go unrecognised for this reason. / Dissertation (MSc(Agric))--University of Pretoria, 2011. / Animal and Wildlife Sciences / unrestricted

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13268号 / 論医博第2182号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 佐藤 俊哉, 教授 今中 雄一, 教授 柳田 素子 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Silk is a natural protein produced by members of the class Arachnida (over 30,000 species of spiders) and by several worms. Silk-based materials have been investigated for medical and biotechnological applications for many years. Although silkworm silk has been studied extensively because of ready availability of the protein, lately the advancements in recombinant technology has made production of spider silk proteins increasingly available. Due to the characteristics like biocompatibility, biodegradability and mechanical strength, silk is highly desirable as a biomaterial for medical purpose. Along with this, techniques for functionalization, has further aided in the development of silk into highly sophisticated material for advanced applications. The main objective of this thesis has been to investigate novel strategies for functionalization of the recombinant spider silk protein Masp2. Two distinct approaches were used, chemical modification and genetic fusion. In the first modification, we created an infection responsive silk nanospheres by chemically grafting a thrombin sensitive peptide to the silk protein encapsulating antibiotic. These particles were then evaluated for in vitro infection responsive drug release and antimicrobial activity. From these assessments, we found that these particles can release the drug effectively in the presence of infection providing the evidence that these particles are enzyme responsive and can be used to formulate targeted drug release. In the second modification, spider silk was genetically modified with a heparin binding peptide to create a fusion protein which can prevent both thrombosis and infection simultaneously. This fusion protein was evaluated for its heparin binding ability and anticoagulant properties in its solution form. Furthermore, due to the similarity in structure of HBP with antimicrobial peptides, it is predicted that the fusion protein will also show antimicrobial property. After establishing these properties, next this fusion protein was utilized as a coating for hemodialysis catheter. Deposition of coating was evaluated after which anticoagulant and anti-infective properties of the protein as a coating material was investigated. This thesis provides evidence of successful production of a recombinant silk-based biopolymer that can be chemically and genetically embedded with a various functional motif to create a hybrid product for different applications.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / OBJECTIVES: The objective of this study was to perform a systematic review and meta-analysis of the literature to evaluate the efficacy and safety of therapies for cerebral toxoplasmosis in HIV-infected adults. The pyrimethamine plus sulfadiazine (P-S) combination is considered the mainstay therapy for cerebral toxoplasmosis and pyrimethamine plus clindamycin (P-C) is the most common alternative treatment. Although trimethoprim-sulfamethoxazole (TMP-SMX) has potential advantages, its use is infrequent. METHODS: We searched PubMed and four other databases to identify randomized controlled trials (RCTs) and cohort studies. Two independent reviewers searched the databases, identified studies and extracted data. Risk ratios (RRs) were pooled across studies using random-effects models. RESULTS: Nine studies were included (five RCTs, three retrospective cohort studies and one prospective cohort study). In comparison to P-S, treatment with P-C or TMP-SMX was associated with similar rates of partial or complete clinical response [P-C: RR 0.87; 95% confidence interval (CI) 0.70-1.08; TMP-SMX: RR 0.97; 95% CI 0.78-1.21], radiological response (P-C: RR 0.92; 95% CI 0.82-1.03), skin rash (P-C: RR 0.81; 95% CI 0.56-1.17; TMP-SMX: RR 0.17; 95% CI 0.02-1.29), gastrointestinal impairment (P-C: RR 5.16; 95% CI 0.66-40.11), and drug discontinuation because of adverse events (P-C: RR 0.32; 95% CI 0.07-1.47). Liver impairment was more frequent with P-S than P-C (P-C vs. P-S: RR 0.48; 95% CI 0.24-0.97). CONCLUSIONS: The current evidence fails to identify a superior regimen in terms of relative efficacy or safety for the treatment of HIV-associated cerebral toxoplasmosis. Use of TMP-SMX as preferred treatment may be consistent with the available evidence and other real-world considerations. Larger comparative studies are needed. / Revisión por pares

The main objective of root canal therapy is cleaning, shaping and obturating the root canal system in three dimensions, as well as preventing reinfection. The objective of this in vitro study was: <ul> <li>to establish the antimicrobial efficacy of nine different root canal irrigation solutions, </li> <li>to determine the smear layer dissolving capabilities of these irrigating solutions, at various levels in straight root canals, and </li> <li>to examine the degree of erosion caused by the irrigation solutions on the root canal walls. </li></ul> Antimicrobial Effects: In this test the antimicrobial activity of nine irrigation solutions against E. faecalis was measured using a disc diffusion test. The antibacterial activity of materials was apparent from circular clear inhibition zones forming around the filtration paper. Effect on Smear Layer: Root canals were prepared by using Pro Taper nickel titanium rotary files. During preparation, the canals were irrigated with copious amounts of 3.5% sodium hypochlorite for 15 minutes. Thereafter, the teeth were randomly divided into nine groups. One group was kept as control. The other eight groups received a final rinse with one of the following irrigation solutions: 18% EDTA, Sterilox, Top Clear 17% EDTA, 2% Chlorhexidine, 10% Citric acid, Biopure MTAD, Ozonated water and Smear Clear. A total of 25ml of each solution was utilized to irrigate each root canal system, and left undisturbed in the root canal for 2 minutes. Biopure MTAD was left in the root canals for 5 minutes. Finally, the irrigation solutions were removed from the root canals with 10ml sterile distilled water. Each root was sectioned horizontally with a diamond disc in sections corresponding with the coronal, middle and apical levels of the root canal system. Each fragment was fractured laterally and prepared according to standard methods for biological SEM evaluation. The absence or presence of the smear layer as well as the amount of erosion of the peritubular dentine was assessed. The One-way ANOVA test was used to determine whether there were any statistical significant differences between the different test groups. No significant inhibition of E. faecalis was observed with sterile water (control) and the undiluted solutions of Sterilox, 10% Citric acid and Ozonated water. However, 3.5% NaOCl, 18% EDTA, Top Clear 17% EDTA, 2% Chlorhexidine, Biopure MTAD and Smear Clear showed significant inhibition of E. faecalis. For the 1/10 diluted solutions no inhibition of E. faecalis was observed with sterile water (control), 3.5% NaOCl, Sterilox, 10% Citric acid and Ozonated water. Biopure MTAD and Smear Clear demonstrated significant inhibition of E. faecalis compared to 18% EDTA, Top Clear 17% EDTA and 2% Chlorhexidine. Biopure MTAD was the only solution that showed significant inhibition of E. faecalis using a 1/100 diluted solution. No inhibition was observed with the 1/1000 diluted test solutions.Scanning electron microscope examination revealed that 3.5% NaOCl and Ozonated water had no visible effect on the smear layer. The 10% Citric acid solution slightly modified the smear layer at the coronal and middle levels of the root canals. There was no visible effect on the smear layer at the apical levels. Sterilox and 2% Chlorhexidine removed the smear layer at the coronal levels, modified it slightly in the middle levels and left the smear layer undisturbed in the apical levels of the root canals. 18% EDTA, Top Clear 17% EDTA, Biopure MTAD and Smear Clear removed the smear layer completely at the coronal levels. At the middle and apical levels of the root canals most of the smear layer was removed. However, there were less open dentinal tubules visible at the apical levels compared to the samples examined at the middle levels of the root canals. It was also noted that Top Clear 17% EDTA caused a significantly high percentage of erosion of the peritubular dentine at the coronal levels of the root canals compared to all the other irrigation solutions. Considering all the results of the present study Biopure MTAD demonstrated the best antimicrobial activity against E. faecalis, and removed most of the smear layer at all three levels of the root canal systems without significant erosion of the peritubular dentine. / Dissertation (MSc(Odont))--University of Pretoria, 2008. / Community Dentistry / unrestricted

Microsporidia are an obligate, intracellular fungal pathogen that can cause devastating, disseminating infections in the immunocompromised. Because of the limitations of current medications, microsporidia’s abundant presence in the environment, and an increasing number of at-risk populations, investigation into decreasing microsporidia infectivity is needed. As an intracellular pathogen, microspridial attachment is a vital first step to infection, and if attachment is reduced, previous work shows that infectivity is mitigated. An in silico analysis of Encephalitozoon intestinalis revealed a predicted protein similar in sequence to ADAM (A Disintegrin And Metalloproteinase) proteins. This predicted protein is termed microsporidia ADAM or MADAM. ADAM proteins contain an integrin binding region, which is well known to bind to integrin proteins. Integrins are important receptors for attachment and cell signaling, and several pathogens utilize host integrins as a receptor to aid in attachment during infection. Immunoelectron microscopy demonstrates that MADAM protein is found on the plasma membrane, anchoring disk, and polar tube of E. intestinalis spores. Our hypothesis is that MADAM is involved in the key role of host cell attachment. To this end, a 17 amino acid long section of the MADAM protein was generated that surrounded the integrin binding domain. During spore adherence assays, pretreating host cells with this small peptide protein, significantly decreased E. intestinalis spore attachment to host cells as compared to control samples. These results suggest E. intestinalis cleverly exploits host integrins as a means to bind to host cells before infection.

The infection process of intracellular opportunistic microsporidia involves the forcible eversion of a coiled hollow polar filament that pierces the host cell membrane, allowing the passage of infectious sporoplasm into the host cell cytoplasm. Although the exact mechanism of spore activation leading to polar filament discharge is unknown, we have shown that spore adherence to host cells, which is mediated by sulfated glycosaminoglycans, may play a vital role. When adherence is inhibited, host cell infection decreases, indicating a direct link between adherence and infection. The goal of this study was to evaluate the effects of exogenous divalent cations on microsporidia spore adherence and infection. Data generated using an in vitro spore adherence assay show that spore adherence is augmented by manganese (Mn2+) and magnesium (Mg2+), but not by calcium (Ca2+). However, each of the three divalent cations contributed to increased host cell infection when included in the assay. Finally, we show that Mn2+ and Mg2+ may activate a constituent on the microsporidia spore, not on the host cell, leading to higher infection efficiency. This report further supports recent evidence that spore adherence to the host cell surface is an important aspect of the microsporidial infection process.

Actinomycotic infections involving the oesophagus are uncommon but have been reported in both immunocompromised and immunocompetent individuals. We report a case of actinomycosis oesophagitis in a patient with lung cancer who received chemo- and radiotherapy. This patient was admitted with severe dysphagia and odynophagia and biopsy from an oesophageal ulcer found on oesophagogastroduodenoscopy (EGD) revealed actinomycosis. The patient was treated with intravenous penicillin G followed by ceftriaxone with clinical improvement and repeat EGD showed reduction in the size of the oesophageal ulcer, but he relapsed due to non-compliance. We review the English literature regarding the clinical features, diagnosis, and management of actinomycotic infections of the oesophagus.

These studies evaluated the effects treatment with glucan phosphate, a soluble polysaccharide immunomodulator, on the inflammatory response induced by burn injury and on resistance to Pseudomonas aeruginosa burn wound infection. Mice were exposed to 35% total body surface area burns and were resuscitated with lactated Ringer's (LR) solution alone or LR supplemented with glucan phosphate (40 mg/kg). Glucan phosphate treatment attenuated burn-induced expression of interleukin (IL)-1β, IL-6, and IL-10 mRNAs in spleen, lung, and heart. Plasma concentrations of IL-1β, IL-6, macrophage inflammatory protein (MIP)-2, and IL-10 were also decreased in burned mice treated with glucan phosphate compared with vehicle-treated controls. Early postburn mortality was not significantly different between control (20%) and glucan phosphate-treated (10%) mice, but there was a small improvement in acid-base balance in the glucan phosphate-treated group. Mice received a second injection of glucan phosphate or LR on day 4 postburn and were infected by topical application of P. aeruginosa to the burn wound on day 5. Glucan phosphate treatment significantly improved survival in mice exposed to P. aeruginosa burn wound infection. The improved survival correlated with lower bacterial burden in the burn wound, attenuated production of proinflammatory cytokines, and enhanced production of Th1 cytokines. These studies show that glucan phosphate treatment attenuates burn-induced inflammation and increases resistance to P. aeruginosa burn wound infection in an experimental model of burn injury.