Allen F. Ryan

Commensal bacteria regulate Toll-like receptor 3― dependent inflammation after skin injury

The normal microflora of the skin includes staphylococcal species that will induce inflammation when present below the dermis but are tolerated on the epidermal surface without initiating inflammation. Here we reveal a previously unknown mechanism by which a product of staphylococci inhibits skin inflammation. This inhibition is mediated by staphylococcal lipoteichoic acid (LTA) and acts selectively on keratinocytes triggered through Toll-like receptor 3(TLR3). We show that TLR3 activation is required for normal inflammation after injury and that keratinocytes require TLR3 to respond to RNA from damaged cells with the release of inflammatory cytokines. Staphylococcal LTA inhibits both inflammatory cytokine release from keratinocytes and inflammation triggered by injury through a TLR2-dependent mechanism. To our knowledge, these findings show for the first time that the skin epithelium requires TLR3 for normal inflammation after wounding and that the microflora can modulate specific cutaneous inflammatory responses.

Distribution of the P2X2 receptor subunit of the ATP‐gated ion channels in the rat central nervous system

The distribution of the P2X2 receptor subunit of the adenosine 5′‐triphosphate (ATP)‐gated ion channels was examined in the adult rat central nervous system (CNS) by using P2X2 receptor‐specific antisera and riboprobe‐based in situ hybridisation. P2X2 receptor mRNA expression matched the P2X2 receptor protein localisation. An extensive expression pattern was observed, including: olfactory bulb, cerebral cortex, hippocampus, habenula, thalamic and subthalamic nuclei, caudate putamen, posteromedial amygdalo‐hippocampal and amygdalo‐cortical nuclei, substantia nigra pars compacta, ventromedial and arcuate hypothalamic nuclei, supraoptic nucleus, tuberomammillary nucleus, mesencephalic trigeminal nucleus, dorsal raphe, locus coeruleus, medial parabrachial nucleus, tegmental areas, pontine nuclei, red nucleus, lateral superior olive, cochlear nuclei, spinal trigeminal nuclei, cranial motor nuclei, ventrolateral medulla, area postrema, nucleus of solitary tract, and cerebellar cortex. In the spinal cord, P2X2 receptor expression was highest in the dorsal horn, with significant neuronal labeling in the ventral horn and intermediolateral cell column. The identification of extensive P2X2 receptor immunoreactivity and mRNA distribution within the CNS demonstrated here provides a basis for the P2X receptor antagonist pharmacology reported in electrophysiological studies. These data support the role for extracellular ATP acting as a fast neurotransmitter at pre‐ and postsynaptic sites in processes such as sensory transmission, sensory‐motor integration, motor and autonomic control, and in neuronal phenomena such as long‐term potentiation (LTP) and depression (LTD). Additionally, labelling of neuroglia and fibre tracts supports a diverse role for extracellular ATP in CNS homeostasis. J. Comp. Neurol. 407:11–32, 1999.

Activation of TLR2 by a Small Molecule Produced by Staphylococcus epidermidis Increases Antimicrobial Defense against Bacterial Skin Infections

Production of antimicrobial peptides by epithelia is an essential defense against infectious pathogens. In this study we evaluated whether the commensal microorganism Staphylococcus epidermidis may enhance production of antimicrobial peptides by keratinocytes and thus augment skin defense against infection. Exposure of cultured undifferentiated human keratinocytes to a sterile nontoxic small molecule of <10 kDa from S. epidermidis conditioned culture medium (SECM), but not similar preparations from other bacteria, enhanced human beta-defensin 2 (hBD2) and hBD3 mRNA expression and increased the capacity of cell lysates to inhibit the growth of group A Streptococcus (GAS) and S. aureus. Partial gene silencing of hBD3 inhibited this antimicrobial action. This effect was relevant in vivo as administration of SECM to mice decreased susceptibility to infection by GAS. Toll-like receptor 2 (TLR2) was important to this process as a TLR2-neutralizing antibody blocked induction of hBDs 2 and 3, and Tlr2-deficient mice did not show induction of mBD4. Taken together, these findings reveal a potential use for normal commensal bacterium S. epidermidis to activate TLR2 signaling and induce antimicrobial peptide expression, thus enabling the skin to mount an enhanced response to pathogens.

The development of auditory function in the cochlea of the mongolian gerbil.

Cochlear microphonic (CM) potentials were recorded throughout the development of auditory function in the Mongolian gerbil. CM responses were first recorded at 12 days after birth (DAB), with thresholds exceeding 103 dB SPL. CM thresholds subsequently improved rapidly in a parallel fashion across the responsive frequency range, achieving adult levels by 18 DAB. There was no evidence from CM thresholds of a preferential rate of maturation for either the high or low frequency ranges. CM responses to suptrathreshold stimulation were also studied throughout development. At all responsive ages, CM input-output functions increased logarithmically to a saturating maximum value and then declined. Maximum CM responses increased continuously throughout development in a parallel fashion across all frequencies. The dynamic range of the CM input-output functions (the intensity interval from CM threshold to maximum CM value) also developed in a parallel manner across frequencies, but reached mature assymptotic values by 16 DAB. The results suggest that throughout the adult CM frequency range cochlear hair cell function develops simultaneously.

N-Glycolylneuraminic Acid Deficiency in Mice: Implications for Human Biology and Evolution

ABSTRACT Humans and chimpanzees share >99% identity in most proteins. One rare difference is a human-specific inactivating deletion in the CMAH gene, which determines biosynthesis of the sialic acid N-glycolylneuraminic acid (Neu5Gc). Since Neu5Gc is prominent on most chimpanzee cell surfaces, this mutation could have affected multiple systems. However, Neu5Gc is found in human cancers and fetuses and in trace amounts in normal human tissues, suggesting an alternate biosynthetic pathway. We inactivated the mouse Cmah gene and studied the in vivo consequences. There was no evidence for an alternate pathway in normal, fetal, or malignant tissue. Rather, null fetuses accumulated Neu5Gc from heterozygous mothers and dietary Neu5Gc was incorporated into oncogene-induced tumors. As with humans, there were accumulation of the precursor N-acetylneuraminic acid and increases in sialic acid O acetylation. Null mice showed other abnormalities reminiscent of the human condition. Adult mice showed a diminished acoustic startle response and required higher acoustic stimuli to increase responses above the baseline level. In this regard, histological abnormalities of the inner ear occurred in older mice, which had impaired hearing. Adult animals also showed delayed skin wound healing. Loss of Neu5Gc in hominid ancestors ∼2 to 3 million years ago likely had immediate and long-term consequences for human biology.

The relationship of local cerebral glucose utilization to optical density ratios

The validity of optical density ratios used in [14C]2-deoxyglucose neuroanatomical mapping experiments is evaluated by comparing local cerebral glucose utilization (LCGU) and optical density (OD) ratios in the same animals. OD ratios are calculated by dividing the optical density of different gray matter structures by the optical density of a single white matter structure in each animal. OD ratios are linearly related to LCGU within a given animal including stimulated, highly activated regions. Anesthesia profoundly affects the relationship between LCGU and OD ratios, however, showing that OD ratios do not provide an accurate index of LCGU between animals in different physiological states. Anesthesia had only a slight effect on OD ratios, however, indicating that OD ratios may be helpful in assessing whether structures are functionally activated between animals in different physiological states.

A Forward Genetics Screen in Mice Identifies Recessive Deafness Traits and Reveals That Pejvakin Is Essential for Outer Hair Cell Function

Deafness is the most common form of sensory impairment in the human population and is frequently caused by recessive mutations. To obtain animal models for recessive forms of deafness and to identify genes that control the development and function of the auditory sense organs, we performed a forward genetics screen in mice. We identified 13 mouse lines with defects in auditory function and six lines with auditory and vestibular defects. We mapped several of the affected genetic loci and identified point mutations in four genes. Interestingly, all identified genes are expressed in mechanosensory hair cells and required for their function. One mutation maps to the pejvakin gene, which encodes a new member of the gasdermin protein family. Previous studies have described two missense mutations in the human pejvakin gene that cause nonsyndromic recessive deafness (DFNB59) by affecting the function of auditory neurons. In contrast, the pejvakin allele described here introduces a premature stop codon, causes outer hair cell defects, and leads to progressive hearing loss. We also identified a novel allele of the human pejvakin gene in an Iranian pedigree that is afflicted with progressive hearing loss. Our findings suggest that the mechanisms of pathogenesis associated with pejvakin mutations are more diverse than previously appreciated. More generally, our findings demonstrate that recessive screens in mice are powerful tools for identifying genes that control the development and function of mechanosensory hair cells and cause deafness in humans, as well as generating animal models for disease.

Fundamental immune mechanisms of the brain and inner ear

Because of the blood-brain and blood-labyrinthine barriers, the brain and inner ear were once thought to be immunoprivileged sites. Although these barriers provide protection from inflammatory damage to the delicate structures of the organs, both sites have since been shown to be capable of active immune responses when appropriately stimulated. In the inner ear, perisacular tissue around the endolymphatic sac hosts resident lymphocytes and serves as a site of immunosurveillance. Lymphocytes also enter the inner ear from the circulation, and in the cochlea this occurs via the spiral modiolar vein. Immune responses can protect the labyrinth from infection, but they can also cause bystander injury. Moreover, the cochlea can itself become the target of immune responses that damage hearing. Such autoimmune sensorineural hearing loss can be site specific, with the primary manifestation of the disorder being hearing loss and dysequilibrium. Some of these cases can be diagnosed by antibody or lymphocyte responses to inner ear antigens. Alternately, systemic autoimmune disorders can result in inner ear dysfunction as part of a broader spectrum of disease. Both forms of immune-mediated inner ear dysfunction may respond to immunosuppressive therapies, including steroids, cytotoxic agents, and plasmapheresis.

Spiral ganglion cell density in young and old gerbils

The Mongolian gerbil, like other mammalian species, has a decreased number of spiral ganglion cells as a function of age. This loss of cells was first seen in 24- to 30-month old animals in the basal end of the ganglion. In the oldest individuals the apical end of the ganglion was also affected. There were approximately 15-25% fewer cells in the affected areas in the 36- to 42-month old animals. In the oldest animals degeneration of the stria vascularis was seen in the apical turn and some degenerative changes in the organ of Corti were seen throughout the length of the cochlear duct. The aging pattern in the gerbil cochlea, is similar to that described for other species. Vacuoles, previously described in the gerbil cochlear nucleus, were also seen in the auditory nerve within the modiolus, but central to the Schwann-glial border in all animals. Vacuoles were not present within the spiral ganglion or the peripheral processes of the ganglion cells. Because the ganglion cell axons should be similar on either side of the Schwann-glial border, but the vacuoles were confined to the central nervous system, it is concluded that the degenerative process affects glial cells as opposed to neurons.

Neural phase-locking properties in the absence of cochlear outer hair cells.

A combined regimen of kanamycin sulfate treatment (175 mg/kg/day) and behavioral evaluation of resulting audiometric threshold shifts was used to produce selective outer hair cells (OHC) loss in chinchillas. This protocol resulted in a 3-7 mm region in the cochlear base in which OHCs were completely absent and inner hair cells (IHCs) were largely resent and normal at both light and electron microscopic levels. Partial OHC loss was associated with audiometric threshold shifts in excess of 15 dB, while complete OHC loss was associated with audiometric threshold shifts in excess of 40 dB. After recovery periods of at least three weeks, phase-locking was examined across frequency for auditory nerve (VIIIth nerve) and ventral cochlear nucleus (VCN) neurons. The frequency range for neural phase-locking in normal subjects extended up to approximately 4 kHz for VIIIth nerve fibers and 3 kHz for VCN neurons. Following kanamycin intoxication, however, the frequency range for neural phase-locking in both of these auditory regions varied with characteristic frequency (CF): neurons whose CF corresponded to normal cochlear regions exhibited phase-locking throughout the normal frequency range; neurons whole CF corresponded to cochlear regions with selective OHC loss exhibited a marked reduction in the frequency range over which they could phase-lock.