The Ultra-Potent and Selective TLR8 Agonist VTX-294 Activates Human Newborn and Adult Leukocytes

Background: Newborns display distinct immune responses that contribute to susceptibility to infection and reduced vaccine responses. Toll-like receptor (TLR) agonists may serve as vaccine adjuvants, when given individually or in combination, but responses of neonatal leukocytes to many TLR agonists are diminished. TLR8 agonists are more effective than other TLR agonists in activating human neonatal leukocytes in vitro, but little is known about whether different TLR8 agonists may distinctly activate neonatal leukocytes. We characterized the in vitro immuno-stimulatory activities of a novel benzazepine TLR8 agonist, VTX-294, in comparison to imidazoquinolines that activate TLR8 (R-848; (TLR7/8) CL075; (TLR8/7)), with respect to activation of human newborn and adult leukocytes. Effects of VTX-294 and R-848 in combination with monophosphoryl lipid A (MPLA; TLR4) were also assessed. Methods: TLR agonist specificity was assessed using TLR-transfected HEK293 cells expressing a NF-κB reporter gene. TLR agonist-induced cytokine production was measured in human newborn cord and adult peripheral blood using ELISA and multiplex assays. Newborn and adult monocytes were differentiated into monocyte-derived dendritic cells (MoDCs) and TLR agonist-induced activation assessed by cytokine production (ELISA) and co-stimulatory molecule expression (flow cytometry). Results: VTX-294 was ∼100x more active on TLR8- than TLR7-transfected HEK cells (EC50, ∼50 nM vs. ∼5700 nM). VTX-294-induced TNF and IL-1β production were comparable in newborn cord and adult peripheral blood, while VTX-294 was ∼ 1 log more potent in inducing TNF and IL-1β production than MPLA, R848 or CL075. Combination of VTX-294 and MPLA induced greater blood TNF and IL-1β responses than combination of R-848 and MPLA. VTX-294 also potently induced expression of cytokines and co-stimulatory molecules HLA-DR and CD86 in human newborn MoDCs. Conclusions: VTX-294 is a novel ultra-potent TLR8 agonist that activates newborn and adult leukocytes and is a candidate vaccine adjuvant in both early life and adulthood

Structure of Metallathiacycles: Planar vs Nonplanar Geometries. A Theoretical and Experimental Investigation

Since the previously reported results of Fenske-Hall molecular orbital calculations suggested that the bent metallacycle geometries observed in transition-metal-inserted thiophene complexes do not result from electronic properties, molecular mechanics calculations were employed to explore how steric factors could influence ring geometries. These calculations have identified steric factors responsible for the observed ring deformations in (C 5 Me 5 )Rh(PMe 3 )(η 2 -C,S-2,5-Me 2 C 4 H 2 S) and benzothiophene and dibenzothiophene analogs. The results of the molecular mechanics calculations have been supported by X-ray structural characterization of the products formed via insertion of the reactive 16-electron metal fragment [(C 5 Me 5 )Rh(PMe 3 )] into the C-S bonds of unsubstituted thiophene, benzothiophene, and dibenzothiophene. The parent thiophene complex, unlike the previously reported ringsubstituted analog (and as predicted by the molecular mechanics calculations) is found to adopt a planar six-membered ring structure. The benzothiophene insertion product shows a moderate degree of bending of the metallathiacycle, whereas the dibenzothiophene molecule is strongly bent

Enhanced immune activation linked to endotoxemia in HIV-1 seronegative MSM

This study assessed cellular and soluble markers of immune activation in HIV-1 seronegative MSM. MSM immune profiles were characterized by an increased expression of CD57 on T cells and endotoxemia. Endotoxin presence was linked to recent high-risk exposure and associated with elevated cytokine levels and decreased CD4+/CD8+ T cell ratios. Taken together, these data show elevated levels of inflammation linked to periods of endotoxemia resulting in a significantly different immune phenotype in a subset of MSM at a high risk of HIV-1 acquisition.National Institutes of Health (U.S.) (Grant P01 AI074415

Innate immune activation in neonatal tracheal aspirates suggests endotoxin-driven inflammation

Background:: Tracheal aspirates (TAs) from critically ill neonates accumulate bacterial endotoxin and demonstrate mobilization of endotoxin-binding proteins, but the potential bioactivity of endotoxin in TAs is unknown. We characterized innate immune activation in TAs of mechanically ventilated neonates. Methods: Innate immune activation in TAs of mechanically ventilated neonates was characterized using a targeted 84-gene quantitative real-time (qRT) PCR array. Protein expression of cytokines was confirmed by multiplex assay. Expression and localization of the endotoxin-inducible antimicrobial protein Calgranulin C (S100A12) was assessed by flow cytometry. Endotoxin levels were measured in TA supernatants using the Limulus amoebocyte lysate assay. Results:: Analyses by qRT-PCR demonstrated expression of pattern recognition receptors, Toll-like receptor-nuclear factor κB and inflammasome pathways, cytokines/chemokines and their receptors, and anti-infective proteins in TA cells. Endotoxin positivity increased with postnatal age. As compared with endotoxin-negative TAs, endotoxin-positive TAs demonstrated significantly greater tumor necrosis factor (TNF), interleukin (IL)-6, IL-10, and serpin peptidase inhibitor, clade E, member 1 (SERPINE1) mRNA, and IL-10, TNF, and IL-1β protein. Expression of S100A12 protein was localized to TA neutrophils. Conclusion:: Correlation of endotoxin with TA inflammatory responses suggests endotoxin bioactivity and the possibility that endotoxin antagonists could mitigate pulmonary inflammation and its sequelae in this vulnerable population

Status of Muon Collider Research and Development and Future Plans

The status of the research on muon colliders is discussed and plans are outlined for future theoretical and experimental studies. Besides continued work on the parameters of a 3-4 and 0.5 TeV center-of-mass (CoM) energy collider, many studies are now concentrating on a machine near 0.1 TeV (CoM) that could be a factory for the s-channel production of Higgs particles. We discuss the research on the various components in such muon colliders, starting from the proton accelerator needed to generate pions from a heavy-Z target and proceeding through the phase rotation and decay ($\pi \to \mu \nu_{\mu}$) channel, muon cooling, acceleration, storage in a collider ring and the collider detector. We also present theoretical and experimental R & D plans for the next several years that should lead to a better understanding of the design and feasibility issues for all of the components. This report is an update of the progress on the R & D since the Feasibility Study of Muon Colliders presented at the Snowmass'96 Workshop [R. B. Palmer, A. Sessler and A. Tollestrup, Proceedings of the 1996 DPF/DPB Summer Study on High-Energy Physics (Stanford Linear Accelerator Center, Menlo Park, CA, 1997)].Comment: 95 pages, 75 figures. Submitted to Physical Review Special Topics, Accelerators and Beam

MR imaging of overuse injuries in the skeletally immature gymnast: spectrum of soft-tissue and osseous lesions in the hand and wrist

In the pediatric gymnast, stress-related physeal injuries have been well described with characteristic imaging findings. However, a spectrum of overuse injuries, some rarely reported in the literature, can be encountered in the gymnast’s hand and wrist. To demonstrate the MR appearance of a spectrum of overuse injuries in the skeletally immature wrist and hand of pediatric gymnasts. A total of 125 MR exams of the hand and wrist in skeletally immature children were performed at our institution during a 2-year period. Clinical histories were reviewed for gymnastics participation. MR studies of that subpopulation were reviewed and abnormalities tabulated. Of the MR studies reviewed, ten gymnasts were identified, all girls age 12–16 years (mean age 14.2 years) who presented with wrist or hand pain. Three of these children had bilateral MR exams. Abnormalities included chronic physeal injuries in three children. Two girls exhibited focal lunate osteochondral defects. Triangular fibrocartilage tears were present in three girls, one of whom had a scapholunate ligament tear. Two girls manifested metacarpal head flattening and necrosis. A variety of soft-tissue and osseous lesions can be encountered in the skeletally immature gymnast. Familiarity with these stress-related injuries is important for accurate diagnosis

Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn's disease

The cause of Crohn's disease (CD) remains poorly understood. Counterintuitively, these patients possess an impaired acute inflammatory response, which could result in delayed clearance of bacteria penetrating the lining of the bowel and predispose to granuloma formation and chronicity. We tested this hypothesis in human subjects by monitoring responses to killed Escherichia coli injected subcutaneously into the forearm. Accumulation of 111In-labeled neutrophils at these sites and clearance of 32P-labeled bacteria from them were markedly impaired in CD. Locally increased blood flow and bacterial clearance were dependent on the numbers of bacteria injected. Secretion of proinflammatory cytokines by CD macrophages was grossly impaired in response to E. coli or specific Toll-like receptor agonists. Despite normal levels and stability of cytokine messenger RNA, intracellular levels of tumor necrosis factor (TNF) were abnormally low in CD macrophages. Coupled with reduced secretion, these findings indicate accelerated intracellular breakdown. Differential transcription profiles identified disease-specific genes, notably including those encoding proteins involved in vesicle trafficking. Intracellular destruction of TNF was decreased by inhibitors of lysosomal function. Together, our findings suggest that in CD macrophages, an abnormal proportion of cytokines are routed to lysosomes and degraded rather than being released through the normal secretory pathway

Differential frequency of NKG2C/KLRC2 deletion in distinct African populations and susceptibility to Trachoma: a new method for imputation of KLRC2 genotypes from SNP genotyping data.

NKG2C is an activating receptor that is preferentially expressed on natural killer (NK) cells. The gene encoding NKG2C (killer cell lectin-like receptor C2, KLRC2) is present at different copy numbers in the genomes of different individuals. Deletion at the NKG2C locus was investigated in a case-control study of 1522 individuals indigenous to East- and West-Africa and the association with the ocular Chlamydia trachomatis infection and its sequelae was explored. The frequency of homozygous KLRC2 deletion was 13.7 % in Gambians and 4.7 % in Tanzanians. A significantly higher frequency of the deletion allele was found in West-Africans from the Gambia and Guinea-Bissau (36.2 % p = 2.105 × 10(-8), 26.8 % p = 0.050; respectively) in comparison to East-African Tanzanians where the frequency of the deletion is comparable to other human populations (20.9 %). We found no evidence for an association between the numbers of KLRC2 gene copies and the clinical manifestations of trachoma (follicular trachoma or conjunctival scarring). A new method for imputation of KLRC2 genotypes from single nucleotide polymorphism (SNP) data in 2621 individuals from the Gambia further confirmed these results. Our data suggest that NKG2C does not play a major role in trachomatous disease. We found that the deletion allele is present at different frequencies in different populations but the reason behind these differences is currently not understood. The new method offers the potential to use SNP arrays from genome wide association studies to study the frequency of KLRC2 deletion in other populations and its association with other diseases