Nanobodies against the myelin enzyme CNPase as tools for structural and functional studies.

2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an abundant constituent of central nervous system non-compact myelin, frequently used as a marker antigen for myelinating cells. The catalytic activity of CNPase, the 3'-hydrolysis of 2',3'-cyclic nucleotides, is well characterised in vitro, but the in vivo function of CNPase remains unclear. CNPase interacts with the actin cytoskeleton to counteract the developmental closure of cytoplasmic channels that travel through compact myelin; its enzymatic activity may be involved in adenosine metabolism and RNA degradation. We developed a set of high-affinity nanobodies recognizing the phosphodiesterase domain of CNPase, and the crystal structures of each complex show that the five nanobodies have distinct epitopes. One of the nanobodies bound deep into the CNPase active site and acted as an inhibitor. Moreover, the nanobodies were characterised in imaging applications and as intrabodies, expressed in mammalian cells, such as primary oligodendrocytes. Fluorescently labelled nanobodies functioned in imaging of teased nerve fibers and whole brain tissue sections, as well as super-resolution microscopy. These anti-CNPase nanobodies provide new tools for structural and functional biology of myelination, including high-resolution imaging of nerve tissue.

Publisher Correction: ß-Synuclein-reactive T cells induce autoimmune CNS grey matter degeneration.

In this Article, owing to an error during the production process, the y-axis label of Fig. 2c should read "Number of Tß-syn cells" rather than "Number of T1ß-syn cells" and the left and right panels of Fig. 4 should be labelled 'a' and 'b', respectively. These errors have been corrected online.

ß-Synuclein-reactive T cells induce autoimmune CNS grey matter degeneration.

The grey matter is a central target of pathological processes in neurodegenerative disorders such as Parkinson's and Alzheimer's diseases. The grey matter is often also affected in multiple sclerosis, an autoimmune disease of the central nervous system. The mechanisms that underlie grey matter inflammation and degeneration in multiple sclerosis are not well understood. Here we show that, in Lewis rats, T cells directed against the neuronal protein ß-synuclein specifically invade the grey matter and that this is accompanied by the presentation of multifaceted clinical disease. The expression pattern of ß-synuclein induces the local activation of these T cells and, therefore, determined inflammatory priming of the tissue and targeted recruitment of immune cells. The resulting inflammation led to significant changes in the grey matter, which ranged from gliosis and neuronal destruction to brain atrophy. In humans, ß-synuclein-specific T cells were enriched in patients with chronic-progressive multiple sclerosis. These findings reveal a previously unrecognized role of ß-synuclein in provoking T-cell-mediated pathology of the central nervous system.

Effector T-cell trafficking between the leptomeninges and the cerebrospinal fluid.

In multiple sclerosis, brain-reactive T cells invade the central nervous system (CNS) and induce a self-destructive inflammatory process. T-cell infiltrates are not only found within the parenchyma and the meninges, but also in the cerebrospinal fluid (CSF) that bathes the entire CNS tissue. How the T cells reach the CSF, their functionality, and whether they traffic between the CSF and other CNS compartments remains hypothetical. Here we show that effector T cells enter the CSF from the leptomeninges during Lewis rat experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. While moving through the three-dimensional leptomeningeal network of collagen fibres in a random Brownian walk, T cells were flushed from the surface by the flow of the CSF. The detached cells displayed significantly lower activation levels compared to T cells from the leptomeninges and CNS parenchyma. However, they did not represent a specialized non-pathogenic cellular sub-fraction, as their gene expression profile strongly resembled that of tissue-derived T cells and they fully retained their encephalitogenic potential. T-cell detachment from the leptomeninges was counteracted by integrins VLA-4 and LFA-1 binding to their respective ligands produced by resident macrophages. Chemokine signalling via CCR5/CXCR3 and antigenic stimulation of T cells in contact with the leptomeningeal macrophages enforced their adhesiveness. T cells floating in the CSF were able to reattach to the leptomeninges through steps reminiscent of vascular adhesion in CNS blood vessels, and invade the parenchyma. The molecular/cellular conditions for T-cell reattachment were the same as the requirements for detachment from the leptomeningeal milieu. Our data indicate that the leptomeninges represent a checkpoint at which activated T cells are licensed to enter the CNS parenchyma and non-activated T cells are preferentially released into the CSF, from where they can reach areas of antigen availability and tissue damage.

Transmission and prevention of HIV among heterosexual populations in Australia.

In Australia, unlike much of the rest of the world, HIV transmission through heterosexual contact remains a relatively rare occurrence. In consequence, HIV-prevention efforts have been firmly focused on male-to-male sex as the most frequent source of HIV transmission. There are emerging signs that this epidemiological landscape may be shifting, which raises questions about current and future HIV prevention strategies. Over the past decade, national surveillance data have shown an increase in HIV notifications for which exposure to HIV was attributed to heterosexual contact. This paper offers an epidemiological and sociocultural picture of heterosexual HIV transmission in Australia. We outline recent trends in heterosexually acquired HIV and discuss specific factors that shape transmission and prevention among people at risk of HIV infection through heterosexual contact. To illustrate the contextual dynamics surrounding HIV in this diverse population, we detail two key examples: HIV among people from minority ethnic backgrounds in New South Wales; and overseas-acquired HIV among men in Western Australia. We argue that, despite their differences, there are significant commonalities across groups at risk of HIV infection through heterosexual contact, which not only provide opportunities for HIV prevention, but also call for a rethink of the dominant HIV response in Australia.

A combination of fluorescent NFAT and H2B sensors uncovers dynamics of T cell activation in real time during CNS autoimmunity.

Multiple sclerosis is an autoimmune disease of the central nervous system (CNS) that is initiated when self-reactive T cells enter the brain and become locally activated after encountering their specific nervous antigens. When and where the disease-relevant antigen encounters occur is unclear. Here we combined fluorescently labeled nuclear factor of activated T cells (NFAT) with histone protein H2B to create a broadly applicable molecular sensor for intravital imaging of T cell activation. In experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis, we report that effector T cells entering the CNS become activated after short contacts with leptomeningeal phagocytes. During established disease, the activation process is extended to the depth of the CNS parenchyma, where the cells form contacts with microglia and recruited phagocytes. We show that it is the activation processes during the preclinical phase rather than during established disease that are essential for the intensity and duration of the disease bout.

Discourses of depression of Australian general practitioners working with gay men.

The data for this article are from a primary health care project on HIV and depression, in which the prevalence, nature, clinical management, and self-management of depression among homosexually active men attending high-HIV-caseload general practice clinics were investigated. One of the qualitative arms consisted of in-depth interviews with general practitioners (GPs) with high caseloads of gay men. The approach to discourse analysis was informed by Halliday's systemic functional linguistics. GPs constructed three discourses of depression: engaging with psychiatric discourse, engaging with the patient's world, and engaging with social structures. When GPs drew on the discourse of psychiatry, this discourse was positioned as only one possible construction of depression. This discourse was also contextualized in the social lives of gay men, and it was explicitly challenged and rejected. Engaging with their patients' social world was considered vital for recognizing depression in gay men. Finally, the GPs' construction of depression was inextricably linked to social disadvantage and marginalization. Depression is highly heterogeneous and constructed in terms of social relationships rather than as an independent entity that resides in the individual. There is a synergy between GPs' constructions of depression and men's experiences of depression, which differs from conventional medical views, and which enables GPs to be highly effective in dealing with the mental health issues of their gay patients.

'The black dog just came and sat on my face and built a kennel': Gay men making sense of 'depression'.

This article reports on in-depth interviews with gay men about their experiences and understanding of depression. It is a key outcome of the collaboration between social researchers, general practitioners and community partners to investigate the management of depression in gay men in primary care settings. As part of the qualitative arm of the project in-depth interviews were conducted with 40 gay men in Sydney and Adelaide (Australia). The approach to discourse analysis is informed by Hallidayan systemic functional linguistics. Six constructions of depression were identified: (1) depression as a constellation of symptoms; (2) symptoms constructed as experience; (3) depression as agent; (4) depression as mental processes; (5) not meeting social expectations; and (6) engaging with psychiatric discourse: constructing alternative positions. Gay men draw on the biomedical model of depression as low mood and loss of pleasure as well as on constructions of depression in terms of social experience. The biomedical model of depression is, however, not positioned as unproblematic. Rather, gay men align or disalign with this discourse according to their own experience, thereby enacting diverse masculinities. Gay men's discourses of depression are inextricably linked to the community activism of gay men and their community organizations in the context of the HIV epidemic, as well as a synergy between gay men and their doctors.

Negotiating treatment for hepatitis C: interpersonal alignment in the clinical encounter.

Antiviral treatment for hepatitis C constitutes a considerable physical and psycho-social challenge without guarantee of treatment success. Using semi-structured in-depth interviews, this article investigates the experiences of people on hepatitis C treatment and the experiences of physicians who care for people with hepatitis C. Given the importance of the interpersonal dimension of the patient-physician relationship for patients accessing treatment, adhering to treatment, dealing with treatment side-effects and completing treatment, the article focuses on the interpersonal dimension of patients' and physicians' accounts. The theoretical foundation is 'appraisal' theory from systemic functional linguistics, which is grounded in Bakhtin's notions of heteroglossia and dialogism. The article describes the intersubjective stances that patients and physicians adopt in accounts of their interactions about hepatitis C treatment, the values they construct and the semantic backdrop against which these meanings are constructed. The article traces the semantic patterns of intersubjective alignment and disalignment between patients and physicians, as well as the semantics of patients' challenging the intersubjective stances taken by their physicians. While the biomedical discourse was intersubjectively at odds with the experiences of some patients, others aligned with it. In fact, understanding and speaking the language of biomedicine enabled some patients to challenge the traditional doctor-patient relationship and to reconfigure it into a partnership approach.

Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer.

Recently, we and others identified the microRNA miR-34a as a target of the tumor suppressor gene product p53. Ectopic miR-34a induces a G(1) cell cycle arrest, senescence and apoptosis. Here we report that miR-34a expression is silenced in several types of cancer due to aberrant CpG methylation of its promoter. 19 out of 24 (79.1%) primary prostate carcinomas displayed CpG methylation of the miR-34a promoter and concomitant loss of miR-34a expression. CpG methylation of the miR-34a promoter was also detected in breast (6/24; 25%), lung (7/24; 29.1%), colon (3/23; 13%), kidney (3/14; 21.4%), bladder (2/6; 33.3%) and pancreatic (3/19; 15.7%) carcinoma cell lines, as well as in melanoma cell lines (19/44; 43.2%) and primary melanoma (20/32 samples; 62.5%). Silencing of miR-34a was dominant over its transactivation by p53 after DNA damage. Re-expression of miR-34a in prostate and pancreas carcinoma cell lines induced senescence and cell cycle arrest at least in part by targeting CDK6. These results show that miR-34a represents a tumor suppressor gene which is inactivated by CpG methylation and subsequent transcriptional silencing in a broad range of tumors.

'It's really a myriad of different signals, not just the textbook': the complexities of diagnosing depression in gay men in general practice.

This paper reports on in-depth interviews with general practitioners (GPs) about their views and experiences of diagnosing depression in gay men - some of whom are living with HIV - and the broader social contexts in which such a diagnosis is located. This analysis is a key outcome of a collaboration between social researchers, primary healthcare researchers, GPs and community partners, to investigate the management of depression in gay men in primary care settings. As the qualitative component of this project, semi-structured in-depth interviews were conducted with 16 GPs with high caseloads of gay men, in three geographical settings in Australia: Sydney, Adelaide and a rural-coastal town. GPs considered the diagnosis and management of depression to be an integral part of primary care, especially in gay male patients. They had a heightened sense of awareness that depression was common in the group of patients they were seeing. Central to diagnosing depression was the ongoing, long-term relationship GPs had with their gay male patients. GPs were vigilant and proactively inquired about depression, taking into account somatic, social and psychological indicators. In their approach to diagnosing depression, GPs considered not only the life circumstances of individual patients but also the broader social context of stigma related to homosexuality, and the effects that the HIV epidemic has had on individuals, especially on gay men who have been living with HIV for a long time.

'If I had my residency I wouldn't worry': negotiating migration and HIV in Sydney, Australia.

OBJECTIVE: To describe the interrelationships between migration and resettlement, the Australian immigration system and living with HIV. METHOD: Data were collected through semi-structured, in-depth interviews with clients of the Multicultural HIV/AIDS and Hepatitis C Service and a sexual health clinic in the Sydney metropolitan area over an 18-month period in 2003-2004. RESULTS: Three major themes interwoven with migration were identified: HIV diagnosis, access to care and support, and forming social relations. Participants who applied for permanent residency in Australia rather than off-shore were usually diagnosed as HIV-positive as part of the health requirement for permanent residency. This jeopardized their prospect of staying in Australia and was at the same time a barrier to returning to the country of birth. It was also a barrier to accessing health care and support services and a major source of uncertainty. The meaning of an HIV-positive diagnosis was grounded in participants' knowledge about HIV from their country of birth: HIV infection was perceived as a terminal illness. Because of the stigma associated with HIV/AIDS, many had little or no contact with their ethnic communities in Australia. At the same time, they found it difficult to form new social relations in the Anglo-Celtic mainstream culture. A further problem was feeling torn between Australia and the promise of a better future, and the close emotional relationships with family and friends in the country of birth. CONCLUSION: New migrants with HIV need to negotiate two major life disruptions and two major uncertainties simultaneously: migration and HIV infection. In the Anglo-Celtic mainstream, language, cultural and financial barriers to health and support services should be removed or minimized. In ethnic communities, HIV-related stigma needs to be addressed to enable new migrants to form social relations in these communities and to rebuild their lives.

Negotiating cultures: disclosure of HIV-positive status among people from minority ethnic communities in Sydney.

Because of the multiple stigma attached to HIV/AIDS, disclosure of HIV-positive serostatus is a considerable social risk for those who disclose. While HIV/AIDS-related stigma affects all HIV-positive people, for people from minority cultures additional cultural factors may play a significant role in self-disclosure. This paper draws on data from semi-structured, in-depth interviews with HIV-positive people from minority cultures in Sydney. Disclosure decisions were influenced by gender, sexual orientation, as well as cultural background. Gay men drew on both collectivist and individualist notions of interdependence and self-reliance in different socio-cultural contexts. This enabled them to accommodate the imperative to maintain harmony with the family and meet their individual needs for support. Heterosexual men who had disclosed voluntarily or involuntarily experienced discrimination and avoidance, and interdependence with family and ethnic community was disrupted. Heterosexual women disclosed to no one outside the health care system and were anxious to avoid any disclosure in the future. For all participants, voluntary and involuntary disclosure caused potential and actual disruption of relationships with their families and ethnic communities. The paper concludes by arguing for an ecological perspective of health in which decisions are not located in rational decision making alone but in the broader context of family and community.

Digital karyotyping reveals frequent inactivation of the dystrophin/DMD gene in malignant melanoma.

Malignant melanoma is still poorly understood at the genomic level. Recently, a new technique for the high-resolution analysis of copy number changes named digital karyotyping was introduced. This approach is derived from SAGE (serial analysis of gene expression) and allows the detection of genomic amplifications and deletions, which are indicative of oncogenes and tumor suppressor genes. Four human melanoma cell lines were subjected to analysis by digital karyotyping. 828,780 genomic tags were generated and analyzed quantitatively. Thereby, we identified a somatic, homozygous deletion of 570 kbp removing exons 3-29 of the dystrophin (DMD, Duchenne muscular dystrophy) gene. Analysis of 51 melanoma cell lines further revealed a homozygous and a hemizygous deletion in DMD. Furthermore, DMD mRNA expression was downregulated with respect to primary melanocytes and accompanied by loss of dystrophin protein expression in 38 of 55 (69%) and significantly reduced in 10 of 55 (18%) melanoma cell lines. Sequence analysis of DMD cDNAs in 37 melanoma cell lines revealed six new sequence variants with a significantly lower frequency than previously described DMD polymorphisms, which may affect dystrophin function. Knock-down of DMD enhanced migration and invasion, whereas re-expression of DMD attenuated migration and induced a senescent phenotype in melanoma cell lines. Therefore, loss of DMD may critically change the migratory and proliferative capacity of melanocytes. Taken together, our results suggest that inactivation of DMD is involved in the pathogenesis of malignant melanoma.

Representations of people with HIV and hepatitis C in editorials of medical journals: discourses and interdiscursive relations.

HIV and hepatitis C are blood-borne viruses that cause chronic diseases and affect (in parts of the developed world) predominantly groups that are marginalized and discriminated against: gay men and injecting drug users, respectively. This paper compares the representation of people with HIV and hepatitis C in editorials of medical journals between 1989 and 2001. Analysis is informed by critical discourse analysis and systemic functional linguistics. Hepatitis C editorials draw almost exclusively on the discourse of biomedicine, and patients are either absent or objects in medical procedures. In HIV editorials, a variety of other discourses are integrated into the discourse of biomedicine, thereby creating multidimensional representations of people with HIV as patients and agents in medical procedures, involved in decision making, affected by economic factors, social and cultural issues. The paper discusses the role of the gay community in discursive change and argues that discursive diversity in the representation of people infected with HIV and hepatitis C in medical journals is necessary for health policy, the professional development of healthcare providers, and media reporting to the general public.

'I didn't think I was at risk': Interdiscursive relations in narratives of sexual practices and exposure to HIV.

This article is concerned with the discursive construction of sexual practices and 'risk' in gay men's accounts of exposure to HIV. The data are in-depth interviews from two Sydney-based studies. While the events reported in both studies were very similar, there are considerable differences in the language choices. Drawing on critical discourse analysis and systemic functional linguistics, this paper shows how language choices in individual narratives resonate intertextually with the public health discourse of safe sex, which emphasizes knowledge of safe sex, control over sexual practices and condom use. While in accounts of exposure to HIV in the context of prevention sexual practice is construed consistently as 'doing' with the speakers as Actor, in accounts of exposure to HIV in the context of transmission it is construed predominantly as 'thinking' and 'being'. There are also differences in the negotiation of alternatives. All narratives resonate with the discourse of safe sex, however, it is not passively reflected in speech but is actively engaged with and shaped to fit the knowledge and understanding of individuals. The narratives also resonate with the private world of intimate relationships and everyday life. This suggests a notion of 'risk' as a hybrid of multiple, potentially conflicting discourses.

Oral Actinomyces isolates forming red colonies on brain heart blood agar can bee unambiguously classified as A odontolyticus by macroscopic examination.

The accurate classification of oral Actinomyces isolates as one species is difficult. Out of 18 Actinomyces isolates forming red colonies on brain heart blood agar, 12 could be straightforwardly assigned as Actinomyces odontolyticus by biochemical, morphological, and chemotaxonomic characteristics. For the remaining six isolates, the results of the different identification methods were inconsistent. By sequencing a 16S ribosomal DNA fragment by a rapid mass spectrometric method, all isolates could be identified unambiguously as A. odontolyticus. This result proves the importance of red colony pigmentation on brain heart blood agar together with the characteristic cell morphology for unequivocal assignment of oral Actinomyces isolates to the species A. odontolyticus.