Arterial immune protein expression demonstrates the complexity of immune responses in Kawasaki disease arteritis.

A more complete understanding of immune-mediated damage to the coronary arteries in children with Kawasaki disease (KD) is required for improvements in patient treatment and outcomes. We recently reported the transcriptional profile of KD coronary arteritis, and in this study sought to determine protein expression of transcriptionally up-regulated immune genes in KD coronary arteries from the first 2 months after disease onset. We examined the coronary arteries of 12 fatal KD cases and 13 childhood controls for expression of a set of proteins whose genes were highly up-regulated in the KD coronary artery transcriptome: allograft inflammatory factor 1 (AIF1), interleukin 18 (IL-18), CD74, CD1c, CD20 (MS4A1), Toll-like receptor 7 (TLR-7) and Z-DNA binding protein 1 (ZBP1). Immunohistochemistry and immunofluorescence studies were performed to evaluate protein expression and co-localization, respectively. AIF1 was expressed transmurally in KD arteritis and localized to macrophages and myeloid dendritic cells. CD74, which interacts with major histocompatibility complex (MHC) class II on antigen-presenting cells, localized to the intima-media. CD1c, a marker of myeloid dendritic cells, was expressed in a transmural pattern, as were IL-18 and CD20. ZBP1 and TLR-7 were up-regulated compared to controls, but less highly compared to the other proteins. These findings provide evidence of antigen presentation and interferon response in KD arteritis. In combination with prior studies demonstrating T lymphocyte activation, these results demonstrate the complexity of the KD arterial immune response.

Synthesis and biological activities of d-chiro-inositol analogues with insulin-like actions.

d-chiro-inositol (DCI, 1) evokes therapeutic actions in diabetes and insulin resistance but has sub-optimal pharmacokinetic profiles. To investigate what positions on the DCI cyclohexanol ring may be amenable to modification to improve pharmaceutical formulations, a series of analogues based on DCI were synthesised. These compounds were then evaluated for their ability to stimulate glucose transport using 3T3-L1 adipocytes as a model system. Positional analyses indicate that the hydroxyl group at position 1 is not essential for activity and can be modified without affecting glucose uptake. Removal of the hydroxyl at position 3 also had minimal effect on activity but this group is sensitive to modification. By comparison, the oxygen at position 2 is crucial to the potency of DCI, although this group can withstand modification without fundamentally affecting activity. These data reveal that positions 1 and 2 on the cyclohexanol ring of DCI offer further scope for modification to develop DCI analogues with desirable pharmacokinetic profiles for the potential treatment of metabolic disease.

Trees for bees.

Controversy over the origins and evolution of social behaviour in the major groups of social bees (the corbiculate bees) has fuelled arguments over different approaches for building evolutionary trees. However, the application of different analytical methodologies does not explain why molecular and morphological data suggest strikingly different hypotheses for the evolution of eusociality in bees. Determining the phylogenetic root is expected to help resolve the question of the social evolution of corbiculate bees. However, this requires that the long branch attraction problem is overcome. This phenomenon affects both molecular and morphological data for corbiculate bees.

Multiple molecular data sets suggest independent origins of highly eusocial behavior in bees (Hymenoptera:Apinae).

Different views of the pattern of social evolution among the highly eusocial bees have arisen as a result of discordance between past molecular and morphology-based phylogenies. Here we present new data and taxa for four molecular data sets and reassess the morphological characters available to date. We show there is no significant character incongruence between four molecular data sets (two nuclear and two mitochondrial), but highly significant character incongruence leads to topological incongruence between the molecular and morphological data. We investigate the effects of using different outgroup combinations to root the estimated tree. We also consider various ways in which biases in the sequence data could be misleading, using several maximum likelihood models, LogDet corrections, and spectral analyses. Ultimately, we concede there is strong discordance between the molecular and morphological data partitions and appropriately apply the conditional combination approach in this case. We also find two equally well supported placements of the root for the molecular trees, one supported by 16S and 28S sequences, the other supported by cytochrome b and opsin. The strength of the evidence leads us to accept two equally well supported hypotheses based on analyses of the molecular data sets. These are the most rigorously supported hypotheses of corbiculate bee relationships at this time, and frame our argument that highly eusocial behavior within the corbiculate bees evolved twice independently.

Rapid compensatory changes in GABA receptor efficacy in rat vestibular neurones after unilateral labyrinthectomy.

1. The inhibitory effects of the GABAA agonist muscimol and the GABAB agonist baclofen on tonically active medial vestibular nucleus (MVN) neurones were recorded in slices of the rat dorsal brainstem in vitro, to determine whether any changes occurred in the functional efficacy of GABAergic inhibition in these cells during the initial rapid stage of 'vestibular compensation', the behavioural recovery that takes place after unilateral labyrinthectomy (UL). These experiments were carried out in preparations where the midline was cut, severing all commissural connections between the two vestibular nuclei. 2. Slices of the MVN were prepared from normal animals and animals that had been unilaterally labyrinthectomised 4 h earlier. The mean in vitro discharge rate of MVN neurones in the rostral region of the ipsi-lesional nucleus after UL was significantly higher than that in control slices, confirming our earlier reports of an increase in intrinsic excitability of these cells in the early stage of vestibular compensation. The in vitro discharge rates of caudal ipsi-lesional MVN cells, and rostral and caudal contra-lesional MVN cells, were not different from controls. 3. Muscimol and baclofen caused reversible, dose-related inhibition of the tonic discharge rate of MVN cells in control slices. In slices prepared from UL animals, MVN cells in the rostral region of the ipsi-lesional nucleus showed a marked downregulation of their response to both muscimol and baclofen, seen as a rightward shift and a decrease in slope of the dose-response relationships for the two agonists. In the contra-lesional nucleus, there was a small but significant upregulation of the responsiveness of both rostral and caudal MVN cells to baclofen, and a marked upregulation of the responsiveness of caudal MVN cells to muscimol. 4. In slices from animals that had undergone bilateral labyrinthectomy 4 h earlier, the downregulation of the functional efficacy of GABA receptors in the rostral MVN cells did not occur. The changes in GABA receptor efficacy after UL are therefore not due to the vestibular de-afferentation itself, but are instead due to the imbalance in excitability of the vestibular nuclei of the lesioned and intact sides, and the enhanced commissural inhibition of the ipsi-lesional MVN cells that follows UL. 5. The downregulation of GABA receptor efficacy in the ipsi-lesional MVN neurones is functionally compensatory, in that their response to commissural and cerebellar inhibitory drive will be significantly reduced after UL. Their intrinsic membrane conductances, and their remaining excitatory synaptic inputs, will consequently be more effective in causing depolarisation and the restoration of resting activity. Simultaneously the upregulation of GABAergic efficacy in the contra-lesional MVN will tend to reduce the hyperactivity on the contralateral side. These adaptive changes therefore represent a plausible cellular mechanism for the recovery of resting discharge in the ipsi-lesional MVN neurones, and the 're-balancing' of the excitability of the vestibular neurones of the lesioned and intact sides, as occurs after UL in vivo. 6. We propose that the adaptive regulation of the functional efficacy of GABA receptors in the MVN neurones may be an important cellular mechanism for the 'homeostasis of bilateral excitability' of the vestibular nuclei of the two sides.

The major opsin in bees (Insecta: Hymenoptera): A promising nuclear gene for higher level phylogenetics.

We report the phylogenetic utility of the nuclear gene encoding the long-wavelength opsin (LW Rh) for tribes of bees. Aligned nucleotide sequences were examined in multiple taxa from the four tribes comprising the corbiculate bees within the subfamily Apinae. Phylogenetic analyses of sequence variation in a 502-bp fragment (approx 40% of the coding region) strongly supported the monophyly of each of the four tribes, which are well established from previous studies of morphology and DNA. Trees estimated from parsimony and maximum likelihood analyses of LW Rh sequences show a strongly supported relationship between the tribes Meliponini and Bombini, a relationship that has been found uniformly in studies of other genes (28S, 16S, and cytochrome b). All of the tribal clades as well as relationships among the tribes are supported by high bootstrap values, suggesting the utility of LW Rh in estimating tribal and subfamily rank for these bees. The sequences exhibit minimal base composition bias. Both 1st + 2nd and 3rd position sites provide information for estimating a reliable tree topology. These results suggest that LW Rh, which has not been reported previously in studies of organismal phylogenetics, could provide important new data from the nuclear genome for phylogeny reconstruction.

Lesion-induced plasticity in rat vestibular nucleus neurones dependent on glucocorticoid receptor activation.

1. We have recently shown that neurones in the rostral region of the medial vestibular nucleus (MVN) develop a sustained increase in their intrinsic excitability within 4 h of a lesion of the vestibular receptors of the ipsilateral inner ear. This increased excitability may be important in the rapid recovery of resting activity in these neurones during 'vestibular compensation', the behavioural recovery that follows unilateral vestibular deafferentation. In this study we investigated the role of the acute stress that normally accompanies the symptoms of unilateral labyrinthectomy (UL), and in particular the role of glucocorticoid receptors (GRs), in the development of the increase in excitability in the rostral MVN cells after UL in the rat. 2. The compensatory increase in intrinsic excitability (CIE) of MVN neurones failed to occur in animals that were labyrinthectomized under urethane anaesthesia and kept at a stable level of anaesthesia for either 4 or 6 h after UL, so that they did not experience the stress normally associated with the vestibular deafferentation syndrome. In these animals, 'mimicking' the stress response by administration of the synthetic GR agonist dexamethasone at the time of UL, restored and somewhat potentiated CIE in the MVN cells. Administration of dexamethasone in itself had no effect on the intrinsic excitability of MVN cells in sham-operated animals. 3. In animals that awoke after labyrinthectomy, and which therefore experienced the full range of oculomotor and postural symptoms of UL, there was a high level of Fos-like immunoreactivity in the paraventricular nucleus of the hypothalamus over 1.5-3 h post-UL, indicating a strong activation of the stress axis. 4. The GR antagonist RU38486 administered at the time of UL abolished CIE in the rostral MVN cells, and significantly delayed behavioural recovery as indicated by the persistence of circular walking. The mineralocorticoid receptor (MR) antagonist spironolactone administered at the time of UL had no effect. 5. Vestibular compensation thus involves a novel form of 'metaplasticity' in the adult brain, in which the increase in intrinsic excitability of rostral MVN cells and the initial behavioural recovery are dependent both on the vestibular deafferentation and on the activation of glucocorticoid receptors, during the acute behavioural stress response that follows UL. These findings help elucidate the beneficial effects of neuroactive steroids on vestibular plasticity in various species including man, while the lack of such an effect in the guinea-pig may be due to the significant differences in the physiology of the stress axis in that species.

Hierarchical analysis of variation in the mitochondrial 16S rRNA gene among Hymenoptera.

Nucleotide sequences from a 434-bp region of the 16S rRNA gene were analyzed for 65 taxa of Hymenoptera (ants, bees, wasps, parasitoid wasps, sawflies) to examine the patterns of variation within the gene fragment and the taxonomic levels for which it shows maximum utility in phylogeny estimation. A hierarchical approach was adopted in the study through comparison of levels of sequence variation among taxa at different taxonomic levels. As previously reported for many holometabolous insects, the 16S data reported here for Hymenoptera are highly AT-rich and exhibit strong site-to-site variation in substitution rate. More precise estimates of the shape parameter (alpha) of the gamma distribution and the proportion of invariant sites were obtained in this study by employing a reference phylogeny and utilizing maximum-likelihood estimation. The effectiveness of this approach to recovering expected phylogenies of selected hymenopteran taxa has been tested against the use of maximum parsimony. This study finds that the 16S gene is most informative for phylogenetic analysis at two different levels: among closely related species or populations, and among tribes, subfamilies, and families. Maximization of the phylogenetic signal extracted from the 16S gene at higher taxonomic levels may require consideration of the base composition bias and the site-to-site rate variation in a maximum-likelihood framework.

Cellular basis of vestibular compensation: changes in intrinsic excitability of MVN neurones.

A systematic survey of the intrinsically generated in vitro discharge rates of rat medial vestibular nucleus (MVN) neurones was carried out in slices from normal animals and animals undergoing vestibular compensation over 48 h after unilateral labyrinthectomy (UL). Isolation of the individual MVN in vitro revealed that the tonic discharge rates of neurones in the rostral MVN ipsilateral to the lesion were not different from control 2 h post-UL, but increased significantly at 4 h post-UL and remained significantly higher until 24 h post-UL. There were no significant changes in the in vitro discharge rates of MVN cells in the contralateral nucleus. The increase in excitability of the ipsilateral MVN cells after UL may be accounted for by a down-regulation of GABA receptors on these cells, following their sustained exposure to excessive commissural inhibition after labyrinthectomy. We suggest that the increased intrinsic excitability of the ipsilateral MVN cells is responsible for the restoration of the resting discharge in these cells after UL and the consequent recovery of static vestibular function.

Morphology of iris vasculopathy in exfoliation glaucoma.

Iris tissue obtained from 26 consecutive patients operated upon for exfoliation glaucoma and control iris tissue from 26 age-matched subjects operated upon for primary open angle glaucoma was used to investigate the iris vasculopathy associated with exfoliation glaucoma. By light microscopy exfoliation material was discerned by increased density of the perivascular matrix in affected vessels. By transmission electron microscopy exfoliation vasculopathy was divided into 4 grades. Grade I was characterized by focal accumulation of exfoliation material without evidence of cellular degeneration. In grade II, exfoliation material accumulation was accompanied by degeneration of vascular supporting cells; endothelial cells were unaffected. In grade III, endothelial cells exhibited degenerative changes and in grade IV, exfoliation material occupied an acellular vascular wall (ghost vessel). It is suggested that in iris vessels the synthesis of exfoliation material can be attributed primarily to the vascular supporting cells.

Multiple origins of advanced eusociality in bees inferred from mitochondrial DNA sequences.

The remarkably high level of colony organization found in the honey bees and stingless bees (family Apidae) is extremely rare among animals. Yet there is controversy over whether these two groups independently evolved advanced eusocial behavior or inherited it from a common ancestor. Phylogenetic analyses of DNA sequence information from the mitochondrial genome (large-subunit ribosomal RNA gene) of representative apid bees suggest that advanced eusocial behavior evolved twice independently within this assemblage. These results depart from previous hypotheses of apid relationships by indicating a close phylogenetic relationship between the primitively eusocial bumble bees and the stingless bees.

Pulsed neodymium-YAG laser trabeculotomy: energy requirements and replicability.

Short pulsed laser trabeculotomy has been shown to reduce intraocular pressure in patients with primary open angle glaucoma. This study seeks to determine the energy levels required to produce a fistula into the canal of Schlemm for four different Q-switched neodymium-YAG lasers. The laser was fired at fixed human trabecular meshwork specimens at a range of energy settings for each laser and the characteristics and replicability of the lesions produced were analysed. Energy levels between 3 and 5 mJ were sufficient to produce fistulae into the canal of Schlemm with an approximately 50% success rate for each instrument.

Short-pulsed neodymium-YAG laser trabeculotomy. An in vivo morphological study in the human eye.

The in vivo response to short-pulsed Nd-YAG laser damage to the trabecular meshwork has not been studied in the human eye. The nature of the response will determine the potential efficacy of this treatment for glaucoma. We have investigated short-pulsed laser trabeculotomy lesions created in the trabecular meshwork of four human eyes within 18 hr prior to enucleation for intraocular melanoma. Scanning electron micrographs showed irregular craters (150-300 micron diameter) in the trabecular meshwork surrounded by trabecular beams which were splayed towards the anterior chamber. The adjacent damage to trabecular and corneal tissues was characterized by denudation of endothelial cells and deposition of debris. Light and transmission electron micrographs of the edge of the trabeculotomy lesions revealed fragmentation of the endothelial cells and splitting of the trabecular beams. Preservation of normal morphology was noted in the deeper tissues within 50 micron of the edge of the crater. Neutrophils were present within 20 min of laser treatment whilst macrophages characterised the inflammatory response at later stages. Perforation of the canal of Schlemm was only obtained with lesions in the middle of the trabecular meshwork but not with lesions placed more anteriorly.

Parameters for neodymium-YAG laser trabeculotomy: an in-vitro study.

A Biophysic Médical Nanolas Q-Switched pulsed neodymium-YAG laser has been used to produce lesions in human trabecular meshwork in vitro. Sectors of corneoscleral tissue containing trabecular meshwork were suspended in a waterbath which had had a Trokel gonioscopic contact lens mounted into one side by means of a watertight seal. The laser was used to produce lesions in the trabecular meshwork on either side of each specimen. The energy levels delivered ranged from 0.5 to 7.0 mJ, and convergence angles of 10 degrees and 18 degrees were employed. The energy levels required to produce discrete lesions into the canal of Schlemm without perforating the underlying sclera were 3.0-5.0 mJ at the 10 degrees convergence angle and 4.0-6.0 mJ at the 18 degrees convergence angle setting. It is recommended that such data be determined for each type of laser prior to attempting short-pulsed laser internal trabeculotomy in patients with glaucoma.

Aiming criteria for neodymium-YAG laser trabeculotomy. A clinico-pathological study.

Recently reported clinical and experimental studies have shown that pulsed neodymium-YAG laser trabeculotomy may reduce intraocular pressure but not in all cases. We report a clinico-pathological study designed to delineate the aiming criteria required to produce a fistula between the anterior chamber and the canal of Schlemm. In summary, the aiming beam must be centred on the reflecting surface of the gonioscopic contact lens, and aimed at the posterior trabecular meshwork. This study also indicates that the energy levels required to produce a discrete trabeculotomy in vivo are similar to those determined by in vitro studies.

Brood care by male bumble bees.

Male Bombus griseocollis (Hymenoptera: Apidae) share in the brood care of nestmates by incubating pupae (usually during the first few days after they emerge as adults). Male posture during incubation of a pupa is identical to that observed for females. Pupae incubated by males were 4 degrees C-6 degrees C above the temperature of unincubated pupae. Although this increase was not as great as that caused by workers or queens, it was an important factor in warming pupae. Incubating males may benefit nestmates incidentally without lowering their own individual fitness.