The active site residue V266 of Chlamydial HtrA is critical for substrate binding during both in vitro and in vivo conditions

HtrA is a complex, multimeric chaperone and serine protease important for the virulence and survival of many bacteria. Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that is responsible for severe disease pathology. C. trachomatis HtrA (CtHtrA) has been shown to be highly expressed in laboratory models of disease. In this study, molecular modelling of CtHtrA protein active site structure identified putative S1-S3 subsite residues I242, I265, and V266. These residues were altered by site-directed mutagenesis, and these changes were shown to considerably reduce protease activity on known substrates and resulted in a narrower and distinct range of substrates compared to wild type. Bacterial two-hybrid analysis revealed that CtHtrA is able to interact in vivo with a broad range of protein sequences with high affinity. Notably, however, the interaction was significantly altered in 35 out of 69 clones when residue V266 was mutated, indicating that this residue has an important function during substrate binding.

Comparison of cervical and blood T-cell responses to human papillomavirus-16 in women with human papillomavirus-associated cervical intraepithelial neoplasia

Human papillomaviruses (HPVs) are obligate epithelial pathogens and typically cause localized mucosal infections. We therefore hypothesized that T-cell responses to HPV antigens would be greater at sites of pathology than in the blood. Focusing on HPV-16 because of its association with cervical cancer, the magnitude of HPV-specific T-cell responses at the cervix was compared with those in the peripheral blood by intracellular cytokine staining following direct ex vivo stimulation with both virus-like particles assembled from the major capsid protein L1, and the major HPV oncoprotein, E7. We show that both CD4 + and CD8 + T cells from the cervix responded to the HPV-16 antigens and that interferon-γ (IFN-γ) production was HPV type-specific. Comparing HPV-specific T-cell IFN-γ responses at the cervix with those in the blood, we found that while CD4 + and CD8 + T-cell responses to L1 were significantly correlated between compartments (P = 0.02 and P = 0.05, respectively), IFN-γ responses in both T-cell subsets were significantly greater in magnitude at the cervix than in peripheral blood (P = 0.02 and P = 0.003, respectively). In contrast, both CD4 + and CD8 + T-cell IFN-γ responses to E7 were of similar magnitude in both compartments and CD8 + responses were significantly correlated between these distinct immunological compartments (P = 0.04). We therefore show that inflammatory T-cell responses against L1 (but not E7) demonstrate clear compartmental bias and the magnitude of these responses do reflect local viral replication but that correlation of HPV-specific responses between compartments indicates their linkage.

Maize streak virus: An old and complex 'emerging' pathogen

Maize streak virus (MSV; Genus Mastrevirus, Family Geminiviridae) occurs throughout Africa, where it causes what is probably the most serious viral crop disease on the continent. It is obligately transmitted by as many as six leafhopper species in the Genus Cicadulina, but mainly by C. mbila Naudé and C. storeyi. In addition to maize, it can infect over 80 other species in the Family Poaceae. Whereas 11 strains of MSV are currently known, only the MSV-A strain is known to cause economically significant streak disease in maize. Severe maize streak disease (MSD) manifests as pronounced, continuous parallel chlorotic streaks on leaves, with severe stunting of the affected plant and, usuallly, a failure to produce complete cobs or seed. Natural resistance to MSV in maize, and/or maize infections caused by non-maize-adapted MSV strains, can result in narrow, interrupted streaks and no obvious yield losses. MSV epidemiology is primarily governed by environmental influences on its vector species, resulting in erratic epidemics every 3-10 years. Even in epidemic years, disease incidences can vary from a few infected plants per field, with little associated yield loss, to 100% infection rates and complete yield loss. Taxonomy: The only virus species known to cause MSD is MSV, the type member of the Genus Mastrevirus in the Family Geminiviridae. In addition to the MSV-A strain, which causes the most severe form of streak disease in maize, 10 other MSV strains (MSV-B to MSV-K) are known to infect barley, wheat, oats, rye, sugarcane, millet and many wild, mostly annual, grass species. Seven other mastrevirus species, many with host and geographical ranges partially overlapping those of MSV, appear to infect primarily perennial grasses. Physical properties: MSV and all related grass mastreviruses have single-component, circular, single-stranded DNA genomes of approximately 2700 bases, encapsidated in 22 × 38-nm geminate particles comprising two incomplete T = 1 icosahedra, with 22 pentameric capsomers composed of a single 32-kDa capsid protein. Particles are generally stable in buffers of pH 4-8. Disease symptoms: In infected maize plants, streak disease initially manifests as minute, pale, circular spots on the lowest exposed portion of the youngest leaves. The only leaves that develop symptoms are those formed after infection, with older leaves remaining healthy. As the disease progresses, newer leaves emerge containing streaks up to several millimetres in length along the leaf veins, with primary veins being less affected than secondary or tertiary veins. The streaks are often fused laterally, appearing as narrow, broken, chlorotic stripes, which may extend over the entire length of severely affected leaves. Lesion colour generally varies from white to yellow, with some virus strains causing red pigmentation on maize leaves and abnormal shoot and flower bunching in grasses. Reduced photosynthesis and increased respiration usually lead to a reduction in leaf length and plant height; thus, maize plants infected at an early stage become severely stunted, producing undersized, misshapen cobs or giving no yield at all. Yield loss in susceptible maize is directly related to the time of infection: Infected seedlings produce no yield or are killed, whereas plants infected at later times are proportionately less affected. Disease control: Disease avoidance can be practised by only planting maize during the early season when viral inoculum loads are lowest. Leafhopper vectors can also be controlled with insecticides such as carbofuran. However, the development and use of streak-resistant cultivars is probably the most effective and economically viable means of preventing streak epidemics. Naturally occurring tolerance to MSV (meaning that, although plants become systemically infected, they do not suffer serious yield losses) has been found, which has primarily been attributed to a single gene, msv-1. However, other MSV resistance genes also exist and improved resistance has been achieved by concentrating these within individual maiz genotypes. Whereas true MSV immunity (meaning that plants cannot be symptomatically infected by the virus) has been achieved in lines that include multiple small-effect resistance genes together with msv-1, it has proven difficult to transfer this immunity into commercial maize genotypes. An alternative resistance strategy using genetic engineering is currently being investigated in South Africa. Useful websites: 〈http://www.mcb.uct.ac.za/MSV/mastrevirus.htm〉; 〈http://www. danforthcenter.org/iltab/geminiviridae/geminiaccess/mastrevirus/Mastrevirus. htm〉. © 2009 Blackwell Publishing Ltd.

Expression of MUC1 and MUC2 mucins in epithelial ovarian tumours

This is the first study to describe the association between expression of MUC1 and MUC2 mucins and prognosis in ovarian cancer. Paraffin sections of epithelial ovarian tumours (n=182: 29 benign, 21 low malignant potential, and 132 invasive tumours) were analysed immunohistochemically for expression of MUC1 and MUC2 mucin core proteins. Most benign, low malignant potential, and invasive tumours showed high MUC1 expression in the cytoplasm. Low cytoplasmic expression of MUC1 was a predictor for good prognosis, particularly within stage III tumours. A minority of benign epithelial tumours, but most low malignant potential and invasive non-mucinous tumours, showed high MUC1 expression on the cell membrane. High apical MUC1 reactivity was associated with non-mucinous tumours. Low expression of MUC1 in the apical membrane was associated with early stage and good outcome for invasive tumours. Most benign and low malignant potential tumours, but only a minority of invasive tumours, showed MUC2 expression. MUC2 was found in non-mucinous as well as in mucinous tumours. The presence of MUC2 was inversely associated with high tumour grade but was not associated with altered survival. These results support experimental evidence that MUC1 influences the metastatic ability of ovarian cancer.

Antenatal ureaplasma infection impairs development of the fetal ovine gut in an IL-1-dependent manner

Ureaplasma infection of the amniotic cavity is associated with adverse postnatal intestinal outcomes. We tested whether interleukin-1 (IL-1) signaling underlies intestinal pathology following ureaplasma exposure in fetal sheep. Pregnant ewes received intra-amniotic injections of ureaplasma or culture media for controls at 3, 7, and 14 d before preterm delivery at 124 d gestation (term 150 d). Intra-amniotic injections of recombinant human interleukin IL-1 receptor antagonist (rhIL-1ra) or saline for controls  were given 3 h before and every 2 d after Ureaplasma injection. Ureaplasma exposure caused fetal gut inflammation within 7 d with damaged villus epithelium and gut barrier loss. Proliferation, differentiation, and maturation of enterocytes were significantly reduced after 7 d of ureaplasma exposure, leading to severe villus atrophy at 14 d. Inflammation, impaired development and villus atrophy of the fetal gut was largely prevented by intra-uterine rhIL-1ra treatment. These data form the basis for a clinical understanding of the role of ureaplasma in postnatal intestinal pathologies.

Effects of oxygen and culture system on in vitro propagation and redifferentiation of osteoarthritic human articular chondrocytes

Regenerative medicine-based approaches for the repair of damaged cartilage rely on the ability to propagate cells while promoting their chondrogenic potential. Thus, conditions for cell expansion should be optimized through careful environmental control. Appropriate oxygen tension and cell expansion substrates and controllable bioreactor systems are probably critical for expansion and subsequent tissue formation during chondrogenic differentiation. We therefore evaluated the effects of oxygen and microcarrier culture on the expansion and subsequent differentiation of human osteoarthritic chondrocytes. Freshly isolated chondrocytes were expanded on tissue culture plastic or CultiSpher-G microcarriers under hypoxic or normoxic conditions (5% or 20% oxygen partial pressure, respectively) followed by cell phenotype analysis with flow cytometry. Cells were redifferentiated in micromass pellet cultures over 4 weeks, under either hypoxia or normoxia. Chondrocytes cultured on tissue culture plastic proliferated faster, expressed higher levels of cell surface markers CD44 and CD105 and demonstrated stronger staining for proteoglycans and collagen type II in pellet cultures compared with microcarrier-cultivated cells. Pellet wet weight, glycosaminoglycan content and expression of chondrogenic genes were significantly increased in cells differentiated under hypoxia. Hypoxia-inducible factor-3alpha mRNA was up-regulated in these cultures in response to low oxygen tension. These data confirm the beneficial influence of reduced oxygen on ex vivo chondrogenesis. However, hypoxia during cell expansion and microcarrier bioreactor culture does not enhance intrinsic chondrogenic potential. Further improvements in cell culture conditions are therefore required before chondrocytes from osteoarthritic and aged patients can become a useful cell source for cartilage regeneration.

Access to cardiac rehabilitation does not equate to attendance

Background/Aims Timely access to appropriate cardiac care is critical for optimizing positive outcomes after a cardiac event. Attendance at cardiac rehabilitation (CR) remains less than optimal (10%–30%). Our aim was to derive an objective, comparable, geographic measure reflecting access to cardiac services after a cardiac event in Australia. Methods An expert panel defined a single patient care pathway and a hierarchy of the minimum health services for CR and secondary prevention. Using geographic information systems a numeric/alpha index was modelled to describe access before and after a cardiac event. The aftercare phase was modelled into five alphabetical categories: from category A (access to medical service, pharmacy, CR, pathology within 1 h) to category E (no services available within 1 h). Results Approximately 96% or 19 million people lived within 1 h of the four basic services to support CR and secondary prevention, including 96% of older Australians and 75% of the indigenous population. Conversely, 14% (64,000) indigenous people resided in population locations that had poor access to health services that support CR after a cardiac event. Conclusion Results demonstrated that the majority of Australians had excellent ‘geographic’ access to services to support CR and secondary prevention. Therefore, it appears that it is not the distance to services that affects attendance. Our ‘geographic’ lens has identified that more research on socioeconomic, sociological or psychological aspects to attendance is needed.

Access to Cardiac Rehabilitation does not Equate to Attendance. (On Behalf of the Cardiac ARIA Project. Winner Nursing/Allied Health Professional Investigator Award)

Background/Aims Timely access to appropriate cardiac care is critical for optimizing positive outcomes after a cardiac event. Attendance at cardiac rehabilitation (CR) remains less than optimal (10%–30%). Our aim was to derive an objective, comparable, geographic measure reflecting access to cardiac services after a cardiac event in Australia. Methods An expert panel defined a single patient care pathway and a hierarchy of the minimum health services for CR and secondary prevention. Using geographic information systems a numeric/alpha index was modelled to describe access before and after a cardiac event. The aftercare phase was modelled into five alphabetical categories: from category A (access to medical service, pharmacy, CR, pathology within 1 h) to category E (no services available within 1 h). Results Approximately 96% or 19 million people lived within 1 h of the four basic services to support CR and secondary prevention, including 96% of older Australians and 75% of the indigenous population. Conversely, 14% (64,000) indigenous people resided in population locations that had poor access to health services that support CR after a cardiac event. Conclusion Results demonstrated that the majority of Australians had excellent ‘geographic’ access to services to support CR and secondary prevention. Therefore, it appears that it is not the distance to services that affects attendance. Our ‘geographic’ lens has identified that more research on socioeconomic, sociological or psychological aspects to attendance is needed.

BRCA1 is an essential mediator of vinorelbine-induced apoptosis in mesothelioma

The rapeutic options for malignant pleural mesothelioma (MPM) are limited despite the increasing incidence globally. The vinca alkaloid vinorelbine exhibits clinical activity; however, to date, treatment optimization has not been achieved using biomarkers. BRCA1 regulates sensitivity to microtubule poisons; however, its role in regulating vinorelbine-induced apoptosis in mesothelioma is unknown. Here we demonstrate that BRCA1 plays an essential role in mediating vinorelbine-induced apoptosis, as evidenced by (1) the strong correlation between vinorelbine sensitivity and BRCA1 expression level; (2) induction of resistance to vinorelbine by BRCA1 using siRNA oligonucleotides; (3) dramatic down-regulation of BRCA1 following selection for vinorelbine resistance; and (4) the re-activation of vinorelbine-induced apoptosis following re-expression of BRCA1 in resistant cells. To determine whether loss of BRCA1 expression in mesothelioma was potentially relevant in vivo, BRCA1 immunohistochemistry was subsequently performed on 144 primary mesothelioma specimens. Loss of BRCA1 protein expression was identified in 38.9% of samples. Together, these data suggest that BRCA1 plays a critical role in mediating apoptosis by vinorelbine in mesothelioma, warranting its clinical evaluation as a predictive biomarker. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Discrimination experienced by HIV/AIDS infected persons and its associations with mental health in an Indian sample

Purpose The purpose of this study was to investigate the nature and prevalence of discrimination against people living with HIV/AIDS in West Bengal, India, and how discrimination is associated with depression, suicidal ideation and suicidal attempts. Method Semi-structured interviews and the Beck Depression Inventory were administered to 105 HIV infected persons recruited by incidental sampling, at an Integrated Counseling and Testing Center (ICTC) and through Networks of People Living with HIV/AIDS, in the West Bengal area. Results Findings showed that 40.8% of the sample has experienced discrimination at least in one social setting – such as family (29.1%), health centers (18.4%), community (17.5%) and workplace (6.8%). About two-fifths (40.8%) reported experiencing discrimination in multiple social settings. Demographic factors associated with discrimination were gender, age, occupation, education, and current residence. More than half of the sample was suffering from severe depression while 8.7% had attempted suicide. Discrimination in most areas was significantly associated with suicidal ideation and suicidal attempts. Conclusions Prevalence of discrimination associated with HIV/AIDS is high in our sample from West Bengal. While discrimination was not associated with depressive symptomatology, discrimination was associated with suicidal ideation and attempts. These findings suggest that there is an urgent need for interventions to reduce discrimination of HIV/AIDS in the West Bengal region.

Organotypic culture of normal, dysplastic and squamous cell carcinoma-derived oral cell lines reveals loss of spatial regulation of CD44 and p75(NTR) in malignancy

Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted, yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence, in this study, we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2), mild dysplasia (DOK), severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44, p75NTR, CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75NTR was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44, p75NTR, CD24 antigens and ALDH activity (ALDEFLUOR® assay), with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer, increased FOXA1 and decreased FOXA2 expression correlated with disease severity, but OCT3/4, Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells.

The predictors of active ageing of older Australians : the triple a study

The concept of older adults contributing to society in a meaningful way has been termed ‘active ageing’. Active ageing reflects changes in prevailing theories of social and psychological aspects of ageing, with a focus on individuals' strengths as opposed to their deficits or pathology. In order to explore predictors of active ageing, the Australian Active Ageing (Triple A) project group undertook a national postal survey of participants over the age of 50 years recruited randomly through their 2004 membership of a large Australia-wide senior's organisation. The survey comprised 178 items covering paid and voluntary work, learning, social, spiritual, emotional, health and home, life events and demographic items. A 45% response rate (2655 returned surveys) reflected an expected balance of gender, age and geographic representation of participants. The data were analysed using data mining techniques to represent generalizations on individual situations. Data mining identifies the valid, novel, potentially useful and understandable patterns and trends in data. The results based on the clustering mining technique indicate that physical and emotional health combined with the desire to learn were the most significant factors when considering active ageing. The findings suggest that remaining active in later life is not only directly related to the maintenance of emotional and physical health, but may be significantly intertwined with the opportunity to engage in on-going learning activities that are relevant to the individual. The findings of this study suggest that practitioners and policy makers need to incorporate older peoples' learning needs within service and policy framework developments.

Learning landscapes 2.0 : warning - assembly required

Twenty first century learners operate in organic, immersive environments. A pedagogy of student-centred learning is not a recipe for rooms. A contemporary learning environment is like a landscape that grows, morphs, and responds to the pressures of the context and micro-culture. There is no single adaptable solution, nor a suite of off-the-shelf answers; propositions must be customisable and infinitely variable. They must be indeterminate and changeable; based on the creation of learning places, not restrictive or constraining spaces. A sustainable solution will be un-fixed, responsive to the life cycle of the components and materials, able to be manipulated by the users; it will create and construct its own history. Learning occurs as formal education with situational knowledge structures, but also as informal learning, active learning, blended learning social learning, incidental learning, and unintended learning. These are not spatial concepts but socio-cultural patterns of discovery. Individual learning requirements must run free and need to be accommodated as the learner sees fit. The spatial solution must accommodate and enable a full array of learning situations. It is a system not an object. Three major components: 1. The determinate landscape: in-situ concrete 'plate' that is permanent. It predates the other components of the system and remains as a remnant/imprint/fossil after the other components of the system have been relocated. It is a functional learning landscape in its own right; enabling a variety of experiences and activities. 2. The indeterminate landscape: a kit of pre-fabricated 2-D panels assembled in a unique manner at each site to suit the client and context. Manufactured to the principles of design-for-disassembly. A symbiotic barnacle like system that attaches itself to the existing infrastructure through the determinate landscape which acts as a fast growth rhizome. A carapace of protective panels, infinitely variable to create enclosed, semi-enclosed, and open learning places. 3. The stations: pre-fabricated packages of highly-serviced space connected through the determinate landscape. Four main types of stations; wet-room learning centres, dry-room learning centres, ablutions, and low-impact building services. Entirely customised at the factory and delivered to site. The stations can be retro-fitted to suit a new context during relocation. Principles of design for disassembly: material principles • use recycled and recyclable materials • minimise the number of types of materials • no toxic materials • use lightweight materials • avoid secondary finishes • provide identification of material types component principles • minimise/standardise the number of types of components • use mechanical not chemical connections • design for use of common tools and equipment • provide easy access to all components • make component size to suite means of handling • provide built in means of handling • design to realistic tolerances • use a minimum number of connectors and a minimum number of types system principles • design for durability and repeated use • use prefabrication and mass production • provide spare components on site • sustain all assembly and material information

Understanding QC and EQA : an authentic learning model for undergraduate students (Conference Abstract)

Introduction QC and EQA are integral to good pathology laboratory practice. Medical Laboratory Science students undertake a project exploring internal QC and EQA procedures used in chemical pathology laboratories. Each student represents an individual lab and the class group represents the peer group of labs performing the same assay using the same method. Methods Using a manual BCG assay for serum albumin, normal and abnormal controls are run with a patient sample over 7 weeks. The QC results are assessed each week using calculated z-scores and both 2S & 3S control rules to determine whether a run is ‘in control’. At the end of the 7 weeks a completed LJ chart is assessed using the Westgard Multirules. Students investigate causes of error and the implications for both lab practice and patient care if runs are not ‘in control’. Twice in the 7 weeks two EQA samples (with target values unknown) are assayed alongside the weekly QC and patient samples. Results from each student are collated and form the basis of an EQA program. ALP are provided and students complete a Youden Plot, which is used to analyse the performance of each ‘lab’ and the method to identify bias. Students explore the concept of possible clinical implications of a biased method and address the actions that should be taken if a lab is not in consensus with the peer group. Conclusion This project is a model of ‘real world’ practice in which student demonstrate an understanding of the importance of QC procedures in a pathology laboratory, apply and interpret statistics and QC rules and charts, apply critical thinking and analytical skills to quality performance data to make recommendations for further practice and improve their technical competence and confidence.