Induction of triggering receptor expressed on myeloid cells (TREM-1) in airway epithelial cells by 1,25(OH)₂ vitamin D₃

The airway epithelium plays a role in host defense through the binding of innate immune receptors, which leads to the activation of inflammatory mediators, including antimicrobial peptides. The active form of vitamin D, 1,25(OH)(2)D(3), induces the expression of the antimicrobial peptide LL-37 in both myeloid cells and airway epithelial cells (AEC). Here, we demonstrate that mRNA encoding triggering receptor expressed on myeloid cells (TREM)-1 was induced up to 12-fold by 1,25(OH)(2)D(3) in normal human bronchial epithelial (NHBE) cells and in well-differentiated cultures of six airway epithelial cell lines from patients with cystic fibrosis and healthy individuals. TREM-2 and DAP12 were also expressed in airway cultures, but not induced by vitamin D. Induction occurs through a vitamin D response element identified in its proximal promoter region, and was regulated by PU.1 expressed in the AEC. Activation of TREM-1 by a cross-linking antibody led to an induction of both human β-defensin-2 and TNF-α mRNA, demonstrating its functionality in these cells. Our results expand on the role played by the airway epithelium in innate immunity and suggest that vitamin D can modulate the innate immune defense of the airway epithelium, and could potentially be developed as an adjunctive therapy for airway infections.     

Impact of the UK colorectal cancer screening pilot studies on incidence, stage distribution and mortality trends

Objective: To assess the impact of the UK colorectal cancer guaiac faecal occult blood test screening pilot studies on incidence trends, stage distribution and mortality trends. Design: Ecological study. Setting: Scotland and the West Midlands. Data: We extracted anonymised colorectal cancer (ICD-10 C18–C20) registration (1982–2006) and death records (1982–2007), along with corresponding mid-year population estimates. Intervention: Residents of the screening pilot areas, in the age group 50–69 years, were offered biennial guaiac faecal occult blood test screening from 2000 onwards. Screening was not offered routinely in non-pilot areas until the start of the roll-out of the national screening programmes in England and in Scotland in 2006 and 2007, respectively. Main outcome measures: We analysed trends in age-specific incidence and mortality rates, and Dukes’ stage distribution. Within each country/region, we compared the screening pilot areas to non-screening pilot (‘control’) areas using Chi square tests and Poisson regression modelling. Results: Following the start of the screening pilots, as expected in the prevalent round of a new screening programme, in the pilot areas there was a short-lived increase in incidence of colorectal cancer among 50–69 year olds except for females in the West Midlands. A trend towards earlier stage and less advanced disease was also observed, with males showing significant increases in Dukes’ A and corresponding decreases in Dukes’ C in the screening pilot areas (all P < 0.03). With the exception of females in the West Midlands, mortality rates for colorectal cancer decreased significantly and at a faster rate in the populations invited for screening. Conclusion: The existence of a natural control population not yet invited for screening provided a unique opportunity to assess whether the benefits of colorectal cancer screening, beyond the setting of a randomised controlled trial, could be detected using routinely collected statistics. Our analysis suggests that screening will fulfil its aim of reducing mortality from colorectal cancer.     

1-Benzyloxy-4,5-dihydro-1H-imidazol-2-yl-amines,a novel class of NR1/2B subtype selective NMDA receptor antagonists

Screening of the Roche compound depository led to the identification of (1-benzyloxy-4,5-dihydro-1H-imidazol-2-yl)-butyl amine 4, a structurally novel NR1/2B subtype selective NMDA receptor antagonist. The structure-activity relationships developed in this series resulted in the discovery of a novel class of potent and selective NMDA receptor blockers displaying activity in vivo.     

Heelstrike and the pathomechanics of osteoarthrosis: a pilot gait study

Involvement of mechanical factors in osteoarthrosis (OA) has been well documented. For OA of the human lower limb, the impulse imparted at heelstrike has been suggested as a pathogenic factor. It has also been reported that there is a large amount of variation in the level of impulse experienced by different individuals, and it is suggested that those who experience large impulses are at a greater risk of developing OA. The current study investigated gait patterns of 12 normal subjects to establish the gait determinants responsible for producing large impulses at heelstrike. The results suggest that subtle variations in the early part of the swing phase pattern are responsible for large differences in the impulse experienced at heelstrike; the usually reported gait variables mask these variations.     

Orientation of collagen at the osteocyte lacunae in human secondary osteons

This work characterizes an aspect of human bone micro-structure, pertinent to fracture initiation and arrest. It addresses how the orientation of elementary components proximate to osteocyte lacunae influences secondary osteon micro-biomechanics. New data at the perilacunar region concerning orientation of collagen-apatite, and prior data on collagen orientation outside the perilacunar region, are incorporated in a novel simulation of osteons to investigate how orientation relates to strains and stresses during mechanical testing. The perilacunar region was observed by confocal microscopy within single lamellar specimens, isolated from osteons. The specimens were separated by extinct or bright appearance in transverse section under circularly polarizing light. This is because synchrotron diffraction and confocal microscopy had established that each type, away from the perilacunar region, corresponds to specific dominant collagen orientation (extinct lamellae's dominant collagen forming small angles with the original osteon axis, while the bright lamellae's forms larger angles). Morphometry of serial confocal images of each perilacunar region showed collagen orientation generally following the orientation of canaliculi, circumambiently-perpendicular to the lacuna. The lacunae tilted relative to the lamellar walls were more numerous in extinct than in bright lamella. Their apices were less likely in extinct than bright lamella to show collagen following the canalicular orientation. The simulation of osteocyte lacunae in osteons, under tension or compression loading, supports the hypothesis that collagen orientation affects strains and stresses at the equatorial perilacunar region in conjunction with the presence of the lacuna. We further conjecture that collagen orientation diverts propagation of micro-cracks initiating from apices.     

Single-chain TNF, a TNF derivative with enhanced stability and antitumoral activity

The inflammatory and proapoptotic cytokine TNF possesses a compelling potential as an antitumoral therapeutic agent. Possible target cells include the malignant cells themselves, the tumor vasculature, or the immune system. As the clinical use of TNF is limited by systemic toxicity, targeting strategies using TNF-based fusion proteins are currently used. A major obstacle, however, is that homotrimeric TNF ligands are prone to activity loss due to dissociation into their monomers. In this study, we report the construction of single-chain TNF molecule, a TNF mutant consisting of three TNF monomers fused by short peptide linkers. In comparison to wild-type TNF, single-chain TNF was found to possess increased stability in vitro and in vivo, displayed reduced systemic toxicity yet slightly enhanced antitumoral activity in mouse models. Creation of single-chain variants is a new approach for improvement of functional activity of therapeutics based on TNF family ligands.     

Cutting edge: FimH adhesin of type 1 fimbriae is a novel TLR4 ligand

Several TLR ligands of bacterial origin induce innate immune responses. Although FimH, the adhesin portion of type 1 fimbria, plays an important role in the pathogenicity of some gram-negative bacteria, its ability to stimulate the innate immune system via TLR signaling remains unclear. In this study we report that FimH induces potent innate responses in a MyD88-dependent fashion. The FimH-induced innate activity was restricted to cells expressing TLR4. In addition, FimH was able to bind directly to TLR4. More importantly, cells unresponsive to LPS were responsive to FimH and the presence or absence of MD-2 and CD14 had no effect on FimH activity. Our data suggest that TLR4 is a functional receptor for the adhesin portion of bacterial type 1 fimbria.     

Role of Fyn and PI3K in H2O2-induced inhibition of apical Cl-/OH- exchange activity in human intestinal epithelial cells

H(2)O(2) is a highly reactive oxygen metabolite that has been implicated as an important mediator of inflammation-induced intestinal injury associated with ischaemia/reperfusion, radiation and inflammatory bowel disease. Previous studies have shown that H(2)O(2) inhibits NaCl absorption and activates Cl(-) secretion in the rat and rabbit colon. To date, however, almost no information is available with respect to its effect on the human intestinal apical anion exchanger Cl(-)/OH(-) (HCO(3)(-)). The present studies were, therefore, undertaken to examine the direct effects of H(2)O(2) on OH(-) gradient-driven DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate)-sensitive (36)Cl(-) uptake utilizing a post-confluent transformed human intestinal epithelial cell line, Caco-2. Our results demonstrate that H(2)O(2) (1 mM for 60 min) significantly inhibited (approx. 60%; P<0.05) Cl(-)/OH(-) exchange activity in Caco-2 cells. H(2)O(2)-mediated inhibition of Cl(-)/OH(-) exchange activity involved the Src kinase Fyn and PI3K (phosphoinositide 3-kinase)-dependent pathways. H(2)O(2) also induced phosphorylation of Fyn and p85 (the regulatory subunit of PI3K) in Caco-2 cells. Moreover, an increased association of Fyn and p85 was observed in response to H(2)O(2), resulting in the activation of the downstream target PLCgamma1 (phospholipase Cgamma1). Elevated intracellular Ca(2+) levels and PKCalpha (protein kinase Calpha) functioned as downstream effectors of H(2)O(2)-induced PLCgamma1 activation. Our results, for the first time, provide evidence for H(2)O(2)-induced Src kinase Fyn/PI3K complex association. This complex association resulted in the subsequent activation of PLCgamma1 and Ca(2+)-dependent PKCalpha, resulting in the inhibition of Cl(-)/OH(-) exchange activity. These findings suggest that H(2)O(2)-induced inhibition of the Cl(-)/OH(-) exchange process may play an important role in the pathophysiology of diarrhoea associated with inflammatory disorders, where the amount of reactive oxygen species is markedly elevated.     

Transplacental toxoplasmosis in naturally-infected white-tailed deer: Isolation and genetic characterisation of Toxoplasma gondii from foetuses of different gestational ages

Clinical toxoplasmosis is most severe in congenitally-infected hosts. In humans, transmission of Toxoplasma gondii from the mother to the foetus is considered to be most efficient during the last trimester of pregnancy but clinical congenital toxoplasmosis is more severe if transmission occurs during the first trimester. However, there are no data on the rate of congenital transmission of T. gondii with respect to gestational age in any host during natural infection. In the present study, attempts were made to isolate T. gondii by bioassay in mice inoculated with tissues from foetuses of 88 naturally-exposed white-tailed deer from Iowa and Minnesota. Viable T. gondii was isolated from foetuses of six of 61 deer in early pregnancy (45-85 days of gestation) from Iowa and foetuses of nine of 27 deer from Minnesota in mid-gestation (130-150 days) of a gestational period of 7 months. The 15 T. gondii isolates obtained from foetal deer were PCR-restriction fragment length polymorphism genotyped using polymorphisms at 10 nuclear markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and an apicoplast marker, Apico. Five genotypes were revealed, including the clonal Type II and III lineages, and three non-clonal genotypes. DNA sequencing analysis of representative isolates at loci SAG2, c22-8, L358 and PK1 revealed that the three non-clonal genotypes are closely related to the clonal Type I, II and III lineages. It is very likely that these non-clonal genotypes were derived from genetic crosses among the three clonal Type I, II and III lineages. The most common genotype was Type II, commonly found in humans in North America and Europe, suggesting the possible link of transmission from game animals to humans.     

Location and function of STIM1 in the activation of Ca2+ entry signals

Store-operated channels (SOCs) mediate Ca(2+) entry signals in response to endoplasmic reticulum (ER) Ca(2+) depletion in most cells. STIM1 senses decreased ER luminal Ca(2+) through its EF-hand Ca(2+)-binding motif and aggregates in near-plasma membrane (PM) ER junctions to activate PM Orai1, the functional SOC. STIM1 is also present in the PM, although its role there is unknown. STIM1-mediated coupling was examined using the stable EF20 HEK293 cell line expressing the STIM1-D76A/E87A EF-hand mutant (STIM1(EF)) deficient in Ca(2+) binding. EF20 cells were viable despite constitutive Ca(2+) entry, allowing study of SOC activation without depleting ER Ca(2+). STIM1(EF) was exclusively in stable near-PM junctions, 3.5-fold larger than formed with STIM1(WT). STIM(EF)-expressing cells had normal ER Ca(2+) levels but substantially reduced ER Ca(2+) leak. Expression of antiapoptotic Bcl-2 proteins (BCl-2, MCL-1, BCL-XL) were increased 2-fold in EF20 cells, probably reflecting survival of EF20 cells but not accounting for decreased ER Ca(2+) leak. Surface biotinylation and streptavidin pull-down of cells expressing STIM1(WT) or STIM1(EF) revealed strong PM interactions of both proteins. Although surface expression of STIM1(WT) was clearly detectable, STIM1(EF) was undetectable at the cell surface. Thus, the Ca(2+) binding-defective STIM1(EF) mutant exists exclusively in aggregates within near-PM junctions but, unlike STIM1(WT), is not trafficked to the PM. Although not inserted in the PM, external application of a monoclonal anti-N-terminal STIM1 antibody blocked constitutive STIM(EF)-mediated Ca(2+) entry, but only in cells expressing endogenous STIM1(WT) and not in DT40 STIM1 knock-out cells devoid of STIM(WT). This suggests that PM-STIM1 may play a regulatory role in SOC activation.