Convergence of sensory pathways in the development of somatic and visceral hypersensitivity

Sensory neurons innervating different tissues converge onto second-order neurons in the spinal cord. We examined whether inflammation or transient overexpression of nerve growth factor (NGF) in one tissue triggers hypersensitivity in referral sites. Thresholds to mechanical and thermal stimulation of the hindpaw, visceromotor responses to colorectal distension, and cystometrograms were performed in appropriate controls and mice with experimentally induced cystitis, inflammation of the hindpaw or front paw, or injection of viral vectors encoding NGF or green fluorescent protein (GFP). Cystitis and NGF but not GFP overexpression in the bladder triggered bladder hyperactivity associated with mechanical and thermal hypersensitivity in cutaneous referral sites and enhanced responses to colorectal distension. Hindpaw inflammation and injection of the NGF- but not GFP-encoding viral vector or front paw inflammation induced mechanical and thermal hyperalgesia in the affected hindpaw and increased responses to colorectal distension without altering the micturition reflex. In conclusion, sensitization of sensory pathways by inflammation or NGF contributes to the development of hypersensitivity in neighboring organs and cutaneous referral sites and provides a potential mechanism underlying the coexistence of pain syndromes in patients with functional diseases.     

Characterization of the γδ T cell response to acute leukemia

Background: Previous work from our center has suggested a correlation between increased donor-derived Vδ1+ γδ T cells and long-term relapse-free survival following bone marrow transplantation for leukemia. Questions remain, however, as to whether this observation can be explained by a γδ T cell-based immune response against primary leukemia. Methods: We examined γδ T cell receptor (TCR) phenotype, cell proliferation, and cytolytic activity following culture with irradiated primary leukemia blasts from a haploidentical first-degree relative. Subsequently, we also studied the γδ TCR phenotype and complimentarity determining region 3 (CDR3) cDNA sequences from 17 newly diagnosed leukemia patients. Results: In 17/28 (61%) of in vitro cultures, γδ T cells proliferated in culture with primary blasts. Vδ1+ T cells were proportionally increased in all cultures and were the predominant cell population in 6/17. In the 7 cultures where cytotoxicity could be assessed, 6 (86%) showed some degree of cytotoxicity to the primary leukemia. Vδ1+ T cells were also the predominant γδ T cell subtype in pre-treatment leukemia patients principally due to loss of Vδ2+ T cells rather than expansion of Vδ1+ cells. The Vδ1 CDR3-region cDNA sequence from these patients revealed exclusive use of the Jδ1 constant region and sequence conservation in 4/11 patients. Conclusions: γδ T cells exhibit an in vitro response to primary leukemia blasts that is manifested by proliferation, an increased proportion of Vδ1+ T cells, and cytotoxicity to the primary leukemia blasts. The Vδ1+ T cell population is also predominant in newly diagnosed leukemia patients likely due to a loss of circulating Vδ2+ T cells. A small proportion of newly diagnosed patients showed Vδ1 CDR3 region similarity. These findings suggest a role for γδ T cells in the immune response to leukemia.Paul F. Meeh and Michelle King are contributed equally to this work.     

Characterisation of the binding of amyloid imaging tracers to rodent Abeta fibrils and rodent-human Abeta co-polymers

Despite the application of amyloid imaging agents such as PIB, SB13, and FDDNP in Alzheimer's disease (AD) patients, the successful use of these agents in transgenic mice models of AD has not been reported to date. As a first step in understanding the behaviour of these ligands in transgenic models of AD, we have investigated in a series of in vitro ligand binding assays the interaction of selected agents, including PIB, FDDNP, SB13, and BSB, with amyloid fibrils produced from rodent Abeta(1-40) (roAbeta) peptide. The data indicate that the ligand binding affinities together with the pattern and number of binding sites on the roAbeta fibrils are broadly conserved with that reported previously for human Abeta(1-40) (huAbeta) fibrils. However, characterisation of huAbeta fibrils formed in the presence of increasing amounts of roAbeta (1, 5, 10% w/w) demonstrated a dose-dependent reduction in the number of high affinity [(3)H]Me-BTA-1 binding sites such that at the highest amount of roAbeta the specific signal was reduced by approximately 95%. These studies suggest that (i) the presence of small amounts of roAbeta in huAbeta fibrils has the potential to cause subtle ultrastructural alterations in the polymers and (ii) the weak binding signal observed in vivo in the transgenic mouse models of AD may in part be due to the decreased number of high affinity binding sites on the Abeta fibrils.     

Retroelement genome painting: cytological visualization of retroelement expansions in the genera Zea and Tripsacum

Divergence of abundant genomic elements among the Zea and Tripsacum genera was examined cytologically and a tool kit established for subsequent studies. The LTR regions from the CRM, Huck, Grande, Prem1, Prem2/Ji, Opie, Cinful-1, and Tekay retroelement families were used as FISH probes on mitotic chromosome spreads from a "trispecies" hybrid containing chromosomes from each of three species: Zea mays (2n = 20), Z. diploperennis (2n = 20), and Tripsacum dactyloides (2n = 36). Except for Tekay, which painted both Zea and Tripsacum chromosomes with nearly equal intensity, the retroelement probes hybridized strongly to the Zea chromosomes, allowing them to be distinguished from those of Tripsacum. Huck and Grande hybridized more intensely to maize than to Z. diploperennis chromosomes. Tripsacum genomic clones containing retroelement sequences were isolated that specifically paint Tripsacum chromosomes. The retroelement paints proved effective for distinguishing different genomes in interspecific hybrids and visualizing alien chromatin from T. dactyloides introgressed into maize lines. Other FISH probes (180-bp knob, TR-1, 5S, NOR, Cent4, CentC, rp1, rp3, and alpha-ZeinA) could be simultaneously visualized with the retroelement probes, emphasizing the value of the retroelement probes for cytogenetic studies of Zea and Tripsacum.     

Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers

Heat shock proteins (HSPs) help maintain cellular function in stressful situations, but the processes controlling their interactions with target proteins are not well defined. This study examined the binding of HSP72, HSP25, and αB-crystallin in skeletal muscle fibers following various stresses. Rat soleus (SOL) and extensor digitorum longus (EDL) muscles were subjected in vitro to heat stress or strongly fatiguing stimulation. Superficial fibers were "skinned" by microdissection and HSP diffusibility assessed from the extent of washout following 10- to 30 min exposure to a physiological intracellular solution. In fibers from nonstressed (control) SOL muscle, >80% of each HSP is readily diffusible. However, after heating a muscle to 40°C for 30 min ～95% of HSP25 and αB-crystallin becomes tightly bound at nonmembranous myofibrillar sites, whereas HSP72 bound at membranous sites only after heat treatment to ≥44°C. The ratio of reduced to oxidized cytoplasmic glutathione (GSH:GSSG) decreased approximately two- and fourfold after heating muscles to 40° and 45°C, respectively. The reducing agent dithiothreitol reversed HSP72 binding in heated muscles but had no effect on the other HSPs. Intense in vitro stimulation of SOL muscles, sufficient to elicit substantial oxidation-related loss of maximum force and approximately fourfold decrease in the GSH:GSSG ratio, had no effect on diffusibility of any of the HSPs. When skinned fibers from heat-treated muscles were bathed with additional exogenous HSP72, total binding increased approximately two- and 10-fold, respectively, in SOL and EDL fibers, possibly reflective of the relative sarco(endo)plasmic reticulum Ca(2+)-ATPase pump densities in the two fiber types. Phosphorylation at Ser59 on αB-crystallin and Ser85 on HSP25 increased with heat treatment but did not appear to determine HSP binding. The findings highlight major differences in the processes controlling binding of HSP72 and the two small HSPs. Binding was not directly related to cytoplasmic oxidative status, but oxidation of cysteine residues influenced HSP72 binding.     

Lateral clustering of TLR3:dsRNA signaling units revealed by TLR3ecd:3Fabs quaternary structure

Toll-like receptor 3 (TLR3) recognizes dsRNA and initiates an innate immune response through the formation of a signaling unit (SU) composed of one double-stranded RNA (dsRNA) and two TLR3 molecules. We report the crystal structure of human TLR3 ectodomain (TLR3ecd) in a quaternary complex with three neutralizing Fab fragments. Fab15 binds an epitope that overlaps the C-terminal dsRNA binding site and, in biochemical assays, blocks the interaction of TLR3ecd with dsRNA, thus directly antagonizing TLR3 signaling through inhibition of SU formation. In contrast, Fab12 and Fab1068 bind TLR3ecd at sites distinct from the N- and C-terminal regions that interact with dsRNA and do not inhibit minimal SU formation with short dsRNA. Molecular modeling based on the co-structure rationalizes these observations by showing that both Fab12 and Fab1068 prevent lateral clustering of SUs along the length of the dsRNA ligand. This model is further supported by cell-based assay results using dsRNA ligands of lengths that support single and multiple SUs. Thus, their antagonism of TLR3 signaling indicates that lateral clustering of SUs is required for TLR3 signal transduction.     

The design, synthesis, and biological evaluation of potent receptor tyrosine kinase inhibitors

Variously substituted indolin-2-ones were synthesized and evaluated for activity against KDR, Flt-1, FGFR-1 and PDGFR. Extension at the 5-position of the oxindole ring with ethyl piperidine (compound 7i) proved to be the most beneficial for attaining both biochemical and cellular potencies. Further optimization of 7i to balance biochemical and cellular potencies with favorable ADME/ PK properties led to the identification of 8h, a compound with a clean CYP profile, acceptable pharmacokinetic and toxicity profiles, and robust efficacy in multiple xenograft tumor models.     

The contribution of Plasmodium chabaudi to our understanding of malaria

Malaria kills close to a million people every year, mostly children under the age of five. In the drive towards the development of an effective vaccine and new chemotherapeutic targets for malaria, field-based studies on human malaria infection and laboratory-based studies using animal models of malaria offer complementary opportunities to further our understanding of the mechanisms behind malaria infection and pathology. We outline here the parallels between the Plasmodium chabaudi mouse model of malaria and human malaria. We will highlight the contribution of P. chabaudi to our understanding of malaria in particular, how the immune response in malaria infection is initiated and regulated, its role in pathology, and how immunological memory is maintained. We will also discuss areas where new tools have opened up potential areas of exploration using this invaluable model system.     

Structure of the Ulster Strain Newcastle Disease Virus Hemagglutinin-Neuraminidase Reveals Auto-Inhibitory Interactions Associated with Low Virulence

Paramyxovirus hemagglutinin-neuraminidase (HN) plays roles in viral entry and maturation, including binding to sialic acid receptors, activation of the F protein to drive membrane fusion, and enabling virion release during virus budding. HN can thereby directly influence virulence and in a subset of avirulent Newcastle disease virus (NDV) strains, such as NDV Ulster, HN must be proteolytically activated to remove a C-terminal extension not found in other NDV HN proteins. Ulster HN is 616 amino acids long and the 45 amino acid C-terminal extension present in its precursor (HN₀) form has to be cleaved to render HN biologically active. Here we show that Ulster HN contains an inter-subunit disulfide bond within the C-terminal extension at residue 596, which regulates HN activities and neuraminidase (NA) domain dimerization. We determined the crystal structure of the dimerized NA domain containing the C-terminal extension, which extends along the outside of the sialidase β-propeller domain and inserts C-terminal residues into the NA domain active site. The C-terminal extension also engages a secondary sialic acid binding site present in NDV HN proteins, which is located at the NA domain dimer interface, that most likely blocks its attachment function. These results clarify how the Ulster HN C-terminal residues lead to an auto-inhibited state of HN, the requirement for proteolytic activation of HN₀ and associated reduced virulence.