Toll-like receptor 9 contributes to recognition of Mycobacterium bovis Bacillus Calmette-Guérin by Flt3-ligand generated dendritic cells

Recognition of mycobacteria by the innate immune system is essential for the development of an adaptive immune response. Mycobacterial antigens stimulate antigen presenting cells (APCs) through distinct Toll-like receptors (TLRs) resulting in rapid activation of the innate immune system. The role of TLRs during infection with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) has been evaluated for TLR2 and TLR4 only. Surprisingly, despite the fact that immune stimulatory CpG-motifs have been originally derived from BCG, for the vaccine strain the role of TLR9 has not been addressed before. To identify the set of TLRs involved in the recognition of BCG, we infected bone marrow-derived macrophages and bone marrow-derived dendritic cells (Flt3-ligand generated DCs) from TLR2, TLR3, TLR4, TLR7, TLR9, MyD88 knockout, TLR2/4 and TLR2/4/9 multiple knockout mice. The degree of activation and stimulation was determined by TNFα, IL-6 and IL-12p40 ELISA. Activation of DCs was measured by surface expression of the costimulatory molecule CD86. We observed the most dramatic reduction of the inflammatory response for TLR2-deficient antigen presenting cells. Both macrophages and DCs produce markedly decreased amounts of TNFα and IL-6 in the absence of TLR2 whereas no significant reduction could be observed for TLR3, 4, 7, 9 single TLR-knockouts. However, IL-12 production in DCs appears not exclusively dependent on TLR2 and only in TLR2/4/9-deficient DCs BCG-induced IL-12 is reduced to background levels. Similarly, up-regulation of CD86 is abolished only in TLR2/4/9-deficient DCs supporting a role of TLR9 in the recognition of M. bovis BCG by murine dendritic cells.     

Assessment of the genetic causes of recessive childhood non-syndromic deafness in the UK - implications for genetic testing

Approximately one in 2000 children is born with a genetic hearing impairment, mostly inherited as a non-syndromic, autosomal recessive trait, for which more than 30 different genes have been identified. Previous studies have shown that one of these genes, connexin 26 (GJB2), accounts for 30-60% of such deafness, but the relative contribution of the many other genes is not known, especially in the outbred UK population. This lack of knowledge hampers the development of diagnostic genetic services for deafness. In an effort to determine the molecular aetiology of deafness in the population, 142 sib pairs with early-onset, non-syndromic hearing impairment were recruited. Those in whom deafness could not be attributed to GJB2 mutations were investigated further for other mapped genes. The genetic basis of 55 cases (38.7%) was established, 33.1% being due to mutations in the GJB2 gene and 3.5% due to mutations in SLC26A4. None of the remaining 26 loci investigated made a significant contribution to deafness in a Caucasian population. We suggest that screening the GJB2 and SLC26A4 genes should form the basis of any genetic testing programme for childhood deafness and highlight a number of important issues for consideration and future work.     

A fast perfusion system for single cell physiology optimized for microscopes with water immersion objectives

A perfusion system was constructed which allows the fast application of different solutes underneath a water immersion objective. The perfusion system is mounted into the immersion objective by milling a slot into the frontal metal plate of the lens holder. It consists of a five-channel pipette fixed to the objective and solution reservoirs gated by computer controlled magnetic valves. Up to five different solutions can be applied to the specimen under study. The solution between objective and specimen is completely exchanged after 1–2 s as determined from fluorescence measurements. This arrangement is optimized for [Ca²⁺] measurements with a fluorescence measurement system in tissue slices, where upright microscopes are required. It offers the advantage of saving a micromanipulator for the perfusion pipette and facilitates a fast, reproducible and precise positioning of the perfusion system.     

Alcoholism and drug abuse in three groups — bipolar I, unipolars and their acquaintances

Objective: Previous work has shown that manic-depressive illness and alcohol abuse are linked. This study further explores the relationship of alcohol and drug abuse in bipolar I patients and unipolar depressives and a comparison group obtained through the acquaintance method. Method: Diagnosis was accomplished according to Research Diagnostic Criteria (RDC): controls=469; bipolars=277; unipolar depressives=678. Systematic data were gathered using the SADS on lifetime and current drug abuse and alcoholism. Both patients and comparison subjects were then followed prospectively for 10 years. First degree family members were interviewed using the RDC family history method. Results: The group of bipolar patients and the group of unipolar patients had higher rates of drug and alcohol abuse than the comparison group when primary and secondary affective disorder patients were combined. However, primary unipolar patients did not have higher rates of alcohol or drug abuse than the comparison group. In contrast, primary bipolar patients had higher rates of alcoholism, stimulant abuse, and ever having abused a drug than the primary unipolar group and the control group. In an evaluation of the bipolar patients, drug abusers were significantly younger at intake and had a significantly younger age of onset of bipolar disorder. There was a significant increase in family history of mania or schizoaffective mania in the drug-abusing bipolar patients as compared to the non-abusing bipolar patients. Limitation: As in all adult samples of patients with affective illness, the chronology of alcohol and substance problems vis-à-vis the onset of illness was determined retrospectively. Conclusions: (1) Alcoholism and drug abuse are more frequent in bipolar than unipolar patients. (2) The drug abuse of bipolar patients tends toward the abuse of stimulant drugs. (3) In a bipolar patient, familial diathesis for mania is significantly associated with the abuse of alcohol and drugs. (4) More provocatively, these findings suggest the hypothesis of a common familial-genetic diathesis for a subtype of bipolar I, alcohol and stimulant abuse. Clinical implications: The present analyses, coupled with two previous ones from the CDS, suggest that drug abuse may precipitate an earlier onset of bipolar I disorder in those who already have a familial predisposition for mania. Furthermore, in dually diagnosed patients with manic-depressive and alcohol/stimulant abuse history, mood stabilization of the bipolar disorder represents a rational approach to control concurrent alcohol and drug problems, and should be studied in systematic controlled trials.     

Caspase inhibitors induce a switch from apoptotic to proinflammatory signaling in CD95-stimulated T lymphocytes

CD95 is a major apoptosis receptor that induces caspase activation and programmed cell death in susceptible cells. CD95-induced apoptosis can be blocked by peptidic caspase inhibitors such as benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone or Ile-Glu-Thr-Asp-fluoromethyl ketone. Here we show that stimulation of CD95 in the presence of these inhibitors induces necrosis and expression of various proinflammatory cytokines in primary T lymphocytes, such as TNF-alpha, IFN-gamma and granulocyte/macrophage colony-stimulating factor. In the absence of caspase inhibition CD95 stimulation did not result in cytokine expression, indicating that this proinflammatory signaling pathway is suppressed by active caspases. Further analysis with A3.01 T cells revealed that the proinflammatory signaling activity of CD95 was mediated by MEK/ERK, p38 and NF-kappaB signaling pathways. These findings point to a pivotal role of caspases not only as mediators of apoptosis but also as enzymes that prevent proinflammatory signaling during CD95-induced apoptosis. Moreover, our findings may be useful for the development of novel pharmacological strategies.     

Transgenic rat model of Huntington's disease

Huntington's disease (HD) is a late manifesting neurodegenerative disorder in humans caused by an expansion of a CAG trinucleotide repeat of more than 39 units in a gene of unknown function. Several mouse models have been reported which show rapid progression of a phenotype leading to death within 3-5 months (transgenic models) resembling the rare juvenile course of HD (Westphal variant) or which do not present with any symptoms (knock-in mice). Owing to the small size of the brain, mice are not suitable for repetitive in vivo imaging studies. Also, rapid progression of the disease in the transgenic models limits their usefulness for neurotransplantation. We therefore generated a rat model transgenic of HD, which carries a truncated huntingtin cDNA fragment with 51 CAG repeats under control of the native rat huntingtin promoter. This is the first transgenic rat model of a neurodegenerative disorder of the brain. These rats exhibit adult-onset neurological phenotypes with reduced anxiety, cognitive impairments, and slowly progressive motor dysfunction as well as typical histopathological alterations in the form of neuronal nuclear inclusions in the brain. As in HD patients, in vivo imaging demonstrates striatal shrinkage in magnetic resonance images and a reduced brain glucose metabolism in high-resolution fluor-deoxy-glucose positron emission tomography studies. This model allows longitudinal in vivo imaging studies and is therefore ideally suited for the evaluation of novel therapeutic approaches such as neurotransplantation.