A locked nucleic acid oligonucleotide targeting microRNA 122 is well-tolerated in cynomolgus monkeys.

MicroRNA 122 (miR-122) is liver specific, fine-tunes lipid metabolism, and is required for hepatitis C virus (HCV) abundance. Miravirsen, an oligonucleotide with locked nucleic acid, binds to miR-122, potently inhibiting its activity. We aimed at determining the safety of the miR-122 antagonism in vivo in 6 to 10 cynomolgus monkeys/group intravenously treated with a range of dose levels twice weekly for 4 weeks. Survival, body weights, clinical signs, and cardiovascular and ophthalmologic parameters were unaffected. Anticipated hypolipidemia due to the inhibition of miR-122 was observed in all treated animals. Only the highest dose level produced distinct transient prolongations of clotting times, slight alternative complement pathway activation, and a reversible increase of hepatic transaminases. Distribution half-life was 10-20 minutes, and accumulation was mainly in the kidney and liver with slow elimination. Microscopic examinations revealed granulated Kupffer cells and lymph node macrophages, cytoplasmic vacuolation in proximal renal tubules, and hepatocytes. The granules were most likely phagolysosomes containing miravirsen. A slightly increased incidence of hepatocyte apoptosis was observed in some monkeys given the highest dose; otherwise, there was no evidence of treatment-related degenerative changes in any organ. In conclusion, the maximal inhibition of miR-122 was associated with limited phenotypic changes, indicating that the clinical assessment of miravirsen as host factor antagonist for treatment of HCV infections is warranted.

The benefits of educational release of hospital-based general practice specialist training registrars into general practice.

In Denmark, specialist training for family medicine consists of 2.5 years training in general practice and 2.5 years training in specific hospital departments. The hospital training programme contains mandatory release time (return days) whereby GP trainees leave their hospitals in order to work with patients in their teaching GP surgeries for one day every month. The goals are to develop and maintain a family medicine perspective during the hospital training and to maintain contact with the family medicine environment. In order to explore the benefits of going back to general practice for one day per month during hospital training, we carried out a qualitative study comprising three focus group interviews with trainees and one focus group with trainers. Return days are important for the development of a professional identity and they can ensure the provision of a useful/necessary breathing space in a turbulent education. If properly organised, return days have the potential to strengthen professional competences due to a stronger focus on the family medicine perspective during training. The process strengthens transferability of skills. A focus on better educational management is needed. Trainers' commitment and trainees' ownership of and responsibility for the educational process are prerequisites for success.

Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection.

The liver-expressed microRNA-122 (miR-122) is essential for hepatitis C virus (HCV) RNA accumulation in cultured liver cells, but its potential as a target for antiviral intervention has not been assessed. We found that treatment of chronically infected chimpanzees with a locked nucleic acid (LNA)-modified oligonucleotide (SPC3649) complementary to miR-122 leads to long-lasting suppression of HCV viremia, with no evidence of viral resistance or side effects in the treated animals. Furthermore, transcriptome and histological analyses of liver biopsies demonstrated derepression of target mRNAs with miR-122 seed sites, down-regulation of interferon-regulated genes, and improvement of HCV-induced liver pathology. The prolonged virological response to SPC3649 treatment without HCV rebound holds promise of a new antiviral therapy with a high barrier to resistance.

Estimation of dose requirements for sustained in vivo activity of a therapeutic human anti-CD20 antibody.

We evaluated the dose requirements for sustained in vivo activity of ofatumumab, a human anti-CD20 antibody under development for the treatment of B cell-mediated diseases. In a mouse xenograft model, a single dose, resulting in an initial plasma antibody concentration of 5 microg/ml, which was expected to result in full target saturation, effectively inhibited human B-cell tumour development. Tumour growth resumed when plasma concentrations dropped below levels that are expected to result in half-maximal saturation. Notably, tumour load significantly impacted antibody pharmacokinetics. In monkeys, initial depletion of circulating and tissue residing B cells required relatively high-dose levels. Re-population of B-cell compartments, however, only became detectable when ofatumumab levels dropped below 10 microg/ml. We conclude that, once saturation of CD20 throughout the body has been reached by high initial dosing, plasma concentrations that maintain target saturation on circulating cells (5-10 microg/ml) are probably sufficient for sustained biological activity. These observations may provide a rationale for establishing dosing schedules for maintenance immunotherapy following initial depletion of CD20 positive (tumour) cells.

Immunological diagnosis of tuberculosis: problems and strategies for success.

Tuberculosis remains a serious social and public health problem, affecting millions of people annually. The bacille Calmette-Guerin (BCG) vaccine, used prophylactically, does not impede the progression of the disease, which usually manifests as decreased cellular immunity. Early diagnosis, together with polychemotherapy, can control the dissemination of the tuberculosis infection. The current diagnostic methods present certain problems. Such problems include the low sensitivity of sputum smear microscopy, the fact that performing microbiological cultures is quite time-consuming, and the low specificity of the skin test with the purified protein derivative of M. tuberculosis. New diagnostic methods, which use specific antigens such as the early secreted antigenic target 6-kDa and culture filtrate protein 10kDa, are being evaluated. The genes that encode these antigens are located in the DNA region of difference 1 of M. tuberculosis, M. africanum and M. bovis. However, they are absent from the M. bovis (BCG) and from most environmental mycobacteria. Diagnostic methods such as QuantiFERON-TB(R) and T SPOT.TB(R), which are based on the production of interferon-gamma by T lymphocytes, in response to those antigens, are being tested and have been found to outstrip the purified protein derivative skin test in the following characteristics: greater sensitivity; lower cross-reactivity due to BCG vaccination or infection with environmental mycobacteria; and execution time. The introduction of diagnostic methods that are more specific and sensitive, together with gaining a better understanding of the molecular and cellular mechanisms that regulate the parasite-host interaction, can increase the efficiency of strategies devised to combat tuberculosis.

Difficulties in avoiding exposure to allergens in cosmetics.

The aim of the study is to describe the ability of patients with allergic contact dermatitis to avoid exposure to allergens in cosmetics. The study is a questionnaire survey among 382 patients with contact allergy to preservatives and fragrances, included from 3 dermatological clinics. The questionnaire included questions about the level of difficulty in reading labels of ingredients on cosmetics and about patients' strategies to avoid substances they were allergic to. It also included questions about eczema severity as well as about educational level. 46% of the patients found it difficult or extremely difficult to read the ingredient labelling of cosmetics, and this finding was significantly related to low educational level. Patients allergic to formaldehyde and methyldibromo glutaronitrile experienced the worst difficulties, while patients with fragrance allergy found ingredient label reading easier than patients with preservative allergy. Reading of ingredient labels is a major problem for patients with contact allergy to allergens in consumer products. It is a general problem for all patients and not restricted to a small group with multiple allergies.

Diagnóstico imunológico da tuberculose: problemas e estratégias para o sucesso / Immunological diagnosis of tuberculosis: problems and strategies for success

A tuberculose continua sendo um grave problema social e de saúde, afetando milhões de pessoas anualmente. A vacina Bacille Calmette-Guerin (BCG), usada no controle profilático, é incapaz de conter a progressão da doença, que usualmente se manifesta através da queda da imunidade celular do indivíduo. O diagnóstico da tuberculose em seus estágios iniciais, aliado à poliquimioterapia, pode contribuir para o controle da disseminação da infecção. Os atuais métodos de diagnóstico apresentam problemas, como: baixa sensibilidade da baciloscopia; longo tempo de realização das culturas microbiológicas; e baixa especificidade do teste cutâneo com o derivado protéico purificado do M. tuberculosis. Novos métodos de diagnóstico que utilizam antígenos específicos (por exemplo, os conhecidos em inglês como o early secreted antigenic target 6-kDa e o culture filtrate protein 10-kDa), estão sendo testados. Os genes que codificam esses antígenos estão localizados na região de diferença 1 do M. tuberculosis, M. africanum e M. bovis, mas estão ausentes no M. bovis (BCG) e na maioria das micobactérias do meio ambiente. Métodos de diagnóstico baseados na produção de interferon-gama por linfócitos T, em resposta a esses antígenos, como o QuantiFERON-TB® e o T SPOT.TB®, estão sendo testados, e superam o teste cutâneo com o derivado protéico purificado nas seguintes características: maior sensibilidade; menor reatividade cruzada devido à vacinação com o BCG ou infecção por micobactérias do meio ambiente; e tempo de execução. A introdução de métodos de diagnóstico mais específicos e sensíveis, assim como um maior entendimento dos mecanismos moleculares e celulares que regulam a interação parasito-hospedeiro, pode contribuir para um eficiente combate à tuberculose.

Tuberculosis remains a serious social and public health problem, affecting millions of people annually. The bacille Calmette-Guerin (BCG) vaccine, used prophylactically, does not impede the progression of the disease, which usually manifests as decreased cellular immunity. Early diagnosis, together with polychemotherapy, can control the dissemination of the tuberculosis infection. The current diagnostic methods present certain problems. Such problems include the low sensitivity of sputum smear microscopy, the fact that performing microbiological cultures is quite time-consuming, and the low specificity of the skin test with the purified protein derivative of M. tuberculosis. New diagnostic methods, which use specific antigens such as the early secreted antigenic target 6-kDa and culture filtrate protein 10kDa, are being evaluated. The genes that encode these antigens are located in the DNA region of difference 1 of M. tuberculosis, M. africanum and M. bovis. However, they are absent from the M. bovis (BCG) and from most environmental mycobacteria. Diagnostic methods such as QuantiFERON-TB® and T SPOT.TB®, which are based on the production of interferon-gamma by T lymphocytes, in response to those antigens, are being tested and have been found to outstrip the purified protein derivative skin test in the following characteristics: greater sensitivity; lower cross-reactivity due to BCG vaccination or infection with environmental mycobacteria; and execution time. The introduction of diagnostic methods that are more specific and sensitive, together with gaining a better understanding of the molecular and cellular mechanisms that regulate the parasite-host interaction, can increase the efficiency of strategies devised to combat tuberculosis.

Prospective evaluation of a whole-blood test using Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP-10 for diagnosis of active tuberculosis.

A new immunodiagnostic test based on the Mycobacterium tuberculosis-specific antigens CFP-10/ESAT-6(QFT-RD1) has been launched as an aid in the diagnosis of latent tuberculosis (TB) infection (LTBI). The aim of this study was to evaluate this test for the diagnosis of active TB. Eighty-two patients with suspicion of TB and 39 healthy BCG-vaccinated persons were enrolled. Forty-eight had active TB, 25 did not, and 9 were excluded. Sensitivity and specificity of the test for active TB were evaluated in a prospective blinded manner in patients suspected of TB. The sensitivity of the QFT-RD1 was 85% (40/48; confidence interval [CI], 75 to 96), and it was higher than the sensitivity of microscopy, 42% (20/48; CI, 27 to 56; P = 0.001), and culture, 59% (27/46; CI, 44 to 73; P = 0.009). Of patients with extrapulmonary TB, 92% (12/13) were QFT-RD1 positive, whereas only 31% (4/13) were positive by microscopy and 42% (5/12) by culture (P < 0.05), and 87% (13/15) of those who were negative by both microscopy and culture were QFT-RD1 positive. By combining microscopy and culture with the QFT-RD1 test, sensitivity increased to 96% (CI, 90 to 102). Ten of 25 (40%) non-TB patients were QFT-RD1 positive, resulting in a specificity of 60%. However, 80% (8/10) of these had risk-factors for TB, indicating latent infection in this group. In healthy controls, only 3% (1/39) were QFT-RD1 positive. In conclusion, the QFT-RD1 test is sensitive for diagnosis of TB, especially in patients with negative microscopy and culture. The accuracy of the QFT-RD1 test will vary with the prevalence of LTBI. We suggest that the QFT-RD1 test could be a very useful supplementary tool for the diagnosis of TB.

Reactivation of tuberculosis during immunosuppressive treatment in a patient with a positive QuantiFERON-RD1 test.

A patient with polymyositis developed tuberculosis during immunosuppressive treatment. Tuberculin Skin Test and chest X-ray failed to demonstrate latent tuberculosis, whereas a blood sample that was tested with a modified QuantiFERON-TB-assay, using the recombinant ESAT-6 and CFP-10, was positive indicating that this patient was latently infected before immunosuppressive therapy. This case indicates the risk of progressing from latent to active tuberculosis given that the subject is RD1 responsive, and we believe that preventive anti-tuberculous treatment could have prevented this case of tuberculosis. We suggest that RD1 based tests are evaluated further in immunocompromised patients.

The hph-1 mouse: a model for dominantly inherited GTP-cyclohydrolase deficiency.

Dominantly inherited guanosine triphosphate (GTP)-cyclohydrolase deficiency, otherwise known as Segawa's disease or dopa-responsive dystonia, has a wide spectrum of phenotypic expression ranging from asymptomatic to very severe. Penetrance is more frequent in women as compared with men, and there is a variable occurrence of diurnal variation in symptom intensity. Biochemical characterization of the disease has demonstrated lower cerebrospinal fluid levels of tetrahydrobiopterin (BH4), neopterin, and homovanillic acid and low levels of tyrosine hydroxylase protein in the striatum. To investigate the pathophysiology, we have begun to characterize biogenic amine and BH4 metabolism in the GTP cyclohydrolase deficient hph-1 mouse. The data show low brain levels of BH4, catecholamines, serotonin, and their metabolites together with low levels of tyrosine hydroxylase protein within the striatum. The hph-1 mouse therefore provides a good model system in which to study the human disease.

Epitope mapping of the immunodominant antigen TB10.4 and the two homologous proteins TB10.3 and TB12.9, which constitute a subfamily of the esat-6 gene family.

The human T-cell recognition of the low-molecular-mass culture filtrate antigen TB10.4 was evaluated in detail. The molecule was strongly recognized by T cells isolated from tuberculosis (TB) patients and from BCG-vaccinated donors. The epitopes on TB10.4 were mapped with overlapping peptides and found to be distributed throughout the molecule. The broadest response was found in TB patients, whereas the response in BCG-vaccinated donors was focused mainly toward a dominant epitope located in the N terminus (amino acids 1 to 18). The gene encoding TB10.4 was found to belong to a subfamily within the esat-6 family that consists of the three highly homologous proteins TB10.4, TB10.3, and TB12.9 (Rv0288, Rv3019c, and Rv3017c, respectively). Southern blot analysis combined with database searches revealed that the three members of the TB10.4 family were present only in strains of the Mycobacterium tuberculosis complex, including BCG, and M. kansasii, whereas other atypical mycobacteria had either one (M. avium, M. intracellulare, and M. marinum) or none (M. scrofulaceum, M. fortuitum, and M. szulgai) of the genes. The fine specificity of the T-cell response to the three closely related esat-6 family members was markedly different, with only a few epitopes shared between the molecules. Minimal differences in the amino acid sequence translated into large differences in recognition by T cells and secretion of gamma interferon. In general, the peptides from TB10.4 stimulated the largest responses, but epitopes unique to both TB10.3 and TB12.9 were found. The relevance of the findings for TB vaccine development and as a potential mechanism for immune evasion is discussed.