Evidence for an accessory protein function for Toll-like receptor 1 in anti-bacterial responses

Members of the Toll-like receptor (TLR) family are components of the mammalian anti-microbial response, signaling with a domain closely related to that of IL-1 receptors. In this report the expression and function of TLR1, a TLR of unknown function, are examined. TLR1 is expressed by monocytes, as demonstrated using a novel mAb. Monocytes also express TLR2. TLR1 transfection of HeLa cells, which express neither TLR1 nor TLR2, was not sufficient to confer responsiveness to several microbial extracts. However, cotransfection of TLR1 and TLR2 resulted in enhanced signaling by HeLa cells to soluble factors released from Neisseria meningitidis relative to the response with either TLR alone. This phenomenon was also seen with high concentrations of some preparations of LPS. The N. meningitidis factors recognized by TLR1/TLR2 were not released by N. meningitidis mutant in the LpxA gene. Although LpxA is required for LPS biosynthesis, because cooperation between TLR1 and TLR2 was not seen with all LPS preparations, the microbial component(s) TLR1/2 recognizes is likely to be a complex of LPS and other molecules or a compound metabolically and chemically related to LPS. The functional IL-1R consists of a heterodimer; this report suggests a similar mechanism for TLR1 and TLR2, for certain agonists. These data further suggest that mammalian responsiveness to some bacterial products may be mediated by combinations of TLRs, suggesting a mechanism for diversifying the repertoire of Toll-mediated responses.     

Structure and polymorphism of the human gene for the interferon-induced p78 protein (MX1): evidence of association with alopecia areata in the Down syndrome region

Alopecia areata (AA) is a chronic inflammatory disease characterised by patchy hair loss with T cell infiltration of hair follicles. AA occurs in approximately 0.1% of the general population, but this is increased to 9% in Down syndrome (DS). DS is associated with an additional copy (full or partial) of chromosome 21, and the DS region may potentially include genes involved in the pathogenesis of AA. MX1 is the gene encoding the interferon-induced p78 protein (MxA). MxA protein confers resistance to influenza viruses, and we have previously shown that MxA protein is strongly expressed in lesional anagen hair bulbs from patients with AA but not in normal follicles. We therefore studied the possible involvement of MX1 in the pathogenesis of AA. To establish markers in the MX1 region which could be screened by PCR-based methods, we defined the human MX1 exon/intron organisation and screened the exons and the introns by conformation-sensitive gel electrophoresis. We found that the MX1 gene contains 17 exons extending over 33 kb. The size and sequence of the region from exon 6 to exon 16 are highly conserved between human and mouse. Screening of 4747 bp within the MX1 gene revealed four single nucleotide polymorphisms in intron 6. These polymorphisms are concentrated within 147 bp and show strong linkage disequilibrium. In a case-control association study for the MX1 (+9959) polymorphism in 165 AA patients and 510 controls we found a significant association of this marker with AA (odds ratio 1.79, 95% CI 1.21-2.66, chi2 = 8.464, P = 0.0036). The risk of disease was greater for patchy AA (mild disease) and with early age at onset (odds ratio 2.34, 95% CI 1.24-4.43, P = 0.0072), providing new evidence of genetic heterogeneity in AA. Our demonstration of genetic association between the MX1 gene and disease supports the hypothesis that this is a new candidate gene in AA.     

Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes.

The CCR5-Delta32 deletion obliterates the CCR5 chemokine and the human immunodeficiency virus (HIV)-1 coreceptor on lymphoid cells, leading to strong resistance against HIV-1 infection and AIDS. A genotype survey of 4,166 individuals revealed a cline of CCR5-Delta32 allele frequencies of 0%-14% across Eurasia, whereas the variant is absent among native African, American Indian, and East Asian ethnic groups. Haplotype analysis of 192 Caucasian chromosomes revealed strong linkage disequilibrium between CCR5 and two microsatellite loci. By use of coalescence theory to interpret modern haplotype genealogy, we estimate the origin of the CCR5-Delta32-containing ancestral haplotype to be approximately 700 years ago, with an estimated range of 275-1,875 years. The geographic cline of CCR5-Delta32 frequencies and its recent emergence are consistent with a historic strong selective event (e.g. , an epidemic of a pathogen that, like HIV-1, utilizes CCR5), driving its frequency upward in ancestral Caucasian populations.     

1-Thio-beta-D-galactofuranosides: synthesis and evaluation as beta-D- galactofuranosidase inhibitors

Beta-D-galactofuranosidase is a good chemotherapeutic target for the design of inhibitors, since beta-D-galactofuranose is a constituent of important parasite glycoconjugates but is not present in the host mammals. With this aim, we have synthesized for the first time alkyl, benzyl and aryl 1-thio-beta-D-galactofuranosides by condensation of penta-O-benzoyl-alpha,beta-D-galactofuranose with the corresponding thiols, in the presence of SnCl4as catalyst. The complete chemical and spectroscopical characterization of these compounds showed that the reaction was stereoselective. Debenzoylation with sodium methoxide afforded the beta-S-galactofuranosides in high yield. The thioglycosides were tested as inhibitors of the beta-D- galactofuranosidase of Penicillium fellutanum, using for the first time 4-nitrophenyl-beta-D-galactofuranoside as chromogenic substrate. The 4- aminophenyl-1-thio-beta-D-galactofuranoside, obtained by catalytic hydrogenation of the nitrophenyl derivative, was the best inhibitor being then an adequate ligand for the preparation of an affinity phase aimed at the isolation of beta-d-galactofuranosidases from different sources. Also the inhibitory activity of d-galactono-1, 4-lactone was shown.      

Regional differences in WT-1 and Tcf21 expression during ventricular development: implications for myocardial compaction

Background

Morphological and functional differences of the right and left ventricle are apparent in the adult human heart. A differential contribution of cardiac fibroblasts and smooth muscle cells (populations of epicardium-derived cells) to each ventricle may account for part of the morphological-functional disparity. Here we studied the relation between epicardial derivatives and the development of compact ventricular myocardium.

Results

Wildtype and Wt1^CreERT2/+ reporter mice were used to study WT-1 expressing cells, and Tcf21^lacZ/+ reporter mice and PDGFRα^-/-;Tcf21^LacZ/+ mice to study the formation of the cardiac fibroblast population. After covering the heart, intramyocardial WT-1+ cells were first observed at the inner curvature, the right ventricular postero-lateral wall and left ventricular apical wall. Later, WT-1+ cells were present in the walls of both ventricles, but significantly more pronounced in the left ventricle. Tcf21-^LacZ + cells followed the same distribution pattern as WT-1+ cells but at later stages, indicating a timing difference between these cell populations. Within the right ventricle, WT-1+ and Tcf21-lacZ+ cell distribution was more pronounced in the posterior inlet part. A gradual increase in myocardial wall thickness was observed early in the left ventricle and at later stages in the right ventricle. PDGFRα^-/-;Tcf21^LacZ/+ mice showed deficient epicardium, diminished number of Tcf21-^LacZ + cells and reduced ventricular compaction.

Conclusions

During normal heart development, spatio-temporal differences in contribution of WT-1 and Tcf21-^LacZ + cells to right versus left ventricular myocardium occur parallel to myocardial thickening. These findings may relate to lateralized differences in ventricular (patho)morphology in humans.     

Phenothiazine-mediated rescue of cognition in tau transgenic mice requires neuroprotection and reduced soluble tau burden

Background

It has traditionally been thought that the pathological accumulation of tau in Alzheimer's disease and other tauopathies facilitates neurodegeneration, which in turn leads to cognitive impairment. However, recent evidence suggests that tau tangles are not the entity responsible for memory loss, rather it is an intermediate tau species that disrupts neuronal function. Thus, efforts to discover therapeutics for tauopathies emphasize soluble tau reductions as well as neuroprotection.

Results

Here, we found that neuroprotection alone caused by methylene blue (MB), the parent compound of the anti-tau phenothiaziazine drug, Rember?, was insufficient to rescue cognition in a mouse model of the human tauopathy, progressive supranuclear palsy (PSP) and fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP17): Only when levels of soluble tau protein were concomitantly reduced by a very high concentration of MB, was cognitive improvement observed. Thus, neurodegeneration can be decoupled from tau accumulation, but phenotypic improvement is only possible when soluble tau levels are also reduced.

Conclusions

Neuroprotection alone is not sufficient to rescue tau-induced memory loss in a transgenic mouse model. Development of neuroprotective agents is an area of intense investigation in the tauopathy drug discovery field. This may ultimately be an unsuccessful approach if soluble toxic tau intermediates are not also reduced. Thus, MB and related compounds, despite their pleiotropic nature, may be the proverbial "magic bullet" because they not only are neuroprotective, but are also able to facilitate soluble tau clearance. Moreover, this shows that neuroprotection is possible without reducing tau levels. This indicates that there is a definitive molecular link between tau and cell death cascades that can be disrupted.

     

<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> inhibits <Emphasis Type="Italic">in-vitro Candida</Emphasis> biofilm development

Background  

Elucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as Candida spp. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species biofilm development.     

Collapsin response mediator protein-2 hyperphosphorylation is an early event in Alzheimer's disease progression

Collapsin response mediator protein 2 (CRMP2) is an abundant brain-enriched protein that can regulate microtubule assembly in neurons. This function of CRMP2 is regulated by phosphorylation by glycogen synthase kinase 3 (GSK3) and cyclin-dependent kinase 5 (Cdk5). Here, using novel phosphospecific antibodies, we demonstrate that phosphorylation of CRMP2 at Ser522 (Cdk5-mediated) is increased in Alzheimer's disease (AD) brain, while CRMP2 expression and phosphorylation of the closely related isoform CRMP4 are not altered. In addition, CRMP2 phosphorylation at the Cdk5 and GSK3 sites is increased in cortex and hippocampus of the triple transgenic mouse [presenilin-1 (PS1)(M146V)KI; Thy1.2-amyloid precursor protein (APP)(swe); Thy1.2tau(P301L)] that develops AD-like plaques and tangles, as well as the double (PS1(M146V)KI; Thy1.2-APP(swe)) transgenic mouse. The hyperphosphorylation is similar in magnitude to that in human AD and is evident by 2 months of age, ahead of plaque or tangle formation. Meanwhile, there is no change in CRMP2 phosphorylation in two other transgenic mouse lines that display elevated amyloid beta peptide levels (Tg2576 and APP/amyloid beta-binding alcohol dehydrogenase). Similarly, CRMP2 phosphorylation is normal in hippocampus and cortex of Tau(P301L) mice that develop tangles but not plaques. These observations implicate hyperphosphorylation of CRMP2 as an early event in the development of AD and suggest that it can be induced by a severe APP over-expression and/or processing defect.