Temporal Variability of the Dominant Fecal Microbiota in Healthy Adult Cats.

While shifts in gut microbiota have been studied in diseased states, the temporal variability of the microbiome in cats has not been widely studied. This study investigated the temporal variability of the feline dysbiosis index (DI) and the abundance of core bacterial groups in healthy adult cats. The secondary aim was to evaluate the relationship between the fecal abundance of Clostridium hiranonis and the fecal concentrations of unconjugated bile acids. A total of 142 fecal samples collected from 17 healthy cats were prospectively included: nine cats with weekly collection over 3 weeks (at least four time points), five cats with monthly collection over 2 months (three time points), and three cats with additional collections for up to 10 months. The DI remained stable within the reference intervals over two months for all cats (Friedman test, p > 0.2), and 100% of the DI values (n = 142) collected throughout the study period remained within the RI. While some temporal individual variation was observed for individual taxa, the magnitude was minimal compared to cats with chronic enteropathy and antibiotic exposure. Additionally, the abundance of Clostridium hiranonis was significantly correlated with the percentage of fecal primary bile acids, supporting its role as a bile acid converter in cats.

Pilot clinical trial: propidium monoazide PCR quantifies reduction of the viable bacterial load after antiseptic preparation of canine oral mucosa.

OBJECTIVE: A pilot clinical study to evaluate the use of propidium monoazide PCR (PMA-PCR) in quantifying a reduction of bacterial load after antiseptic use on the canine oral mucosa and skin, comparison of quantitative PCR (qPCR) to PMA-PCR, and comparison of patterns seen between PCR methods and bacterial culture. ANIMALS: Client-owned dogs (n = 10) undergoing general anesthesia and intravenous catheter placement. PROCEDURES: The oral mucosa and antebrachial skin of each dog underwent swabs for culture, qPCR, and PMA-PCR before and after antiseptic preparation of each site. Reduction in bacterial load between sampling times was evaluated for each quantification method. RESULTS: All testing methods found a significant decrease in bacterial load from oral mucosa after antiseptic preparation (culture P = .0020, qPCR P = .0039, PMA-PCR P = .0039). PMA-PCR had a significantly greater reduction of bacterial load after preparation than qPCR (P = .0494). Only culture detected a significant reduction after preparation of the skin (culture P = .0039, qPCR P = .3125, PMA-PCR P = .0703). CLINICAL RELEVANCE: PMA-PCR was able to quantify a reduction of bacterial load after antiseptic preparation of the high-bacterial load environment, with a pattern similar to that of culture, and was more specific than qPCR for detecting viable bacterial load. The results of this study support the use of PMA-PCR for antiseptic effectiveness studies performed on a high-bacterial load environment, such as canine oral mucosa.

Dysbiosis index to evaluate the fecal microbiota in healthy cats and cats with chronic enteropathies.

OBJECTIVES: Previous studies have identified various bacterial taxa that are altered in cats with chronic enteropathies (CE) vs healthy cats. Therefore, the aim of this study was to develop a targeted quantitative molecular method to evaluate the fecal microbiota of cats. METHODS: Fecal samples from 80 client-owned healthy cats and 68 cats with CE were retrospectively evaluated. A panel of quantitative PCR (qPCR) assays was used to measure the fecal abundance of total bacteria and seven bacterial taxa: Bacteroides, Bifidobacterium, Clostridium hiranonis, Escherichia coli, Faecalibacterium, Streptococcus and Turicibacter. The nearest centroid classifier algorithm was used to calculate a dysbiosis index (DI) based on these qPCR abundances. RESULTS: The abundances of total bacteria, Bacteroides, Bifidobacterium, C hiranonis, Faecalibacterium and Turicibacter were significantly decreased, while those of E coli and Streptococcus were significantly increased in cats with CE (P <0.027 for all). The DI in cats with CE was significantly higher compared with healthy cats (P <0.001). When the cut-off value of the DI was set at 0, it provided 77% (95% confidence interval [CI] 66-85) sensitivity and 96% (95% CI 89-99) specificity to differentiate the microbiota of cats with CE from those of healthy cats. Fifty-two of 68 cats with CE had a DI >0. CONCLUSIONS AND RELEVANCE: A qPCR-based DI for assessing the fecal microbiota of cats was established. The results showed that a large proportion of cats with CE had an altered fecal microbiota as evidenced by an increased DI. Prospective studies are warranted to evaluate the utility of this assay for clinical assessment of feline CE.

Prevalence and clinical characteristics of allergic rhinitis in the elderly Korean population.

PURPOSE: The proportion of elderly people aged ≥ 65 years is increasing worldwide. Although the reported prevalence of sinonasal disease can vary according to the diagnostic methods used, differences in allergic rhinitis prevalence in the elderly according to diagnostic method have not been reported. We thus aimed to evaluate allergic rhinitis prevalence in the elderly according to diagnostic criteria obtained from questionnaires, physician diagnoses, and allergy tests. METHODS: We compared the allergic rhinitis prevalence in the elderly aged ≥ 65 years with adults aged 19-64 years, using data from the Korean National Health and Nutrition Examination Survey 2008-2012. Total serum IgE and IgE levels specific to allergens of Dermatophagoides farina, cockroach, and dog dander were examined, and factors affecting specific IgE levels were investigated. RESULTS: Allergic rhinitis prevalence according to the questionnaire responses, physician diagnoses, and allergy test results was 35.02%, 14.89%, and 17.56%, respectively. The prevalence based on all diagnostic methods assessed was significantly lower in the elderly than in the general adult group (p < 0.001). Rhinorrhea incidence was significantly increased in the elderly (p = 0.018). Sensitization to Dermatophagoides farina was significantly decreased in the elderly (p = 0.006) and did not correlate with socioeconomic status and/or general health factors. CONCLUSIONS: The elderly population has a distinct clinical presentation, including a low prevalence of allergic rhinitis, and an increased incidence of rhinorrhea symptoms, compared with the general adult population. The management of allergic rhinitis in elderly patients may therefore require a different therapeutic approach to improve rhinorrhea rather than nasal obstruction.

Comparison of the clinical characteristics of bilateral and unilateral fungal balls in Korea.

OBJECTIVE: Fungal rhinosinusitis occurs in different forms depending on race and region. While allergic fungal rhinosinusitis is common in Caucasians, fungal ball (FB) is more common in Asians. However, most cases are reported as unilateral, and clinical data on bilateral FB (BFB) are rare. Therefore, the purpose of this study was to analyze and to compare the clinical characteristics of BFB and unilateral FB (UFB) in Koreans. METHODS: We retrospectively analyzed medical records and computed tomography (CT) images of 434 patients diagnosed with FB. The patients were divided into two groups: BFB and UFB. Demographic data, multiple allergen simultaneous test including total or specific immunoglobulin E (IgE) levels, symptoms, CT findings, treatment, and outcomes were analyzed. RESULTS: Among the patients, 26 had BFB and 408 had UFB. Hypertension was noted in 61.5% of the BFB and 39% of the UBF individuals (p = 0.023). While total IgE levels were similar between the two groups, Dermatophagoides pteronyssinus (p = 0.004), Cladosporium (p = 0.017), and Aspergillus-specific IgE positivity (p = 0.025) were significantly higher in the BFB than in the UFB group. Not only symptoms such as postnasal drip (p = 0.013), mucopurulent rhinorrhea (p = 0.009), and foul odor (p = 0.037), but also sphenoid sinus involvement on CT images were more common in the BFB than in the UFB group (p = 0.011). CONCLUSION: Patients with BFB in Korea showed more common hypertension and symptoms of foul odor, mucopurulent rhinorrhea, and postnasal drip with allergy positivity compared to those with UFB. Therefore, understanding clinical characteristics of BFB will allow clinicians to approach BFB more appropriately.

Brain abscess caused by chronic invasive actinomycosis in the nasopharynx: A case report and literature review.

RATIONALE: Actinomycosis is a rare anaerobic, gram-positive bacterial infection caused by Actinomyces, which is part of the normal flora in the oral cavity and respiratory and female genitourinary tracts. The cervicofacial area is the most common site of involvement, and involvement of the central nervous system is rare. PATIENT CONCERNS: We report a case involving a 51-year-old woman who developed an actinomycotic brain abscess 15 months after the treatment of noninvasive nasopharyngeal actinomycosis, which recurred as an invasive form. DIAGNOSES: Histopathological examination of the surgical specimens revealed actinomycosis. INTERVENTIONS: The patient was treated by surgical drainage of the brain abscess and long-term antibiotic treatment. OUTCOMES: Follow-up brain imaging performed 12 months after surgery showed complete resolution of the brain abscess, and there were no further signs or symptoms of infection. LESSONS: Physicians should be aware of the typical clinical presentations of cervicofacial actinomycosis. Moreover, they should know that actinomycosis may mimic the process of malignancy at various anatomical locations.

Chicken embryos as a potential new model for early onset type I diabetes.

Diabetic retinopathy (DR) is the leading cause of blindness among the American working population. The purpose of this study is to establish a new diabetic animal model using a cone-dominant avian species to address the distorted color vision and altered cone pathway responses in prediabetic and early diabetic patients. Chicken embryos were injected with either streptozotocin (STZ), high concentration of glucose (high-glucose), or vehicle at embryonic day 11. Cataracts occurred in varying degrees in both STZ- and high glucose-induced diabetic chick embryos at E18. Streptozotocin-diabetic chicken embryos had decreased levels of blood insulin, glucose transporter 4 (Glut4), and phosphorylated protein kinase B (pAKT). In STZ-injected E20 embryos, the ERG amplitudes of both a- and b-waves were significantly decreased, the implicit time of the a-wave was delayed, while that of the b-wave was significantly increased. Photoreceptors cultured from STZ-injected E18 embryos had a significant decrease in L-type voltage-gated calcium channel (L-VGCC) currents, which was reflected in the decreased level of L-VGCCα1D subunit in the STZ-diabetic retinas. Through these independent lines of evidence, STZ-injection was able to induce pathological conditions in the chicken embryonic retina, and it is promising to use chickens as a potential new animal model for type I diabetes.

Reactive oxygen species prevent imiquimod-induced psoriatic dermatitis through enhancing regulatory T cell function.

Psoriasis is a chronic inflammatory skin disease resulting from immune dysregulation. Regulatory T cells (Tregs) are important in the prevention of psoriasis. Traditionally, reactive oxygen species (ROS) are known to be implicated in the progression of inflammatory diseases, including psoriasis, but many recent studies suggested the protective role of ROS in immune-mediated diseases. In particular, severe cases of psoriasis vulgaris have been reported to be successfully treated by hyperbaric oxygen therapy (HBOT), which raises tissue level of ROS. Also it was reported that Treg function was closely associated with ROS level. However, it has been only investigated in lowered levels of ROS so far. Thus, in this study, to clarify the relationship between ROS level and Treg function, as well as their role in the pathogenesis of psoriasis, we investigated imiquimod-induced psoriatic dermatitis (PD) in association with Treg function both in elevated and lowered levels of ROS by using knockout mice, such as glutathione peroxidase-1(-/-) and neutrophil cytosolic factor-1(-/-) mice, as well as by using HBOT or chemicals, such as 2,3-dimethoxy-1,4-naphthoquinone and N-acetylcysteine. The results consistently showed Tregs were hyperfunctional in elevated levels of ROS, whereas hypofunctional in lowered levels of ROS. In addition, imiquimod-induced PD was attenuated in elevated levels of ROS, whereas aggravated in lowered levels of ROS. For the molecular mechanism that may link ROS level and Treg function, we investigated the expression of an immunoregulatory enzyme, indoleamine 2,3-dioxygenase (IDO) which is induced by ROS, in PD lesions. Taken together, it was implied that appropriately elevated levels of ROS might prevent psoriasis through enhancing IDO expression and Treg function.

Activity of the calcium channel pore Cch1 is dependent on a modulatory region of the subunit Mid1 in Cryptococcus neoformans.

Calcium (Ca(2+))-mediated signaling events in fungal pathogens such as Cryptococcus neoformans are central to physiological processes, including those that mediate stress responses and promote virulence. The Cch1-Mid1 channel (CMC) represents the only high-affinity Ca(2+) channel in the plasma membrane of fungal cells; consequently, cryptococci cannot survive in low-Ca(2+) environments in the absence of CMC. Previous electrophysiological characterization revealed that Cch1, the predicted channel pore, and Mid1, a binding partner of Cch1, function as a store-operated Ca(2+)-selective channel gated by depletion of endoplasmic reticulum (ER) Ca(2+) stores. Cryptococci lacking CMC did not survive ER stress, indicating its critical role in restoring Ca(2+) homeostasis. Despite the requirement for Mid1 in promoting Ca(2+) influx via Cch1, identification of the role of Mid1 remains elusive. Here we show that the C-terminal tail of Mid1 is a modulatory region that impinges on Cch1 channel activity directly and mediates the trafficking of Mid1 to the plasma membrane. This region consists of the last 24 residues of Mid1, and the functional expression of Mid1 in a human embryonic cell line (HEK293) and in C. neoformans is dependent on this domain. Substitutions of arginine (R619A) or cysteine (C621A) in the modulatory region failed to target Mid1 to the plasma membrane and prevented CMC activity. Interestingly, loss of a predicted protein kinase C (PKC)-phosphorylated serine residue (S605A) had no effect on Mid1 trafficking but did alter the kinetics of Cch1 channel activity. Thus, establishment of Ca(2+) homeostasis in C. neoformans is dependent on a modulatory domain of Mid1.

Cch1 restores intracellular Ca2+ in fungal cells during endoplasmic reticulum stress.

Pathogens endure and proliferate during infection by exquisitely coping with the many stresses imposed by the host to prevent pathogen survival. Recent evidence has shown that fungal pathogens and yeast respond to insults to the endoplasmic reticulum (ER) by initiating Ca(2+) influx across their plasma membrane. Although the high affinity Ca(2+) channel, Cch1, and its subunit Mid1, have been suggested as the protein complex responsible for mediating Ca(2+) influx, a direct demonstration of the gating mechanism of the Cch1 channel remains elusive. In this first mechanistic study of Cch1 channel activity we show that the Cch1 channel from the model human fungal pathogen, Cryptococcus neoformans, is directly activated by the depletion of intracellular Ca(2+) stores. Electrophysiological analysis revealed that agents that enable ER Ca(2+) store depletion promote the development of whole cell inward Ca(2+) currents through Cch1 that are effectively blocked by La(3+) and dependent on the presence of Mid1. Cch1 is permeable to both Ca(2+) and Ba(2+); however, unexpectedly, in contrast to Ca(2+) currents, Ba(2+) currents are steeply voltage-dependent. Cch1 maintains a strong Ca(2+) selectivity even in the presence of high concentrations of monovalent ions. Single channel analysis indicated that Cch1 channel conductance is small, similar to that reported for the Ca(2+) current I(CRAC). This study demonstrates that Cch1 functions as a store-operated Ca(2+)-selective channel that is gated by intracellular Ca(2+) depletion. The inability of cryptococcal cells that lacked the Cch1-Mid1 channel to survive ER stress suggests that Cch1 and its co-regulator, Mid1, are critical players in the restoration of Ca(2+) homeostasis.

The functional expression of toxic genes: lessons learned from molecular cloning of CCH1, a high-affinity Ca2+ channel.

Some genes cannot be cloned by conventional methods because in most cases the genes or gene products are toxic to Escherichia coli. CCH1 is a high-affinity Ca(2+) channel present in the plasma membrane of Cryptococcus neoformans and other fungi. Like many toxic genes, the molecular cloning of CCH1 has been a major challenge; consequently, direct studies of CCH1 channel activity in heterologous expression systems have been impossible. We have devised a straightforward approach that resulted in the molecular cloning and functional expression of CCH1 by exploiting homologous recombination both in vitro and in vivo. This approach precluded the standard enzyme digestion-mediated ligation reactions and the subsequent isolation of plasmids from E. coli. The shuttle plasmid carrying CCH1-GFP, which was prepared in vitro and propagated in yeast, was successfully expressed in the mammalian cell line HEK293 (human embryonic kidney 293). CCH1 transcripts were detected only in HEK293 cells transfected with the plasmid DNA. Fluorescence microscopy studies revealed the expression of CCH1-GFP fusion protein on the cell surface of HEK293 cells, similar to the localization pattern of a well-characterized plasma membrane-associated K(+) channel. This approach will be particularly useful for genes that encode ion channels and transporters that cannot be cloned by conventional techniques requiring E. coli.

Molecular determinants of cardiac K(ATP) channel activation by epoxyeicosatrienoic acids.

We have previously reported that epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase metabolites of arachidonic acid, are potent stereospecific activators of the cardiac K(ATP) channel. The epoxide group in EET is critical for reducing channel sensitivity to ATP, thereby activating the channel. This study is to identify the molecular sites on the K(ATP) channels for EET-mediated activation. We investigated the effects of EETs on Kir6.2delta C26 with or without the coexpression of SUR2A and on Kir6.2 mutants of positively charged residues known to affect channel activity coexpressed with SUR2A in HEK293 cells. The ATP IC50 values were significantly increased in Kir6.2 R27A, R50A, K185A, and R201A but not in R16A, K47A, R54A, K67A, R192A, R195A, K207A, K222A, and R314A mutants. Similar to native cardiac K(ATP) channel, 5 microM 11,12-EET increased the ATP IC50 by 9.6-fold in Kir6.2/SUR2A wild type and 8.4-fold in Kir6.2delta C26. 8,9- and 14,15-EET regioisomers activated the Kir6.2 channel as potently as 11,12-EET. 8,9- and 11,12-EET failed to change the ATP sensitivity of Kir6.2 K185A, R195A, and R201A, whereas their effects were intact in the other mutants. 14,15-EET had a similar effect with K185A and R201A mutants, but instead of R195A, it failed to activate Kir6.2R192A. These results indicate that activation of Kir6.2 by EETs does not require the SUR2A subunit, and the region in the Kir6.2 C terminus from Lys-185 to Arg-201 plays a critical role in EET-mediated Kir6.2 channel activation. Based on computer modeling of the Kir6.2 structure, we infer that the EET-Kir6.2 interaction may allosterically change the ATP binding site on Kir6.2, reducing the channel sensitivity to ATP.

Effects of lysophosphatidic acid on sodium currents in rat dorsal root ganglion neurons.

Lysophosphatidic acid (LPA), a simple phospholipid, induces pain. To elucidate an involvement of ion channel mechanism in the LPA-induced pain, its effects on sodium currents in rat dorsal root ganglion (DRG) neurons were investigated. LPA suppressed tetrodotoxin-sensitive (TTX-S) sodium current, but increased tetrodotoxin-resistant (TTX-R) sodium current, when currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV. In both types of currents, LPA produced a hyperpolarizing shift of both activation and inactivation voltages. LPA had a negligible effect on the maximal conductance of TTX-S current, but increased that of TTX-R current. The results suggest that the enhancement of TTX-R current may contribute to the LPA-induced pain.

Effects of free fatty acids on sodium currents in rat dorsal root ganglion neurons.

Free fatty acids (FFAs), especially polyunsaturated fatty acids (PUFAs), are potent modulators of muscle-type sodium channels. It is not known if they also modulate sodium channels of sensory neurons. In this study, we investigated the effects of FFAs on the fast tetrodotoxin-sensitive (fTTX-S) and the slow tetrodotoxin-resistant (sTTX-R) sodium currents in rat dorsal root ganglion neurons. At a holding potential of -80 mV, PUFAs potently inhibited fTTX-S current, but monounsaturated fatty acids (MUFAs) and saturated fatty acids (SFAs) to a lesser extent. All FFAs initially increased sTTX-R current, and then decreased it slightly. PUFAs and MUFAs produced a hyperpolarizing shift of the steady-state inactivation voltage for both types of sodium currents. The shift generally increased with the number of unsaturated bonds. FFAs did not change the maximum amplitude of fTTX-S current, but increased that of sTTX-R current. Most FFAs shifted the activation voltage for fTTX-S current in the hyperpolarizing direction, which was not dependent on the degree of unsaturation. MUFAs and SFAs shifted the activation voltage for sTTX-R current in the hyperpolarizing direction, but PUFAs were without effect. The modulation of sodium currents by FFAs, especially PUFAs, may have considerable impact on the excitability of sensory neurons.

ATP modulation of sodium currents in rat dorsal root ganglion neurons.

The modulation of tetrodotoxin-sensitive (TTX-S) and slow tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion neurons by ATP was studied using the whole-cell patch-clamp method. The effects of ATP on two types of sodium currents were either stimulatory or inhibitory depending on the kinetic parameters tested. At a holding potential of -80 mV ATP suppressed TTX-S sodium currents when the depolarizing potential was positive to -30 mV but it increased them when the depolarizing potential was negative to -30 mV. At the same holding potential slow TTX-R sodium currents were always increased by ATP regardless of the depolarizing potential. In both types of sodium currents ATP shifted both the conductance-voltage relationship curve and the steady-state inactivation curve in the hyperpolarizing direction, and accelerated the time-dependent inactivation. ATP decreased the maximum conductance of TTX-S sodium currents but increased that of slow TTX-R sodium currents. The results suggest that ATP would decrease the excitability of neurons with TTX-S sodium channels but would increase that of neurons with slow TTX-R sodium channels. The effects of ATP on sodium currents were preserved in the presence of a G-protein inhibitor, GDP-beta-S, or purinergic antagonists, suramin and Reactive Blue-2, suggesting that purinergic receptors might not be involved in ATP modulation of sodium currents.