Scientific Publications - Work Done by Microbiology Reader Bioscreen C

1st ABEP/Oxford Centre for Brazilian Studies Conference, St Anthony’s College, Oxford, UK – 19/03/2004

Life Sciences, Abstracts.

Prevalence of Dental Erosion and Dental Caries Among Schoolchildren in the UK - A Pilot Study

Sheyla Marcia Auad (1)(2), Paula Moynihan (1), Paula Waterhouse (1)

(1)University of Newcastle upon Tyne,

(2)Universidade Federal de Minas Gerais - CAPES Scholarship

Abstract: Dental erosion appears to be emerging as a condition affecting a growing number of children and adolescents, but there are scant data for its prevalence worldwide. Aims: To assess the practicalities for future fieldwork including a larger Brazilian sample and to calibrate the principal investigator in the use of the chosen dental indices. Objectives: To determine the prevalence of dental erosion and dental caries among schoolchildren in the UK.

Design: Twenty-five schoolchildren (mean age=13.08yrs, range 11-14 years) were examined for both dental erosion and dental caries at one school in Tyne and Wear. Parents and students were asked for their written consent to participating in the study. Ethical approval was granted by the local ethics committee. The WHO criteria were used for assessing dental caries. Dental erosion was evaluated using the criteria of the National Diet and Nutrition Survey 2000.

Methods: Oral examination took place in a school room, using a head mounted light. Clinical photographs were obtained from a sample of the schoolchildren.

Results: Fifteen subjects showed some evidence of dental erosion and 44% had caries experience. From the 15 students with erosion, one had enamel and dentine involvement, requiring clinical intervention. The other 14 subjects had erosion affecting enamel, indicating the need for preventive counselling, but not restorative treatment. Erosion was most commonly diagnosed on the palatal surfaces of the upper central permanent incisors. Only two students had erosion on the occlusal surfaces of the lower first permanent molars. However, among those with caries experience, the first permanent molars were the teeth most affected by dental caries. A statistically significant association was not found between the prevalence of dental erosion and dental caries. Moreover, in this small sample size, gender did not have statistically significant influence in the prevalence of either dental erosion or dental caries.

Conclusion: The high prevalence of dental erosion observed in this sample emphasizes the need for further studies, aiming to assess whether it can be considered a new dental public health problem and to provide a base on which to build strategies for its prevention.

Keywords: Prevalence; Dental Erosion; Dental Caries; Schoolchildren.

Meningococcal Disease in Brazil: Clinical Characteristics, Outcome and Bacterial Load

Jailson B. Correia (1)(2), Maria C. M. B. Duarte (2), Luis E. Cuevas (3), Malcolm Guiver (4), E. B. Kaczmarski (4), C.A. Hart (1).

(1)Department of Medical Microbiology, University of Liverpool, UK

(2)Instituto Materno Infantil de Pernambuco, Recife, Brazil

(3)Liverpool School of Tropical Medicine, UK

(4)Meningococcal Reference Unit, Manchester, UK

Abstract: The clinical spectrum of meningococcal disease includes meningitis and septicaemia (or both) and blood bacterial load correlates with disease severity in septicaemic cases. This study aims to describe clinical characteristics, outcome and bacterial loads in blood and cerebrospinal fluid (CSF) of Brazilian cases of meningococcal disease. Consecutive cases with a clinical picture of bacterial meningitis or meningococcal septicaemia were enrolled in the city of Recife, Brazil (population 1.4 million), assessed on admission and followed daily during hospital stay. Cases were confirmed by culture or polymerase chain reaction (PCR) in blood or CSF. Bacterial loads were measured by amplification of the ctrA gene in an automated Taqman PCR. From 142 suspected cases initially enrolled, 69 had confirmed meningococcal infection, being 59 (86%) type B and 10 (14%) type C. Nine (12%) of the confirmed cases have died. Meningitis alone was present in 22 (32%) cases, whereas 5 (7%) had only septicaemia and 42 (61%) had both forms. In blood samples, the median bacterial load was significantly higher in non-survivors (1.6 x 10⁷, inter-quartile range: 4.4 x 10⁵ to 3.7 x 10⁷) than in those who survived (2.0 x 10⁴, 3.4 x 10³ to 3.3 x 105 , p=0.001. In CSF, however, median levels in survivors (5.8 x 10⁶, 9.5 x 10⁴ to 9.2 x 10) and non-survivors (2.8 x 10⁶, 1.2 x 10⁶ to 1.3 x 10⁸) were not significantly different. These findings, which expand previous analysis of this cohort of patients, confirm the association of blood bacterial loads with poor outcome in meningococcal disease and show that bacterial loads in CSF do not seem to be associated with higher mortality.

Keywords: Meningococcal Infection; Bacterial Load; Mortality.

Bacterial Load and Inflammatory Response in African Bacterial Meningitis

Jailson B. Correia (1), Mohammed A. Yassin (2), Ali Hassen (3), Luis E. Cuevas (2), Malcolm Guiver (4), E. B. Kaczmarski (4), C.A. Hart (1).

(1)Department of Medical Microbiology, University of Liverpool, UK

(2)Liverpool School of Tropical Medicine, UK

(3)Yirgalem Hospital, Ethiopia

(4)Meningococcal Reference Unit, Manchester, UK

Abstract: Studies from developed countries show that pro and anti-inflammatory cytokines and chemokines play a leading role in the pathogenesis of bacterial meningitis and meningococcal septicaemia. Little is known about patterns of host response in the epidemic-prone African meningitis belt, where meningococcal infection presents mainly as meningitis. This study aims to describe bacterial load and the pattern of host inflammatory response to bacterial meningitis in sub-Saharan Africa. Between January and February 2003, consecutive cases with a clinical picture of meningitis were enrolled at Yirgalem Hospital, Southern Region of Ethiopia, where patients were assessed on admission and followed daily during their hospital stay. Cases were confirmed by PCR and bacterial loads were measured by amplification of the ctrA gene by Taqman PCR. Cytokines and chemokines were measured by ELISA. From 31 suspected cases initially enrolled, 21 were confirmed by PCR and are described here. Eighteen had group A meningococci and 3, pneumococci. The median age was 14 years (range 6 to 60), 10 were male, none had petechiae or purpuric rash and 2 died during hospital stay. The median bacterial load in the CSF of meningococcal cases was 2.3 x 10⁷ copies/mL (range, 4.4 x 10⁴ to 4.7 x 10⁹). Bacterial loads correlated positively with CSF TNF-α (p=0.01) and RANTES (p=0.018) and with plasma IL6 and IL8 (p<001 for both). This first description of bacterial loads and their association with cytokine and chemokine production in African bacterial meningitis provides insights into the role of these substances in the pathophysiology of bacterial meningitis in this population. Larger studies to identify its association with outcome are warranted.

Keywords: Meningococcal Infection; Bacterial Load; Mortality.

Personality Type and its Implications with Success at Code-Review

Alessandra Devito da Cunha (1), David Greathead (1) (1) Newcastle University

Abstract: This study was part of an interdisciplinary research project called DIRC (Interdisciplinary Research Collaboration on Dependability) which is interested in finding solutions and techniques to develop dependable software and systems. For this, a study of the research regarding personality factors involved in software development was necessary prior to conducting the main body of the research.

The literature shows that it is possible to notice large variations in individual performance, it was then suggested that some innate human trait could be behind such variations. Although personality has already been studied as an important factor in the process of software development, it usually treats programming as a single process, ignoring its different parts (which may require different skills). The aim of this study then, was to identify which personality types (according to Myers Briggs Type Indicator – MBTI) are related to success at a code-review task. To examine this hypothesis, ₂nd year computing science students at Newcastle University were asked to complete a set of three tasks.

The first task was a questionnaire designed to obtain extra information about the students. They were able to rate their knowledge in Java (the language adopted for the code-review task); their abilities to find bugs in their own and others’ programs and last but not least their preferred part of the programming process.

The second task was a code-review exercise (the bugs in the code have to be indicated – but not corrected). The students received a 4 page Java program, its manual and API. They were told that all the bugs were semantic but they were not told about the number of bugs in the code (16 bugs were added manually).

The final task was a personality assessment. The students received the MBTI booklets and were asked to answer the questions in the way which made them feel most comfortable.

There were an extra set of data which was the students’ first year marks in modules related to Java. While analysing the data obtained (by Pearson’s correlations and T-Tests), it was found that students with an “Intuitive” preference had a higher performance in code-review than the non-intuitive students.

Keywords: Personality; Code Review; Performance; Software Development.

Wildlife Use and Conservation in the Amana Sustainable Development Reserve, Brazilian Amazon

Leonardo Colombo Fleck (1)

(1) Durrell Institute of Conservation & Ecology, University of Kent

Abstract: Subsistence hunting is an important source of protein for traditional Amazonian communities, but may threaten animal populations if not undertaken sustainably, putting both wildlife existence and people’s subsistence at risk. Sustainable Development Reserves (SDRs) were established in the Brazilian Amazon in the 90’s to address both natural resources conservation and people subsistence. However there is still little knowledge about the extent, importance and implications of different forms of resource use in these reserves. Therefore there is an urgent need to assess the sustainability of these practices based on sound scientific knowledge which will allow the development of frameworks for the proper management of these resources. This study will be carried out in one of the largest communities around the black water Amana Lake, located in the recently established Amana SDR (1998). This community is the focus of a pilot program for assessing natural resources use and conservation in the reserve’s upland forests. The two main objectives of this study are to assess: (1) the conservation status of wildlife and the sustainability of hunting, and (2) the roles (economic, nutritional) of wildlife to traditional people. To accomplish these objectives, (1) current harvests and density estimates of wildlife (in lightly and heavily hunted sites) will be used to feed different sustainability models and the results will be used to evaluate the sustainability of current hunting practices. Harvest patterns will be used to assess the role of bushmeat in fulfilling basic human protein needs, and the valuation method of shadow pricing will be used to estimate the non-market value of bushmeat to these people. A Participatory Hunting Management Plan will be the major output of this project, which is likely to be used later in a larger scale around the whole reserve.

Keywords: Hunting; Sustainability; Amazon; Wildlife; Valuation.

A Cell Motility Structure That Should Not Be There

Catarina Gadelha (1)(2), Bill Wickstead (2), Wanderley de Souza (1), Narcisa L. da Cunha-e-Silva (1), Keith Gull (2)

(1)Sir Willian Dunn School of Pathology, University of Oxford

(2)Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro

Abstract: Eukaryotes have developed ways of moving themselves through their environment, ordering organelles within the cell, and coordinating cell division. Central to these fundamental biological processes is a network of interconnected filaments called the cytoskeleton. Each eukaryotic cell type arranges its cytoskeleton according to its own requirements and some groups have evolved their own unique cytoskeletal structures.

The order Kinetoplastida comprises a group of flagellated unicellular eukaryotes that include parasitic as well as free-living species. These cells contain one of these unique cytoskeletal structures, called the paraflagellar rod (PFR). The PFR is an intricate lattice-like construction of protein filaments running inside the flagellum, and actively involved in cell motility. However, one group of kinetoplastids was thought to lack this PFR structure although they swim in the same way as PFR-containing species. This group also harbours a symbiotic bacterium in their cytoplasm and shows other interesting features that classify them as being a monophyletic group.

We investigated how these organisms are able to swim despite the apparent lack of the PFR found in other kinetoplastids. We have found in these endosymbiont-bearing kinetoplastids a gene for one of the 2 major proteins of the PFR. This gene encodes an expressed protein that is able to rescue cells of a PFR-containing species which had been artificially manipulated to disrupt the PFR, showing that the protein is functional. Moreover, when we carefully re-examined the ultrastructure of the flagellum of endosymbiont-bearing kinetoplastids, we discovered that these organisms do indeed possess a PFR, if somewhat reduced. This demonstrates the existence in the endosymbiont-bearing kinetoplastids of a cell motility structure that had been missed in previous published analysis.

Keywords: Kinetoplastida; Paraflagellar Rod; Cytoskeleton; Motility.

Insights Into Flagellar Function And Pathology From Studies With Trypanosomes

Andrea Baines (1), Laura Briggs (1), Jacqueline Davidge (1), Helen Dawe (1), Helen Farr (1), Catarina Gadelha (1, 2), Michael Ginger (1), Neil Portman (1), Sue Vaughan (1) and Keith Gull (1)

(1)Sir Willian Dunn School of Pathology, University of Oxford

(2)Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro

Abstract: Motile cilia and flagella have important functions in the reproductive and respiratory tracts of humans – but we know little about the molecular composition of these important organelles. As ciliated/flagellated cells are terminally differentiated in higher eukaryotes they are difficult to study at the molecular and biochemical level.

In contrast, flagellated protozoa offer enormous advantages – they can be cultured in large quantities for biochemical study and a range of advanced post-genomic technologies are available for gene function investigations. Cilia and flagella of eukaryotic cells are constructed from a highly conserved structure – the axoneme, a complex arrangement of over 250 structural proteins. Both the structural and molecular composition of the axoneme have been preserved through evolution – many of the same proteins that build the flagella of lower eukaryotes, such as trypanosomes, are also found in human cilia/flagella.

Our aim is to elucidate the molecular basis of diverse flagellar functions in trypanosomes and relate this information to the structure and function of human cilia/flagella.

Keywords: Trypanosomes, Cilium, Flagellum, Motility.

Specialist Reply Letters in the Referral System: the Views and Practices of Secondary Level Medical Specialists in the Municipality of Camaragibe, PE, Brazil: a Qualitative Study

Matthew James Harris (1), Ana Farias Ferreira (2), Isabela Celeste de Moraes (2), Fernanda Maria Rabello de Andrade Lira (2), Deise Ferreira de Souza (2), Monica Franch (3).

(1)London School of Hygiene and Tropical Medicine, London, UK.

(2)Escola de Saúde Publica de Pernambuco, Recife, PE, Brazil.

(3)Departamento de Antropologia, Universidade Federal de Pernambuco, Recife, Brazil.

Abstract: This qualitative study, performed by health professionals of the Family Health Programme (FHP) from the municipality of Camaragibe (Pernambuco), aimed to identify possible factors that explain the low reply rates, from specialists at the secondary level to generalists at the primary health care level (FHP), observed in the municipality of Camaragibe, metropolitan region of Recife, Pernambuco. Semi-structured interviews were carried out with 12 secondary level specialists between September 2003 and December 2003. Interviews were categorized according to intrinsic factors (perceptions, opinions and attitudes) and extrinsic factors (human and material resources) that may explain the low reply rates. Initial analysis demonstrates a range of comprehension and attitudes regarding the use of reply letters. In general, deficiencies in understanding its importance, a lack of participation in municipal health system organization and deficient work processes at the secondary level were observed. Identification of limiting factors that interfere with reply-letter writing may help to direct strategies that improve continuity of care, inter-professional respect and outpatient services.

Keywords: Interdisciplinary Communication [MeSH]; Referral and Consultation [MeSH]; Correspondence [MeSH].

Predictive Forest Species in Guaraque~aba, Atlantic Rain Forest - Brazil

Gracie Abad Maximiano (1) (1) University of Reading

Abstract: The Guaraquecaba Environmental Protection Area - EPA, which is located in Parana State in southern Brazil, is part of the Atlantic Forest, one of the two kinds of rain forest in Brazil. Situated along the Brazilian coastal area, the Atlantic Forest is an ecosystem with unusually high biological diversity stretching across more than 20 degrees of latitude. With high mountains forming a coastal range and a distinct climate, this geographical diversity encourages a rich variety of species.

Occupied by mankind since 1500, today most of these ecosystems are found in small and separated fragments. The biggest fragment of Atlantic forest is encountered in Parana State where the national Government has created an Environmental Protected Area (EPA) in the region of Guaraquecaba. This decision was designed to protect the remaining ecosystem fragment in this area.

The aim of the project is to predict different kinds of forest communities using a modelling approach in a Geographical Information System - GIS. The specific objectives were to:

·                    Establish a predictive model for forest species mapping

·                    Test the validity of the model in the field

·                    Refine the model

·                    Use the model outputs to determine ecological quality

·                    Monitor impact of human activity from sequential satellite imagery

·                    Determine the threat to areas of high ecological quality from human activity

Keywords: Rain Forest; Predictive Vegetation Mapping; GIS; Remote Sensing.

Azole Cross-resistance in Oral Candida Flora from Brazilian HIV 1-Infected Child

Nadja R. Melo (1), M. Marluce S. Vilela (2), Vitoria V. P. Culhari (2),

Hideaki Taguchi (3), Makoto Miyaji (3), Philip Groeneveld (1), Andrew Warrilow (1), Diane Kelly (1), and Steven L. Kelly (1).

(1)University of Wales Aberystwyth, Wales, UK

(2)Center of Paediatric Investigation, University of Campinas’ State, Brazil,

(3)Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Japan

Abstract: Case report - A 5-years old HIV 1-infected child who acquired HIV vertically has been monitored at the Paediatric Immunodeficiency Outpatient Service, University of Campinas’ State, Brazil. The child started HIV antiretroviral therapy since 1999 and never received antifungal therapy. During a screening to investigate Candida colonization and antifungal susceptibility in 52 brazilian HIV 1- infected children we isolated Candida albicans-B from this child. Susceptibility of the isolate to amphotericin B, fluconazole, miconazole, and itraconazole was determined by a microdilution method adopted by the National Committee for Clinical Laboratory Standards. The isolate showed low susceptibility to all azoles tested. To investigate the possible cross-resistance to 8 azoles, the effect of azoles on the maximum specific growth rate of isolate was determined in aerobic batch cultures by a Bioscreen C Analyser; a high-throughput system in 200 separate cultures. Voriconazole, fluconazole, itraconazole, ketoconazole, miconazole, econazole, clotrimazole, imidazole, additionally amphotericin B, and nystatin were tested in different concentrations. The isolate showed cross-resistance (Table 1) to the several azoles tested. The voriconazole, a recent antifungal agent used in the treatment of recurrent oropharingeal candidosis, inhibited only 65% and 84.7 % of growth respectively in the concentrations of 32µg/ml and 64µg/ml. Fluconazole inhibited only 57.9% and 65% of growth in the concentrations of 32µg/ml and 64µg/ml respectively. However clotrimazole showed growth rate inhibition percentage of 90.8%, amphotericin B 89.3%, and nystatin 95.3%. The emergence of cross-resistance to antifungal azoles in paediatric patients, including azoles not previously used in children, does therefore occur. This study represents the first azole cross-resistance case concerning oral Candida flora in Brazilian HIV-infected child.

Keywords: HIV1-infected children; cross-resistance; Candida; Brasil.

Follow up Study - Oral Candida Flora from Brazilian HIV 1-Infected Children in the HAART Era

Nadja R. Melo (1), M. Marluce S. Vilela (2), Vitoria V. P. Culhari (2),

Hideaki Taguchi (3), Ayako Sano (3) and Makoto Miyaji (3)

(4)University of Wales Aberystwyth, Wales, UK

(5)Center of Pediatric Investigation, University of Campinas State, Brazil,

(6)Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Japan

Absctract: HIV-infected patients often suffer from severe forms of oropharyngeal candidosis, mainly caused by Candida albicans. This study analyzed the HIV Protease Inhibitor (IP) influence over oral Candida flora from 52 Brazilian HIV-infected children, comparing the Candida species identified before the IP using (GI) and under IP therapy (GII). There was a significant increase of non- albicans isolates from 9.6% to 28.8% (p=0.005) between GI and GII groups respectively. In the GII group the second most frequent species was C. tropicalis (n=9) followed by C. parapsilosis (n=8). Rare species found in the GII group included C. dubliniensis, C. novergensis, C. humicula and C. rugosa. All isolates in this investigation were susceptible to amphotericin B. Most of C. albicans and non-albicans isolates were susceptible to fluconazole, itraconazole and ketoconazole. However amongst C. tropicalis isolates (n=7) one of them was resistant to FLCZ (MIC > 64 µl/ml) and one C. albicans-B isolate showed cross-resistance to all azoles tested. The influence of the IP using over the virulence factors particularly in Candida albicans isolates, and emergent non-albicans species with low susceptibility to antifungal agents, represent a serious problem in the medical field. This study represents the first follow up investigation concerning oral Candida flora and antifungal susceptibility in Brazilian HIV-infected children.

Keywords: HIV1-infected children; oral Candida; Brasil.

Genetic Polymorphisms Related to Predisposition to Drug Addiction and

Personality Disorders

Fabio Miyajima (1); Sally John (1); J.F.William Deakin (2)

(1)   Centre for Integrated Genome for Medical Research – School of Epidemiology and Health Sciences - University of Manchester – Manchester - UK

(2)   Neuroscience and Psychiatry Unit – University of Manchester – Manchester - UK

Abstract: Genetic evidences have been connected to interindividual differences in susceptibility to drug addiction as well as to some personality disorders. Several authors have reported that genetic factor could play a relevant role estimated at 40-60% of the total risk, and up to 40% of the interindividual differences in drug metabolism and response according to a Swedish study. In some in vitro experiments, it has been seen very large variations in the metabolism of some selective substrates, ranging rates as high as 2000-fold for example, due to genetically polymorphisms in the several genes loci across the genome.

Given the high complexity of our Central Nervous System (CNS), the variation in the metabolism of either endogenous or exogenous substrates, including drugs of abuse, may play unpredictable effects, including vulnerability to addiction and the appearance of some behavior disorders. For instance, same doses of heroine, which has negligible pharmacological activity, develop different ranges of addictive effects among individuals depending on its rate of conversion to 6-mono-acetylmorphine (the active ingredient), and this peculiarity is pointed to the variance in certain genes involved in its metabolism which have been referred as polymorphics. The majority of CNS disorders are considered as complex genetic traits, consequently the main focus in this kind of study has been to provide reasonable evidences and ways forward by moving from loci to haplotypes and allelic variants; however this has proved to be one of the biggest challenges nowadays.

Our project intends to screen polymorphic candidate genes that mediate some pathways in the metabolism of substances of abuse by establishing an association study between case and controls individuals. Samples will be taken from drug dependents on a variety of illicit drugs, such as opioid, cocaine, amphetamine and marijuana. Samples from control individuals will be collected at least in the same amount of the target population. The experiment will also make use of methodological techniques to quantify the frequency of drug-taking, the behavior, the social impact, the psychological effects and objective and subjective consequences of drug use. Family history and environmental contribution will also be assessed by the use of questionnaires.

The information from this may provide a more precise definition of the genetic factors involved with susceptibility to drug addiction and its correlation to some personality disorders. In a future it might be feasible to design treatments to be administered based on the genetic profile of the patients.

Keywords: Drug addiction; Personality Disorder; Genetic Predisposition; Haplotype.

The Genius Monkeys: Tool Use by Cebus apella libidinosus Living in the Caatinga Dry Forest

Antonio C. de A. Moura (1)

(1) Department of Biological Anthropology, University of Cambridge, Downing Street, Cambridge CB2 3DZ

Abstract: Tool use was one of the evolutionary causal forces in the disproportionate brain enlargement and extensive technology observed in Homo sapiens. However, the prime movers for the origin and evolution of human tool technology, which probably boosted an increase in brain size, remain the focus of debate. Cebus spp. monkeys abilities to use tools and to solve problems are notable and make them excellent models for understanding evolution of tool use. Yet, despite thousands of hours of field observation on four different species until now there has been an almost complete absence of observations of tool use in the wild. This lack of tool use, by contrast with their abilities in captivity, has been explained by low terrestriality or by a lesser intelligence in relation to great apes. I suggested that low abundance of food resources could act as a strong pressure for tool use. I observed a group of C. apella for one year and half at the Serra da Capivara National Park in the dry Caatinga forest of Southeast Piaui, Brazil. Despite frequent droughts, an extended dry season and seasonal scarcity in fruit availability, capuchins are found at moderate densities in this habitat. The monkeys were observed to use stones to dig for underground storage organs, and to crack open dry branches, cactus pith, seeds and dry Manihot tubers. The use of stones to pound the ground, helping to dig up tubers/roots or locate insects, is a remarkable type of tool use as yet undescribed for non-human primates in captivity or the wild. They also used branches to probe tree holes and rock crevices. These twigs were often modified by removing leaves or stems. The two most accepted hypothesis (social intelligence and frugivory­mental maps) hypothesis for the expansion of the neocortex in higher primates excludes tool use. I suggested that the extensive use of tools by Cebus can be linked to their extractive foraging style and perhaps the ancestor of hominids and great apes had a similar foraging style.

Keywords: Tool Use; Dry Forest; Brain Evolution.

Chemokine Gene Expression in Leishmania major Infected Toll-like Receptor Competent and Deficient Mice.

Simone Antoniazi Pereira (1)(2), Helen P Price (2), Pascale Kropf (1), Marina Freudenberg (3), Chris Galanos (3), Deborah Smith (2), Ingrid Muller (1).

(1)   Imperial College London, School of Medicine, Immunology Department, St Mary's, London, UK

(2)   Wellcome Trust Laboratories for Molecular Parasitology, Department of Biological Sciences, Imperial College London, South Kensington, London, UK.

(3)   Max-Plank-Institute for Immunobiology, Freiburg, Germany.

Abstract: Diseases of the immune system, the Leishmaniases, are caused by the intracellular protozoan parasite Leishmania, occur in 88 countries worldwide including Brazil, and the endemic areas of disease are still spreading (http://www.who.int/tdr/diseases/leish/). These parasites cause a heterogeneous group of clinical manifestations ranging from self-healing cutaneous lesions to severe visceral forms of the disease, which are lethal if left untreated. To date, there is no vaccine against Leishmania in routine use anywhere in the world, and treatment for the disease is largely dependent on the use of costly, highly toxic drugs. The aim of this study was to characterize the contribution of Toll-like receptors (TLRs) to the induction of chemokine expression in host defence to Leishmania infection. Activation of TLRs by various microbial products triggers the expression and subsequent production of specific cytokines and chemokines. Although the essential role of TLRs during bacterial infections has been well documented, little information is available on their role in parasitic infections. Studies on TLR signalling pathways have emphasised the role of specific chemokines in linking innate and adaptive immunity. Chemokines are molecules that mediate the recruitment of immune cells by chemotaxis to the site of infection, and can also cause activation, influencing Th1/Th2 differentiation and anti-protozoan activity. The involvement of chemokines in Leishmaniasis has been demonstrated in both cutaneous and visceral forms of the disease, with a correlation between MCP-1 and IP-10 chemokine expression and healing of cutaneous lesions. In this work, the expression of a subset of chemokines was studied in TLR4 competent (TLR4^+/+) and deficient mice (TLR4^0/0), following infection with Leishmania major. Chemokine expression at the site of infection (the footpad), in the draining lymph node and in the spleen of infected animals was determined using two different methods of analysis. The results indicate that L. major infection causes an overall upregulation of RANTES, MIP-1α, IP-10 and MCP-1 in the footpads and lymph nodes, while expression of these chemokines is constitutive in the spleens of TLR4^+/+ and TLR4^0/0. In conclusion, different patterns of expression were detected depending on the time post infection, but there was little variation in the expression of these four chemokines in the presence or absence of TLR4. These data contribute to our understanding of the role played by chemokines in the development of the inflammatory and immune response to Leishmania infection.

Keywords: Leishmania major; Toll-like receptor; Chemokines.

Development and Optimisation of an Intranasal Scopolamine Microcapsule Formulation

Marlise Araujo dos Santos (1), Christopher Marriott (1), Marc Brown (1)

(1) King’s College London, Department of Pharmacy, Drug Delivery and Formulation Science Research Group

Abstract: The aims of this project are to develop and optimise suitable microcapsule formulations containing scopolamine for nasal delivery, which can be used by astronauts to prevent motion sickness. To reach these aims, scopolamine base was produced and subjected to TLC analysis using Dragendorff’s reagent to aid the identification of the active compound and any degradants. Empty microcapsules as well as those containing scopolamine were prepared by complex coacervation. An ultraviolet spectrophotometric method was developed to analyse whether any scopolamine had been entrapped in the microcapsules, and an HPLC method was developed to quantify any drug entrapped.

No degradation products were found to be formed by the treatment process on the evidence of TLC analysis. The ultraviolet spectrophotometric method of analysis was shown not be sufficiently sensitive to quantify scopolamine in microcapsules made by the complex coacervation method, following acid or alkaline digestion. The quantification of scopolamine in the microcapsules was however possible using an HLPC method. After acid and alkaline digestion, scopolamine was found to be entrapped. In addition, when alkaline digestion was used, all scopolamine underwent degradation, while scopolamine was only partially degraded using acid digestion. It was therefore decided that it would be necessary to use a different method of microcapsule production and spray-dying is now being evaluated.

In conclusion, the complex coacervation method was shown to not be suitable to prepare scopolamine microcapsules. Ultraviolet spectrophotometric analysis was not sensitive enough to quantify scopolamine in the microcapsules but the HPLC method was found to be sufficiently sensitive.

Keywords: Scopolamine; Microcapsule Formulation; Motion Sickness; HPLC.

Diversity and Activity of Sulphate-Reducing Bacteria (SRB) in Freshwater and Brackish Sediments

Silvana Q. Silva (1)(2), Kevin J. Purdy (3), Martin Embley (4), David B. Nedwell (2)

(1)Natural History Museum, London, UK

(2)University of Essex, UK

(3)University of Reading, UK

(4)University of Newcastle-upon-Tyne, UK

Abstract: Microbial ecology is the field of science that investigates the relationship between microorganisms and their environment. For many years studies of microbial ecology were carried out exclusively using traditional techniques of enrichment and isolation, which is known to be suitable for a minor fraction of microorganisms. However, recent approaches based on molecular biology allowed studies of microorganisms that cannot be easily cultivated, leading to a best picture of the microbial community in situ. It is known that microbes are widespread in the environment and play an essential role in the biogeochemical cycles and degradation of pollutants. Microbial ecosystems are usually highly heterogeneous, comprising members with specialized metabolic capacities, such as sulphate-reduction carried out by sulphate-reducing bacteria (SRB). This group of anaerobic microorganisms has been extensively investigated in marine sediments, where they dominate the terminal anaerobic oxidation of organic matter. In contrast, SRB are poorly studied in freshwater systems, mainly because of the belief that their activity is limited by sulphate. The aim of this research was to compare the role of SRB in freshwater and brackish sediments from the River Colne estuary, Essex, U.K. Sulphate reduction rates (SRR) were measured with ^35SO2- ₄, whereas diversity and abundance of SRB were investigated by 16S rRNA-based techniques. In addition, methane production measurements and slurry experiments amended with metabolic inhibitors were used to investigate competition between SRB and methanogenic archaea. The results indicated that sulphate reduction in the freshwater sediment was found to be 7-folder higher than in brackish sediment, despite the lower sulphate concentrations. The relative contribution of sulphate reduction to terminal carbon mineralization was up to 100% in the brackish site and 68% in the freshwater site. Genotypic diversity of SRB in both sites was apparently similar, being dominated by members of Desulfosarcina group. It is hypothesised that tight coupling between sulphate reduction and sulphide re-oxidation maintained high SRR in this low-sulphate environment. The very high SRR at the freshwater site compared to the brackish site also suggested high availability of electron donors, possible because of subsurface exudation of labile organic molecules from plant roots, as shown in other similar environments.

Keywords: SRB; Anaerobic Sediments; Sulphate Reduction; Molecular Biology; Microbial Ecology.

(Abstract online - PDF)

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