Quetiapine and its metabolite norquetiapine: translation from in vitro pharmacology to in vivo efficacy in rodent models.

BACKGROUND AND PURPOSE: Quetiapine has a range of clinical activity distinct from other atypical antipsychotic drugs, demonstrating efficacy as monotherapy in bipolar depression, major depressive disorder and generalized anxiety disorder. The neuropharmacological mechanisms underlying this clinical profile are not completely understood; however, the major active metabolite, norquetiapine, has been shown to have a distinct in vitro pharmacological profile consistent with a broad therapeutic range and may contribute to the clinical profile of quetiapine. EXPERIMENTAL APPROACH: We evaluated quetiapine and norquetiapine, using in vitro binding and functional assays of targets known to be associated with antidepressant and anxiolytic drug actions and compared these activities with a representative range of established antipsychotics and antidepressants. To determine how the in vitro pharmacological properties translate into in vivo activity, we used preclinical animal models with translational relevance to established antidepressant-like and anxiolytic-like drug action. KEY RESULTS: Norquetiapine had equivalent activity to established antidepressants at the noradrenaline transporter (NET), while quetiapine was inactive. Norquetiapine was active in the mouse forced swimming and rat learned helplessness tests. In in vivo receptor occupancy studies, norquetiapine had significant occupancy at NET at behaviourally relevant doses. Both quetiapine and norquetiapine were agonists at 5-HT1A receptors, and the anxiolytic-like activity of norquetiapine in rat punished responding was blocked by the 5-HT1A antagonist, WAY100635. CONCLUSIONS AND IMPLICATIONS: Quetiapine and norquetiapine have multiple in vitro pharmacological actions, and results from preclinical studies suggest that activity at NET and 5-HT1A receptors contributes to the antidepressant and anxiolytic effects in patients treated with quetiapine.

Vaginal microbiota of spayed dogs with or without recurrent urinary tract infections.

BACKGROUND: Limited information is available regarding the vaginal microbiota of normal spayed dogs and spayed dogs with recurrent UTIs. Vaginal lactic acid-producing bacteria (LAB) have been associated with decreased frequency of recurrent urinary tract infection in women and may have a protective role within the urinary tract of female dogs. HYPOTHESIS/OBJECTIVES: Spayed dogs with historical recurrent UTI will have decreased prevalence of LAB and increased prevalence of uropathogenic bacterial populations in the vaginal microbiota when compared with the vaginal microbiota of healthy, spayed dogs. ANIMALS: Twenty-one client-owned adult spayed female dogs with historical recurrent UTI and 23 healthy, spayed female dogs without a history of recurrent UTI. METHODS: Dogs were placed into a recurrent UTI group or control group in this prospective study. Bacterial populations were isolated and characterized from vaginal swabs obtained from each dog. RESULTS: The most common bacterial isolates obtained from the vaginal tract of all dogs were Escherichia coli (11/44) and S. pseudintermedius (13/44). E. coli was isolated from the vaginal tract of 8 of 21 (38%) dogs in the rUTI group and 3 of 23 (13%) dogs in the control group (P = .08). LAB were isolated from 7 of the 44 dogs. Two of these 7 dogs were in the rUTI group and 5 of the 7 dogs were in the control group. CONCLUSIONS AND CLINICAL IMPORTANCE: The vaginal microbiota of spayed female dogs with recurrent UTI was similar to the control population of normal, spayed female dogs.

The effect of an oral probiotic containing lactobacillus, bifidobacterium, and bacillus species on the vaginal microbiota of spayed female dogs.

BACKGROUND: Recurrent urinary tract infections (UTIs) are often difficult to treat. Vaginal colonization with lactic acid-producing bacteria (LAB) is associated with reduced frequency of recurrent UTIs in women. Oral probiotics might help increase the prevalence of vaginal LAB and decrease the frequency of recurrent UTIs in dogs. HYPOTHESIS: Administration of an oral probiotic supplement containing Lactobacillus, Bifidobacterium, and Bacillus species will increase the prevalence of LAB in the vagina of dogs. ANIMALS: Thirty-five healthy, spayed female dogs without history of recurrent UTIs. METHODS: Prospective, controlled study. Enrolled dogs received an oral probiotic supplement for 14 or 28 days. A vaginal tract culture was obtained from each dog before and after oral probiotic administration. Twenty-three dogs received the oral probiotic supplement daily for a period of 14 days and 12 dogs received the oral probiotic supplement daily for a period of 28 days. RESULTS: Lactic acid-producing bacteria were isolated from 7 of 35 dogs prior to probiotic administration. After the treatment course, 6 of 35 dogs had LAB isolated. Only one of these dogs had LAB (Enterococcus canintestini) isolated for the first time. Enterococcus canintestini was the most common LAB isolated from all dogs in this study, although it was not included in the probiotic supplement. CONCLUSIONS AND CLINICAL IMPORTANCE: Lactic acid-producing bacteria are not a common isolate from the vaginal vault of dogs. Administration of this oral probiotic supplement for a 2- or 4-week period did not increase the prevalence of vaginal LAB in dogs.

Uropathogenic E. coli promote a paracellular urothelial barrier defect characterized by altered tight junction integrity, epithelial cell sloughing and cytokine release.

The urinary bladder is a common site of bacterial infection with a majority of cases attributed to uropathogenic Escherichia coli. Sequelae of urinary tract infections (UTIs) include the loss of urothelial barrier function and subsequent clinical morbidity secondary to the permeation of urine potassium, urea and ammonia into the subepithelium. To date there has been limited research describing the mechanism by which this urothelial permeability defect develops. The present study models acute uropathogenic E. coli infection in vitro using intact canine bladder mucosa mounted in Ussing chambers to determine whether infection induces primarily a transcellular or paracellular permeability defect. The Ussing chamber sustains tissue viability while physically separating submucosal and lumen influences, so this model is ideal for quantitative measurement of transepithelial electrical resistance (TER) to assess alterations of urothelial barrier function. Using this model, changes in both tissue ultrastructure and TER indicated that uropathogenic E. coli infection promotes a paracellular permeability defect associated with the failure of umbrella cell tight junction formation and umbrella cell sloughing. In addition, bacterial interaction with the urothelium promoted secretion of cytokines from the urinary bladder with bioactivity capable of modulating epithelial barrier function including tumour necrosis factor-α, interleukin (IL)-6 and IL-15. IL-15 secretion by the infected bladder mucosa is a novel finding and, because IL-15 plays key roles in reconstitution of tight junction function in damaged intestine, this study points to a potential role for IL-15 in UTI-induced urothelial injury.

Semi-implantable electromagnetic middle ear hearing device for moderate to severe sensorineural hearing loss.

Despite the many improvements in hearing aid technology, conventional hearing aids continue to have significant limitations, which has led to increased interest in implantable hearing devices. The SOUNDTEC Direct Drive Hearing System for moderate to moderately severe sensorineural hearing loss is one such device. In this article the authors present results on five individuals enrolled in a Food And Drug Administration Phase I feasibility study.

Mass loading on the ossicles and middle ear function.

The middle ear as a levered vibrating system for sound transmission from the external to the inner ear is affected by changes in ossicular chain mass. Mass loading of the ossicles may impair ossicular dynamics and sound transmission to the inner ear. It is incumbent on otologic surgeons and researchers of middle ear mechanics to consider the mass loading effect on middle ear function in clinical and physiological applications. The residual hearing and frequency response can change after surgery or implantation of middle ear prostheses. We conducted experiments on mass loading effects on the middle ear transfer functions by using laser Doppler interferometry and a human temporal bone model. Two implant mass loading conditions were tested on 17 fresh or fresh-frozen temporal bones and compared with the unloaded condition for the frequencies 250 to 8,000 Hz. The results show that the linearity of the middle ear function did not change, although displacement of the stapes footplate decreased after the increased masses were placed on the incudostapedial joint. The greater the mass of the implant, the less displacement was measured at the stapes footplate. We conclude that there is a quantitative limit to increased mass on the ossicular chain above which the mass will remarkably impair hearing thresholds.

Early clinical results: SOUNDTEC implantable hearing device phase II study.

OBJECTIVE: To assess the safety and efficacy of a new semi-implantable electromagnetic hearing device, the SOUNDTEC Direct Drive Hearing System (DDHS), and to compare its performance with that of subjects' previously worn, optimally fit hearing aids. Preliminary results for the first 10 subjects are presented. STUDY DESIGN: The protocol specified in the Investigational Device Exemption is used in this ongoing FDA phase II 100-subject multicenter clinical trial. METHODS: For baseline, each subject is tested wearing his or her own optimally fit hearing aid in the ear to be implanted. Six-month postoperative outcome measures using the SOUNDTEC DDHS are compared with the baseline. Multiple objective and subjective outcomes (as listed under Results) were measured. RESULTS: When compared with the subjects' optimally fit hearing aids, the SOUNDTEC DDHS provided an average improvement of 52% in functional gain (250-6000 Hz), 22% in aided thresholds, 3.8% for speech discrimination in quiet, 17% for speech in noise, 13.1% in articulation index scores, 28% in aided benefit, 27.3% in sound quality of speech, and a 16.7% increase in overall subject satisfaction. In addition, with the SOUNDTEC DDHS, subjects reported absence of acoustic feedback, little or no occlusive effects, and more natural sound perception. CONCLUSION: Analysis of data on the first 10 subjects using the SOUNDTEC DDHS indicates positive outcomes regarding safety and efficacy, although the small sample size is not sufficient to permit valid statistical inferences to be drawn from our preliminary data. Results also demonstrate improvement in performance compared with the subjects' optimally fit hearing aids and an improvement in quality of life as demonstrated by objective and subjective tests and measures.

Inflammatory cytokines enhance muscarinic-mediated arachidonic acid release through p38 mitogen-activated protein kinase in A2058 cells.

The human melanoma cell line A2058 expresses the Gq-coupled M5 subtype of muscarinic receptor. Stimulation with the cholinergic agonist, carbachol, induces a dose-dependent increase in arachidonic acid release. The carbachol-induced arachidonate release is potentiated two- to threefold by pretreatment of A2058 cells with either of the inflammatory cytokines, tumor necrosis factor-alpha or interleukin-1beta . Cytokine-induced enhancement of muscarinic-mediated arachidonic acid release peaks near 1 h. Western analysis suggests that both cytokines are capable of activating the nuclear factor-kappaB (NF-kappaB) and p38 mitogen-activated protein kinase (MAPK) pathways. Anisomycin (1 microM) treatment mimics the cytokine-induced enhancement of arachidonic acid production and activates the p38 MAPK pathway, but does not activate the NF-kappaB pathway. Furthermore, pre-treatment of A2058 cells with the putative p38 MAPK inhibitor, SB202190, ablates the cytokine-dependent augmentation without interfering with the muscarinic-mediated arachidonic acid release in untreated cells. Moreover, cytokine treatment does not affect other M5-coupled pathways (e.g., phospholipase C activity or intracellular Ca2+ mobilization), suggesting that p38 MAPK activation principally modulates muscarinic-mediated phospholipase A2 activity. Finally, in primary cultures of cells taken from rat cerebellum, key aspects of this finding are repeated in cultures enriched for glia, but not in cultures enriched for granule neurons.

Identification of SopE2, a Salmonella secreted protein which is highly homologous to SopE and involved in bacterial invasion of epithelial cells.

Type III secreted Sop protein effectors are delivered into target eukaryotic cells and elicit cellular responses underlying Salmonella pathogenicity. In this work, we have identified another secreted protein, SopE2, and showed that SopE2 is an important invasion-associated effector. SopE2 is encoded by the sopE2 gene which is present and conserved in pathogenic strains of Salmonella. SopE2 is highly homologous to SopE, a protein encoded by a gene within a temperate bacteriophage and present in only some pathogenic strains.

The secreted effector protein of Salmonella dublin, SopA, is translocated into eukaryotic cells and influences the induction of enteritis.

Salmonella-induced enteritis is associated with the induction of an acute intestinal inflammatory response and net fluid secretion into the lumen of infected mucosa. Proteins secreted by the Inv/Spa type III secretion system of Salmonella play a key role in the induction of these responses. We have demonstrated recently that the Inv/Spa-secreted SopB and SopD effector proteins are translocated into eukaryotic cells via a Sip-dependent pathway and act in concert to mediate inflammation and fluid secretion in infected ileal mucosa. Mutations of both sopB and sopD significantly reduced, but did not abrogate, the enteropathogenic phenotype. This indicated that other virulence factors are involved in the induction of enteritis. In this work, we characterize SopA, a secreted protein belonging to the family of Sop effectors of Salmonella dublin. We demonstrate that SopA is translocated into eukaryotic cells and provide evidence suggesting that SopA has a role in the induction of enteritis.

A mutation in the glycine binding pocket of the N-methyl-D-aspartate receptor NR1 subunit alters agonist efficacy.

Alanine 714 of the NMDA receptor NR1 subunit resides in the glycine binding pocket. The Ala714Leu mutation substantially shifts glycine affinity, but here no effect on antagonism by DCK is detected. Ala714Leu is also found to limit the efficacy of a partial agonist without altering its apparent affinity. The differential sensitivity of Ala714Leu to glycine agonists suggests that alanine 714 may be an intermediary in transducing the ligand binding signal.

Secreted effector proteins of Salmonella dublin act in concert to induce enteritis.

The ability of enteropathogenic salmonellae to recruit inflammatory cells and induce secretory responses in the infected ileum is considered to be a main feature in Salmonella-induced enteritis. Interactions between the pathogen and intestinal epithelial cells result in a variety of cellular responses mediating inflammation and fluid secretion. It is becoming apparent that proteins secreted by the Inv-Spa type III secretion system of Salmonella spp. play a key role in the induction of these responses. We have recently demonstrated that the SopB effector protein is translocated into eukaryotic cells via a Sip-dependent pathway and mediates inflammation and fluid secretion in infected ileal mucosa. However, SopB did not appear to be the only effector involved, as inactivation of the sopB gene only partially impaired enteropathogenicity. We suggested that at least some of such protein effectors are likely to be proteins of the same class as SopB, i.e., secreted effector proteins translocated into eukaroyotic cells via a Sip-dependent pathway. In this work, we identify SopD, another secreted protein belonging to the family of Sop effectors of Salmonella dublin. Using the cya reporter system we showed that SopD is translocated into eukaroyotic cells. We assessed the potential involvement of SopD in enteropathogenicity and found that inactivation of sopD has an additive effect in relation to the sopB mutation.

Identification of a pathogenicity island required for Salmonella enteropathogenicity.

Salmonella spp. interact with ileal mucosa and disrupt normal intestinal function, which results in an acute inflammatory cell influx, fluid secretion and enteritis. We have recently characterized SopB, a novel secreted effector protein of Salmonella dublin, and presented evidence that SopB is translocated into eukaryotic cells via a sip-dependent pathway to promote fluid secretion and inflammatory responses. Here, we show that sopB is located on a large DNA fragment unique to the Salmonella chromosome. This locus is conserved in Salmonella and maps at approximately 20 centisome of the S. typhimurium chromosome. Sequence analysis revealed that this Salmonella-specific DNA fragment is flanked by DNA sequences with significant sequence similarity to the Escherichia coli K-12 genes, tRNA1ser (serT) on one side and copS/copR on the other. Thus, this Salmonella-specific DNA fragment has features characteristic of 'pathogenicity islands' and, therefore, it was denoted SPI-5 (Salmonella pathogenicity island-5). SPI-5 was sequenced and was found to contain five novel genes, pipA, pipB, pipC, pipD (pathogenicity island-encoded proteins) and orf, in addition to sopB. The effect of mutations in pipA, pipB and pipD on the induction of fluid secretion and an acute inflammatory cell influx was assessed in bovine ligated ileal loops. The effect of mutations in SPI-5-encoded genes on systemic salmonellosis was assessed in mice. The results of these experiments suggest that SPI-5-encoded genes contribute to enteric but not to systemic salmonellosis.

A secreted effector protein of Salmonella dublin is translocated into eukaryotic cells and mediates inflammation and fluid secretion in infected ileal mucosa.

Enteritis induced by non-typhoid pathogenic Salmonella is characterized by fluid secretion and inflammatory responses in the infected ileum. The inflammatory response provoked by Salmonella initially consists largely of a neutrophil (PMN) migration into the intestinal mucosa and the gut lumen. The interactions between Salmonella and intestinal epithelial cells are known to play an essential role in inducing the inflammatory response. Upon interaction with epithelial cells salmonellae are able to elicit transepithelial signalling to neutrophils. This signalling is recognized as a key virulence feature underlying Salmonella-induced enteritis. However, the nature and mechanism of such signalling has not been clarified to date. Here, we characterize SopB, a novel secreted effector protein of Salmonella dublin, and present data implying that SopB is translocated into eukaryotic cells via a sip-dependent pathway to promote fluid secretion and inflammatory responses in the infected ileum.

Implantable hearing device performance measured by laser Doppler interferometry.

Recent application of the Doppler principle laser interferometry to audiology, acoustics and otology has facilitated the development of implantable hearing devices (IHDs). During the design and testing of two different electromagnetic middle ear implants for sensorineural hearing loss, we used single-point laser Doppler interferometry (LDI). A commercially available interferometer, internally calibrated and validated against a National Institute for Standards and Technology (NIST) standard, was used with both mechanical fixtures and fresh temporal bones to evaluate implant mass, shape and orientation, attachment, electromagnetic coupling and acoustic properties. At both Hough Ear Institute and Symphonix Devices, Inc., we have shown that high fidelity and amplitudes can be recorded in vitro over a frequency range of 500 Hz to 10 kHz. These data can provide greater assurance of safety and efficacy to regulatory agencies before entering clinical trials. We propose that LDI be considered as an international standard for accurate, consistent comparison of performances of all IHDs during development. Furthermore, the future availability of human IHD data will allow for the extrapolation of a mechanical bench model of the middle ear transfer function for use in quality control during manufacturing and diagnosis of failure in IHDs.

An alanine residue in the M3-M4 linker lines the glycine binding pocket of the N-methyl-D-aspartate receptor.

While attempting to map a central region in the M3-M4 linker of the N-methyl-D-aspartate receptor NR1 subunit, we found that mutation of a single position, Ala-714, greatly reduced the apparent affinity for glycine. Proximal N-glycosylation localized this region to the extracellular space. Glycine affinities of additional Ala-714 mutations correlated with side chain volume. Substitution of alanine 714 with cysteine did not alter glycine sensitivity, although this mutant was rapidly inhibited by dithionitrobenzoate. Glycine protected the A714C mutant from modification by dithionitrobenzoate, whereas the co-agonist L-glutamate was ineffective. These experiments place Ala-714 in the glycine binding pocket of the N-methyl-D-aspartate receptor, a determination not predicted by previous structural models based on bacterial periplasmic binding protein homology.

SopE, a secreted protein of Salmonella dublin, is translocated into the target eukaryotic cell via a sip-dependent mechanism and promotes bacterial entry.

The entry of Salmonella into cultured epithelial cells is dependent on genes located in several adjacent chromosomal loci. One of these loci encodes the recently identified secretory proteins, denoted Sips (Salmonella invasion proteins). SipB, C,D proteins are essential for the ability of the pathogen to invade epithelial cells. To examine if additional invasion-associated proteins were secreted by Salmonella dublin, the genes encoding already characterized secretory proteins were inactivated to facilitate this analysis. The proteins produced and secreted by a double fIIM/polar sipB mutant of S. dublin were analysed; this revealed a set of novel secreted proteins. These proteins, which we denoted Sops (Salmonella outer proteins), formed large filamentous aggregates in the medium of bacterial culture growing at 37 degrees C. These aggregates contained five predominant proteins. Here we report the identification and characterization of one of these proteins, SopE, which is a novel invasion-associated secretory protein of S. dublin. A specific sopE mutant of S. dublin was found to be defective for invasion into epithelial cells. Upon interaction of Salmonella with HeLa cells, SopE was found to be translocated into the cytoplasm of the target cell by extracellular bacteria. The translocation of SopE was shown to be dependent on the Sip proteins because a polar sipB mutant did not translocate SopE across the HeLa cell membrane.

The 5'-untranslated region of the N-methyl-D-aspartate receptor NR2A subunit controls efficiency of translation.

The N-methyl-D-aspartate (NMDA) receptor plays a central role in such phenomena as long term potentiation and excitotoxicity. This importance in defining both function and viability suggests that neurons must carefully control their expression of NMDA receptors. Whereas the NR1 subunit of the NMDA receptor is ubiquitously transcribed throughout the brain, transcription of NR2 subunits is spatially and temporally controlled. Since heteromeric assembly of both subunits is required for efficient functional expression, post-transcriptional modification of either subunit would affect NMDA receptor activity. Here it is demonstrated that the 5'-untranslated region (5'-UTR) of the NR2A subunit severely restricts its protein translation in both Xenopus oocytes and in an in vitro translation system. Mutational analysis of the 5'-UTR implicates secondary structure as the major translational impediment, while the five alternate start codons play minor roles. An important biological role for the 5'-UTR of NR2A is further suggested by the unusually high level of sequence conservation between species. In contrast, the 5'-UTR of NR1 does not inhibit translation and is not consrved. Taken together, these findings suggest a mechanism for modulation of NMDA receptor activity through the control of translational efficiency of a single subunit.

Structural conservation of ion conduction pathways in K channels and glutamate receptors.

Single channel recordings demonstrate that ion channels switch stochastically between an open and a closed pore conformation. In search of a structural explanation for this universal open/close behavior, we have uncovered a striking degree of amino acid homology across the pore-forming regions of voltage-gated K channels and glutamate receptors. This suggested that the pores of these otherwise unrelated classes of channels could be structurally conserved. Strong experimental evidence supports a hairpin structure for the pore-forming region of K channels. Consequently, we hypothesized the existence of a similar structure for the pore of glutamate receptors. In ligand-gated channels, the pore is formed by M2, the second of four putative transmembrane segments. A hairpin structure for M2 would affect the subsequent membrane topology, inverting the proposed orientation of the next segments, M3. We have tested this idea for the NR1 subunit of the N-methyl-D-aspartate receptor. Mutations that affected the glycosylation pattern of the NR1 subunit localize both extremes of the M3-M4 linker to the extracellular space. Whole cell currents and apparent agonist affinities were not affected by these mutations. Therefore it can be assumed that they represent the native transmembrane topology. The extracellular assignment of the M3-M4 linker challenged the current topology model by inverting M3. Taken together, the amino acid homology and the new topology suggest that the pore-forming M2 segment of glutamate receptors does not transverse the membrane but, rather, forms a hairpin structure, similar to that found in K channels.

The implantable hearing device for sensorineural hearing impairment. The Hough Ear Institute experience.

This article describes one group's experience in developing and implanting rare earth magnets for conductive hearing loss and also for sensorineural hearing loss. Success and failures from over a decade of work are highlighted.