Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d.

Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αß T cell receptors (TCR) and recognize α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells), but our knowledge of the antigens for type II NKT cells is limited. An early study identified a nonlipidic NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa drugs, but its mechanism of NKT cell activation remained unknown. Here, we demonstrate that a range of pentamethylbenzofuransulfonates (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug-like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d tetramers identified type II NKT cell populations expressing αßTCRs and γδTCRs, including those with variable and joining region gene usage (TRAV12-1-TRAJ6) that was conserved across donors. By trapping a CD1d-type II NKT TCR complex for direct mass-spectrometric analysis, we detected molecules that allow the binding of CD1d to TCRs, finding that both selected PBF family members and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that nonlipidic small molecules, which resemble sulfa drugs implicated in systemic hypersensitivity and drug allergy reactions, are targeted by a polyclonal population of type II NKT cells in a CD1d-restricted manner.

Endoplasmic reticulum chaperones stabilize ligand-receptive MR1 molecules for efficient presentation of metabolite antigens.

The antigen-presenting molecule MR1 (MHC class I-related protein 1) presents metabolite antigens derived from microbial vitamin B2 synthesis to activate mucosal-associated invariant T (MAIT) cells. Key aspects of this evolutionarily conserved pathway remain uncharacterized, including where MR1 acquires ligands and what accessory proteins assist ligand binding. We answer these questions by using a fluorophore-labeled stable MR1 antigen analog, a conformation-specific MR1 mAb, proteomic analysis, and a genome-wide CRISPR/Cas9 library screen. We show that the endoplasmic reticulum (ER) contains a pool of two unliganded MR1 conformers stabilized via interactions with chaperones tapasin and tapasin-related protein. This pool is the primary source of MR1 molecules for the presentation of exogenous metabolite antigens to MAIT cells. Deletion of these chaperones reduces the ER-resident MR1 pool and hampers antigen presentation and MAIT cell activation. The MR1 antigen-presentation pathway thus co-opts ER chaperones to fulfill its unique ability to present exogenous metabolite antigens captured within the ER.

A Genus Definition for Bacteria and Archaea Based on a Standard Genome Relatedness Index.

Genus assignment is fundamental in the characterization of microbes, yet there is currently no unambiguous way to demarcate genera solely using standard genomic relatedness indices. Here, we propose an approach to demarcate genera that relies on the combined use of the average nucleotide identity, genome alignment fraction, and the distinction between type- and non-type species. More than 3,500 genomes representing type strains of species from >850 genera of either bacterial or archaeal lineages were tested. Over 140 genera were analyzed in detail within the taxonomic context of order/family. Significant genomic differences between members of a genus and type species of other genera in the same order/family were conserved in 94% of the cases. Nearly 90% (92% if polyphyletic genera are excluded) of the type strains were classified in agreement with current taxonomy. The 448 type strains that need reclassification directly impact 33% of the genera analyzed in detail. The results provide a first line of evidence that the combination of genomic indices provides added resolution to effectively demarcate genera within the taxonomic framework that is currently based on the 16S rRNA gene. We also identify the emergence of natural breakpoints at the genome level that can further help in the circumscription of taxa, increasing the proportion of directly impacted genera to at least 43% and pointing at inaccuracies on the use of the 16S rRNA gene as a taxonomic marker, despite its precision. Altogether, these results suggest that genomic coherence is an emergent property of genera in Bacteria and ArchaeaIMPORTANCE In recent decades, the taxonomy of Bacteria and Archaea, and therefore genus designation, has been largely based on the use of a single ribosomal gene, the 16S rRNA gene, as a taxonomic marker. We propose an approach to delineate genera that excludes the direct use of the 16S rRNA gene and focuses on a standard genome relatedness index, the average nucleotide identity. Our findings are of importance to the microbiology community because the emergent properties of Bacteria and Archaea that are identified in this study will help assign genera with higher taxonomic resolution.

A single-domain bispecific antibody targeting CD1d and the NKT T-cell receptor induces a potent antitumor response.

Antibody-mediated modulation of major histocompatibility complex (MHC) molecules, or MHC class I-like molecules, could constitute an effective immunotherapeutic approach. We describe how single-domain antibodies (VHH), specific for the human MHC class I-like molecule CD1d, can modulate the function of CD1d-restricted T cells and how one VHH (1D12) specifically induced strong type I natural killer T (NKT) cell activation. The crystal structure of the VHH1D12-CD1d(α-GalCer)-NKT T-cell receptor (TCR) complex revealed that VHH1D12 simultaneously contacted CD1d and the type I NKT TCR, thereby stabilizing this interaction through intrinsic bispecificity. This led to greatly enhanced type I NKT cell-mediated antitumor activity in in vitro, including multiple myeloma and acute myeloid leukemia patient-derived bone marrow samples, and in vivo models. Our findings underscore the versatility of VHH molecules in targeting composite epitopes, in this case consisting of a complexed monomorphic antigen-presenting molecule and an invariant TCR, and represent a generalizable antitumor approach.

Total Synthesis of Mycobacterium tuberculosis Dideoxymycobactin-838 and Stereoisomers: Diverse CD1a-Restricted T Cells Display a Common Hierarchy of Lipopeptide Recognition.

Mycobacterium tuberculosis produces dideoxymycobactin-838 (DDM-838), a lipopeptide that potently activates T cells upon binding to the MHC-like antigen-presenting molecule CD1a. M. tuberculosis produces DDM-838 in only trace amounts and a previous solid-phase synthesis provided sub-milligram quantities. We describe a high-yielding solution-phase synthesis of DDM-838 that features a Mitsunobu substitution that avoids yield-limiting epimerization at lysine during esterification, and amidation conditions that prevent double-bond isomerization of the Z-C20:1 acyl chain, and provides material with equivalent antigenicity to natural DDM-838. Isomers of DDM-838 that varied in stereochemistry at the central lysine and the C20:1 acyl chain were compared for their ability to be recognised by CD1a-restricted T cell receptors (TCRs). These TCRs, derived from unrelated human donors, exhibited a similar spectrum of reactivity towards the panel of DDM-838 isomers, highlighting the exquisite sensitivity of lipopeptide-reactive T cells for the natural DDM stereochemistry.

Acute renal failure as an initial manifestation of acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Acute renal failure is a well-recognized complication of ALL after initiation of chemotherapy. Renal failure as the primary manifestation of ALL is rare. Here, we report three children who presented with acute renal failure and hyperuricemia and were subsequently diagnosed to have ALL.

A comparison of tactile, visual, and auditory warnings for rear-end collision prevention in simulated driving.

OBJECTIVE: This study examined the effectiveness of rear-end collision warnings presented in different sensory modalities as a function of warning timing in a driving simulator. BACKGROUND: The proliferation of in-vehicle information and entertainment systems threatens driver attention and may increase the risk of rear-end collisions. Collision warning systems have been shown to improve inattentive and/or distracted driver response time (RT) in rear-end collision situations. However, most previous rear-end collision warning research has not directly compared auditory, visual, and tactile warnings. METHOD: Sixteen participants in a fixed-base driving simulator experienced four warning conditions: no warning, visual, auditory, and tactile. The warnings activated when the time-to-collision (TTC) reached a critical threshold of 3.0 or 5.0 s. Driver RT was captured from a warning below critical threshold to brake initiation. RESULTS: Drivers with a tactile warning had the shortest mean RT. Drivers with a tactile warning had significantly shorter RT than drivers without a warning and had a significant advantage over drivers with visual warnings. CONCLUSION: Tactile warnings show promise as effective rear-end collision warnings. APPLICATION: The results of this study can be applied to the future design and evaluation of automotive warnings designed to reduce rear-end collisions.

The distribution of caveolin-3 immunofluorescence in skeletal muscle fibre membrane defined by dual channel confocal laser scanning microscopy, fast Fourier transform and image modelling.

Membrane domains rich in caveolin-3 overlie sarcomeric actin in skeletal muscle. The membrane exhibits a regular array of caveolin-3 immunofluorescence using confocal laser scanning microscopy (CLSM). Fourier analysis of tissue imaged by CLSM accurately defines a repeating intensity with a long-axis spacing of 1.48 microm confirmed by measurement of direct images. Reverse fast Fourier transform (FFT) and image-modelling allow reconstruction of the pattern. Mathematical modelling has allowed replication of several features of the FFT, including the second order maxima that confirm the relatively high information content of the original images. Measurements of membrane-pattern primary long-axis spacings are consistent with our measurements of the I-band sarcomere repeat in similarly prepared specimens labelled with fluorescent phalloidin or imaged using differential interference contrast microscopy. Dual-channel CLSM analysis of the sarcomeric banding pattern of actin and the repeating pattern of muscle fibre membrane caveolin showed that caveolae overlie the I-band. The anti-caveolin immunofluorescence is deficient over the Z-disc and maximal toward each of the I-band extremities. A mechanism of membrane shape change in which membrane-lipid molecules are interposed between more stable anchored rafts associated with caveolae can be envisaged. Thus, increasing girth and reducing length of the sarcolemma in rapid contraction may be explained.

Use of an infusion-baited gravid trap to collect adult Ochlerotatus japonicus.

Ochlerotatus japonicus adults have proven difficult to collect in significant numbers in the USA. Standard trapping and collection methods have been ineffective. Infusion-baited gravid traps, typically intended to attract Culex spp., have provided the greatest number and most consistent supply of Oc. japonicus adults collected in New Jersey.

Ochlerotatus japonicus collected from natural rockpools in New Jersey.

Ochlerotatus japonicus larvae were collected from natural rockpools in New Jersey within the Delaware Water Gap National Recreational Area. Previously reported collections of Oc. japonicus in the United States have been limited to artificial containers and an occasional treehole.

Use of an AC induction motor system for producing finger movements in human subjects.

This report describes the set-up and evaluation of a novel system for producing precise finger movements, for tests of movement perception. The specifications were to construct a system using commercially available components that were easy to use but which offered both flexibility and also high precision control. The system was constructed around an industrial AC induction motor with an optical encoder, controlled by an AC servo digital control module that could be programmed using a simple, high-level language. This set-up fulfilled the requirements regarding position and velocity control for a range of movements and also the facility for the subject to move the joint voluntarily while still attached to the motor. However a number of problems were encountered, the most serious being the level of vibration and the inability to vary the torque during movements. The vibration was reduced to the point where it did not affect the subject, by the introduction of mechanical dampening using an anti-vibration coupling and a pneumatic splint. The torque control could not be modified during rotation and so the system could only be operated using constant torque for any given movement.

Tendon organ sensitivity to steady-state isotonic contraction of in-series motor units in feline peroneus tertius muscle.

1. Measurements have been made of the sensitivity of tendon organs to steady-state, isotonic contractions of single and groups of in-series motor units in the peroneus tertius muscle of the cat hindlimb. 2. Linear relationships were found between the Ib afferent discharge and the contractile tension generated by tetanic stimulation of single motor units. These relationships held for the fast, fatiguable (FF) units and for all but the lowest tensions generated by the slow (S) and some fast, fatigue resistant (FR) units. The sensitivity of the organs was independent of the contractile properties of the units. 3. Groups of three motor units were stimulated isotonically at low rates (around 30 Hz), but asynchronously to produce a smooth tension profile. Again, linear relationships pertained between the discharge rate and the tension, and the sensitivity was the same for different motor unit types. 4. Under isotonic conditions, therefore, the tendon organs showed linear responses to the tension with similar sensitivities, indicating that tendon organs may have the capacity to signal faithfully steady-state contractile tensions.

The dynamic response of feline Golgi tendon organs during recovery from nerve injury.

The dynamic response of tendon organs to isometric contractions of activating motor units has been examined during recovery from nerve crush or nerve transection followed by suture repair. After nerve crush the characteristic response was rapidly restored, although the early and late phases of the dynamic response were altered differentially. Following nerve transection, recovery was much poorer and many responses were abnormal. Normal responses were only observed in a minority of tendon organ-motor unit interactions but every tendon organ studied did respond normally to at least one motor unit, with a range of dynamic sensitivities similar to normal. This suggests that the abnormalities observed reflect changes in the mechanical input to the organ, due to motor unit reorganisation, rather than abnormalities of the transduction process.

Beta innervation and recurrent inhibition: a hypothesis for manipulatory and postural control.

The innervation of muscle spindles by skeletofusimotor (beta) axons in functionally different muscles of the cat forelimb has been correlated with the neural circuitry of the respective motor nuclei, morphological characteristics of their motoneurones and the innervated muscle fibres. In long digt extensor muscles a high degree of beta-innervation (more than 70%) and of fast contracting muscle fibres (more than 70% IIB fibres) correlates with specific projections of their Ia muscle spindle afferents, with the absence of a recurrent inhibitory system, with cell body diameters in the range of small alpha-motoneurones and with a short duration of their after-hyperpolarisations. In contrast, the investigated elbow muscles display a low degree of beta-innervation (41-47%) irrespective of their fibre type composition, their Ia afferent fibres show a divergent projection pattern and their motor nuclei a distinct recurrent inhibitory system. We suggest that for the distal muscles the specific combination of these different characteristics serves the control of manipulative movements, whereas for the proximal muscles the contrasting characteristics serve the control of posture and locomotion. This hypothesis is discussed in view of the phylogenetic development of motor control.

The static sensitivity of tendon organs during recovery from nerve injury.

An investigation has been carried out into the physiological properties of tendon organs and their interactions with motor units following two types of nerve injury: nerve crush and nerve transection followed by suture repair. Recovery from nerve crush was very successful: 6 weeks after the injury 60% of the tendon organ-motor unit interactions (n = 62) evoked normal or near-normal patterns of afferent discharge but with reduced firing rates. After 10 weeks of recovery 81% of the interactions (n = 43) were normal. The main abnormality observed was a phasic-only pattern of discharge. The overall reductions in firing rate during early recovery may be attributable to the lower contractile forces generated by the reinnervated muscle units, while the phasic-only responses may also represent immaturity of the transduction mechanism of the regenerated afferent axons. Following nerve transection the quality of recovery was much lower and a range of abnormal, as well as normal patterns of response were observed. For all the afferents studied, both types of response were recorded, suggesting that although there may be changes in the sensitivity of the afferents to muscle contraction, the abnormal responses more probably reflect changes in the form of the mechanical input rather than abnormalities of the transduction process.

The dynamic response of Golgi tendon organs to tetanic contraction of in-series motor units.

Golgi tendon organs (TOs) display a dynamic response related to the rate of tension development by the motor units (MUs) which activate each TO. When several MUs are activated together the response increases non-linearly with respect to tension. The dynamic response has been re-examined by recording the responses of TOs, in the adult cat, to tetanic, isometric contractions of one or more MUs and has been found to comprise two components: for each MU-TO pair the instantaneous frequency between the first two impulses of the response was linearly related to the rate of rise of the tension, the slope of the relationship being greatest for the slow units. Similarly, when activating MUs were stimulated together, the relationships between frequency and the initial rate of tension rise were linear. When the relationships were expressed in terms of relative tension rate the organs were found to be most strongly activated by the IIb muscle fibres, the responsiveness at the level of the whole muscle being reduced by unloading effects. The later component of the response showed an exponential relationship between discharge frequency and rate of tension development, the slope of the relationship between frequency and the logarithm of tension rate being independent of MU size. Although absolute tension increases during a contraction, this did not appear to impinge on the response which was dominated by the dynamic parameters of the contraction.

Skeletofusimotor (beta) innervation of proximal and distal forelimb muscles of the cat.

The innervation of muscle spindles by skeletofusimotor (beta) axons has been compared in two long digit extensor muscles with three elbow extensor muscles of the cat forelimb. The proportion of muscle spindle poles with p1-endplates was analysed in silver impregnated teased material. The proximal and distal muscles displayed a significant difference in their proportion of muscle spindle poles with p1-endplates. The distal muscles had an estimated proportion of more than 70% beta innervation of their muscle spindles, the proximal muscles of 41-47%. This difference is discussed with respect to the different circuitry of the motoneurones. We suggest that the high proportion of beta-innervated spindles in distal muscles obtained with the absence of a recurrent inhibitory system in the innervating motoneurones [18] serves the execution of manipulative movements.

Small-signal sensitivity of muscle spindles reinnervated after long-term denervation.

After long periods of denervation regenerated muscle spindle afferents show functional recovery despite gross histological abnormalities in their ending structure. This study prevents results examining the responses of such afferents to small amplitude stimuli, within the linear range of the spindle. The majority of regenerated afferents responded to the stimuli with a sensitivity similar to that of normal afferents.

Evaluation of a fluorescence feedback system for guidance of laser angioplasty.

BACKGROUND AND OBJECTIVE: Laser-induced fluorescence spectroscopy (LIFS) may be capable of guiding laser angioplasty by discriminating normal and atherosclerotic artery and by determining catheter-tissue environment. Previous optical multichannel analyzer based LIFS systems have been expensive and cumbersome. To simplify LIFS, a system based on photomultiplier tubes was developed and evaluated. STUDY DESIGN/MATERIALS AND METHODS: Tissue fluorescence was induced by a helium cadmium laser (wavelength = 325 nm, power = 0.2-0.5 mW), collected by clinical multifiber laser angioplasty catheters and directed through one of two filters (10 nm bandpass, 380 nm or 440 nm peak transmission) to a photomultiplier tube. An LIFS ratio was defined as the relative intensity at 380:440 nm after calibration with an elastin fluorescence spectrum; 157 coronary artery cadaveric specimens were evaluated spectroscopically and histologically. To evaluate the utility of LIFS to optimize catheter position by determining catheter-tissue contact, by determining saline dilution of blood, and by orienting eccentric multifiber catheters a new variable, the total fluorescence intensity (TFI) was defined as the sum of arterial fluorescence intensities at 380 nm and 440 nm. TFI was recorded in vitro through multifiber catheters from 20 arterial specimens in vitro in blood and evaluated as a function of the catheter-to-tissue distance (d) over a range from 0 to 400 mu. RESULTS: Defining normal specimens as those with an intimal thickness < or = 200 mu, and atherosclerotic as those with an intimal thickness > 200 mu, 47/50 (94%) normal and 85/107 (79%) atherosclerotic specimens were correctly classified using a threshold LIFS ratio of 2.0. Mean (+/- SE) normal ratio was 1.76 +/- 0.02 and mean atherosclerotic ratio was 2.78 +/- 0.08 (P < or = 0.01). The classification accuracy of atherosclerotic specimens increased with intimal thickness so that 95% of atherosclerotic specimens (69/73) with intimal thickness > or = 400 mu were correctly classified. TFI was capable of determining catheter-tissue contact as maximal TFI was recorded with the catheter in contact with the tissue (d = 0 mu) and decreased markedly with distance (to 52 +/- 6% at d = 100 mu, 19 +/- 4% at d = 200 mu, and 3 +/- 1% at d = 300 mu). TFI was recorded from ten arterial specimens in blood/saline mixtures ranging in hematocrit from 0% (saline) to 50% (whole blood). TFI was capable of detecting saline hemodilution of blood as TFI decreased markedly at higher hematocrits such that TFI could only by recorded at hematocrits < 10% for catheter-to-tissue distances > or = 300 mu. TFI was recorded through ecentric multifiber catheters from 25 arterial specimens and eval-uated as a function of the degree of catheter-tissue overlap. TFI was capable of detecting maximal catheter-tissue overlap as TFI correlated linearly with the area (A) of overlap (TFI = 1.12 A + .07, r = 0.92). CONCLUSIONS: By discriminating atherosclerotic from normal tissue and by confirming catheter-tissue contact and saline hemodilution, fluorescence feedback should minimize irradiation of normal tissue and/or blood and enhance the safety and efficacy of laser angioplasty.