Protection against diabetes and improved NK/NKT cell performance in NOD.NK1.1 mice congenic at the NK complex

The NK1.1 cell surface receptor, which belongs to the NKR-P1 gene cluster, has been bred onto nonobese diabetic (NOD) mice for two purposes. The first was to tag NK and NKT cells for easier experimental identification of those subsets and better analysis of their implication in type 1 diabetes. The second was to produce a congenic strain carrying Idd6, a susceptibility locus that has been repeatedly mapped in the vicinity of the NKR-P1 gene cluster and the NK complex, to explore the impact of this locus upon autoimmune diabetes. NOD.NK1.1 mice express the NK1.1 marker selectively on the surface of their NK and NKT cell subsets. In addition, the mice manifest reduced disease incidence and improved NK and NKT cell performance, as compared with wild-type NOD mice. The association of those two features in the same congenic strain constitutes a strong argument in favor of Idd6 being associated to the NK complex. This could explain at the same time the multiple alterations of innate immunity reported in NOD mice and the fact that disease onset can be readily modified by boosting the innate immune system of the mouse.     

Diversification and Distribution of Ruminant Chlamydia abortus Clones Assessed by MLST and MLVA

Chlamydia abortus, an obligate intracellular bacterium, is the most common infectious cause of abortion in small ruminants worldwide and has zoonotic potential. We applied multilocus sequence typing (MLST) together with multiple-locus variable-number tandem repeat analysis (MLVA) to genotype 94 ruminant C. abortus strains, field isolates and samples collected from 1950 to 2011 in diverse geographic locations, with the aim of delineating C. abortus lineages and clones. MLST revealed the previously identified sequence types (STs) ST19, ST25, ST29 and ST30, plus ST86, a recently-assigned type on the Chlamydiales MLST website and ST87, a novel type harbouring the hemN_21 allele, whereas MLVA recognized seven types (MT1 to MT7). Minimum-spanning-tree analysis suggested that all STs but one (ST30) belonged to a single clonal complex, possibly reflecting the short evolutionary timescale over which the predicted ancestor (ST19) has diversified into three single-locus variants (ST86, ST87 and ST29) and further, through ST86 diversification, into one double-locus variant (ST25). ST descendants have probably arisen through a point mutation evolution mode. Interestingly, MLVA showed that in the ST19 population there was a greater genetic diversity than in other STs, most of which exhibited the same MT over time and geographical distribution. However, the evolutionary pathways of C. abortus STs seem to be diverse across geographic distances with individual STs restricted to particular geographic locations. The ST30 singleton clone displaying geographic specificity and represented by the Greek strains LLG and POS was effectively distinguished from the clonal complex lineage, supporting the notion that possibly two separate host adaptations and hence independent bottlenecks of C. abortus have occurred through time. The combination of MLST and MLVA assays provides an additional level of C. abortus discrimination and may prove useful for the investigation and surveillance of emergent C. abortus clonal populations.     

Staying cool in a changing landscape: the influence of maximum daily ambient temperature on grizzly bear habitat selection

To fulfill their needs, animals are constantly making trade-offs among limiting factors. Although there is growing evidence about the impact of ambient temperature on habitat selection in mammals, the role of environmental conditions and thermoregulation on apex predators is poorly understood. Our objective was to investigate the influence of ambient temperature on habitat selection patterns of grizzly bears in the managed landscape of Alberta, Canada. Grizzly bear habitat selection followed a daily and seasonal pattern that was influenced by ambient temperature, with adult males showing stronger responses than females to warm temperatures. Cutblocks aged 0–20 years provided an abundance of forage but were on average 6 °C warmer than mature conifer stands and 21- to 40-year-old cutblocks. When ambient temperatures increased, the relative change (odds ratio) in the probability of selection for 0- to 20-year-old cutblocks decreased during the hottest part of the day and increased during cooler periods, especially for males. Concurrently, the probability of selection for 21- to 40-year-old cutblocks increased on warmer days. Following plant phenology, the odds of selecting 0- to 20-year-old cutblocks also increased from early to late summer while the odds of selecting 21- to 40-year-old cutblocks decreased. Our results demonstrate that ambient temperatures, and therefore thermal requirements, play a significant role in habitat selection patterns and behaviour of grizzly bears. In a changing climate, large mammals may increasingly need to adjust spatial and temporal selection patterns in response to thermal constraints.     

In vivo and in vitro analyses of toxic mutants of HET-s: FTIR antiparallel signature correlates with amyloid toxicity

The folding and interactions of amyloid proteins are at the heart of the debate as to how these proteins may or may not become toxic to their host. Although little is known about this issue, the structure seems to be clearly involved with effects on molecular events. To understand how an amyloid may be toxic, we previously generated a yeast toxic amyloid (mutant 8) from the nontoxic HET-s_(218-289) prion domain of Podospora anserina. Here, we performed a comprehensive structure-toxicity study by mutating individually each of the 10 mutations found in mutant 8. The study of the library of new mutants generated allowed us to establish a clear link between Fourier transform infrared antiparallel signature and amyloid toxicity. All of the mutants that form parallel β-sheets are not toxic. Double mutations may be sufficient to shift a parallel structure to antiparallel amyloids, which are toxic to yeast. Our findings also suggest that the toxicity of antiparallel structured mutants may be linked to interaction with membranes.     

Anti-cetuximab IgE ELISA for identification of patients at a high risk of cetuximab-induced anaphylaxis

Cetuximab, a chimeric mouse-human IgG1 monoclonal antibody against the epidermal growth factor receptor, has proven effective in the treatment of metastatic colorectal cancer and squamous cell carcinoma of the head and neck. However, a high incidence of immediate hypersensitivity reactions (HSR) to cetuximab after the first infusion has been observed. We have developed a test for identification of patients likely to show treatment-related HSR to cetuximab. An enzyme-linked immunosorbent assay (ELISA) for detecting anti-cetuximab IgEs was developed and tested on serum samples collected from cancer patients before start of cetuximab treatment, and from healthy blood donors. Similar levels of anti-cetuximab IgE were detected in pre-treatment patient sera (24/92, 26.1%) and sera from healthy blood donors (33/117, 28.2%). HSR were observed in 14 out of the 92 patients (15.2%), and 8 of these (57.1%) were grade 3–4. Anti-cetuximab IgEs were detected in 7/8 of the patients (87.5%) with severe HSRs as compared with 14/78 patients (17.9%) with no HSR (p = 0.0002). Predictive value of the anti-cetuximab IgE test for HSR events of grades 3–4 was calculated using Receiver Operating Characteristics analysis. With a cut-off value of 29 arbitrary units for the anti-cetuximab IgE, the ELISA test showed a sensitivity of 87.5%, specificity of 82.1%, positive predictive value of 33.3% and negative predictive value of 98.5%. Anti-cetuximab IgE ELISA detection could be a valuable tool to help the physician anticipate an anaphylaxis episode following cetuximab infusion and opt for a suitable alternative treatment.Key words: anti-cetuximab antibodies, ELISA, hypersensitivity, therapeutic monoclonal antibody, ROC     

Bisphosphonated fluoroquinolone esters as osteotropic prodrugs for the prevention of osteomyelitis

Osteomyelitis is a difficult to treat bacterial infection of the bone. Delivering antibacterial agents to the bone may overcome the difficulties in treating this illness by effectively concentrating the antibiotic at the site of infection and by limiting the toxicity that may result from systemic exposure to the large doses conventionally used. Using bisphosphonates as osteophilic functional groups, different forms of fluoroquinolone esters were synthesized and evaluated for their ability to bind bone and to release the parent antibacterial agent. Bisphosphonated glycolamide fluoroquinolone esters were found to present a profile consistent with effective and rapid bone binding and efficient release of the active drug moiety. They were assessed for their ability to prevent bone infection in vivo and were found to be effective when the free fluoroquinolones were not.     

Proteomic analysis of polymeric salivary mucins: no evidence for MUC19 in human saliva

MUC5B is the predominant polymeric mucin in human saliva [Thornton, Khan, Mehrotra, Howard, Veerman, Packer and Sheehan (1999) Glycobiology 9, 293-302], where it contributes to oral cavity hydration and protection. More recently, the gene for another putative polymeric mucin, MUC19, has been shown to be expressed in human salivary glands [Chen, Zhao, Kalaslavadi, Hamati, Nehrke, Le, Ann and Wu (2004) Am. J. Respir. Cell Mol. Biol. 30, 155-165]. However, to date, the MUC19 mucin has not been isolated from human saliva. Our aim was therefore to purify and characterize the MUC19 glycoprotein from human saliva. Saliva was solubilized in 4 M guanidinium chloride and the high-density mucins were purified by density-gradient centrifugation. The presence of MUC19 was investigated using tandem MS of tryptic peptides derived from this mucin preparation. Using this approach, we found multiple MUC5B-derived tryptic peptides, but were unable to detect any putative MUC19 peptides. These results suggest that MUC19 is not a major component in human saliva. In contrast, using the same experimental approach, we identified Muc19 and Muc5b glycoproteins in horse saliva. Moreover, we also identified Muc19 from pig, cow and rat saliva; the saliva of cow and rat also contained Muc5b; however, due to the lack of pig Muc5b genomic sequence data, we were unable to identify Muc5b in pig saliva. Our results suggest that unlike human saliva, which contains MUC5B, cow, horse and rat saliva are a heterogeneous mixture of Muc5b and Muc19. The functional consequence of these species differences remains to be elucidated.