Characteristic and Otopathogenic Analysis of a Vibrio alginolyticus Strain Responsible for Chronic Otitis Externa in China.

Vibrio alginolyticus, a Gram-negative rod bacterium found in marine environments, is known to cause opportunistic infections in humans, including ear infections, which can be difficult to diagnose. We investigated the microbiological and otopathogenic characteristics of a V. alginolyticus strain isolated from an ear exudate specimen obtained from a patient with chronic otitis externa to provide a basis for the future diagnosis of V. alginolyticus-associated infections. The identification of V. alginolyticus was accomplished using a combination of matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), classical biochemical identification methods, and the use of Vibrio-selective media and advanced molecular identification methodologies. Antimicrobial susceptibility testing revealed that the strain was resistant to ampicillin and sensitive to ß-lactam, aminoglycosides, fluoroquinolones, and sulfonamide antibiotics. The potential otopathogenic effects of V. alginolyticus were determined through the performance of cell viability, cell apoptosis, and cell death assays in tympanic membrane (TM) keratinocytes and HEI-OC1 cells treated with V. alginolyticus-conditioned medium using cell-counting kit (CCK)-8 assay, a wound-healing migration assay, Annexin V/propidium iodide (PI) flow cytometric analysis, and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL staining). The results indicated that the identified V. alginolyticus strain exerts cytotoxic effects on keratinocytes and HEI-OC1 cells by inhibiting cell proliferation and migration and inducing apoptosis and cell death. To evaluate the ototoxicity of V. alginolyticus, the cell density and morphological integrity of hair cells (HCs) and spiral ganglion neurons (SGNs) were analyzed after exposing cochlear organotypic explants to the bacterial supernatant, which revealed the pre-dominant susceptibility and vulnerability of HCs and SGNs in the basal cochlear region to the ototoxic insults exerted by V. alginolyticus. Our investigation highlights the challenges associated with the identification and characteristic analysis of the Vibrio strain isolated in this case and ultimately aims to increase the understanding and awareness of clinicians and microbiologists for the improved diagnosis of V. alginolyticus-associated ear infections and the recognition of its potential otopathogenic and ototoxic effects.

Adult lead exposure increases blood-retinal permeability: A risk factor for retinal vascular disease.

Low-to-moderate level developmental and adult lead exposure produces retinal dysfunction and/or degeneration in humans and experimental animals. Although high level in vivo or in vitro lead disrupts blood-brain-barrier tight junctions and increases its permeability, the blood-retinal-barrier (BRB) has not been examined. There were four overall goals. First, generate environmentally relevant dose-response models of short-term lead exposure in adult rats. Second, assess retinal histology and functional integrity of the BRB. Third, investigate the transmembrane proteins occludin and claudin-5 as targets mediating the increased BRB permeability. Fourth, examine the contribution of the PI3K-Akt signaling pathway as a mechanism underlying increased BRB permeability. Young adult rats were given water, 0.01% or 0.02% lead drinking solutions for six weeks. In control, 0.01% and 0.02% groups the six week mean blood [Pb] were 1, 12.5 and 19µg/dl, respectively. We employed histology, stereology, quantitative image analysis, immunoblots and densitometry, and pharmacology techniques. Major findings were that adult lead exposure produced dose-dependent 1) decreases in outer and inner nuclear layer thickness, 2) increases in BRB permeability, 3) decreases in occludin and claudin-5 expression, 4) increases in pAkt (Ser473), but not pAkt (Thr308), expression, and 5) wortmannin partially or completely blocked the increased BRB permeability and changes in protein expression. These results indicate that lead-induced increases in PI3K-Akt signaling partially underlie the increased BRB permeability and advance our knowledge about lead-induced retinotoxicity. Furthermore, they suggest that environmental and occupational lead exposures are risk factors for increased BRB permeability in diseases such as age-related macular degeneration, diabetes and stroke.

Noise alters guinea pig's blood-labyrinth barrier ultrastructure and permeability along with a decrease of cochlear Claudin-5 and Occludin.

BACKGROUND: Noise exposure (NE) is a severe modern health hazard that induces hearing impairment. However, the noise-induced ultrastructural changes of blood-labyrinth barrier (BLB) and the potential involvements of tight junction proteins (TJP) remain inconclusive. We investigated the effects of NE on not only the ultrastructure of cochlea and permeability of BLB but also the expression of TJP within the guinea pig cochlea. RESULTS: Male albino guinea pigs were exposed to white noise for 4 h or 2 consecutive days (115 dB sound pressure level, 6 hours per day) and the hearing impairments and light microscopic change of BLB were evaluated with auditory brainstem responses (ABR) and the cochlear sensory epithelia surface preparation, respectively. The cochlear ultrastructure and BLB permeability after NE 2d were revealed with transmission electron microscope (TEM) and lanthanum nitrate-tracing techniques, respectively. The potential alterations of TJPs Claudin-5 and Occludin were quantified with immunohistochemistry and western blot. NE induced significant hearing impairment and NE 2d contributed to significant outer hair cell (OHC) loss that is most severe in the first row of outer hair cells. Furthermore, the loosen TJ and an obvious leakage of lanthanum nitrate particles beneath the basal lamina were revealed with TEM. Moreover, a dose-dependent decrease of Claudin-5 and Occludin was observed in the cochlea after NE. CONCLUSIONS: All these findings suggest that both decrease of Claudin-5 and Occludin and increased BLB permeability are involved in the pathologic process of noise-induced hearing impairment; however, the causal relationship and underlying mechanisms should be further investigated.

Correlation between serum IGF-1 and blood lead level in short stature children and adolescent with growth hormone deficiency.

UNLABELLED: This study aimed to investigate correlation between serum insulin-like growth factor-1 (IGF-1) and blood lead level in short stature children with growth hormone deficiency (GHD), and IGF-1 signal molecules were investigated in lead exposed rats. Our findings may provide evidence for clarifying pathogenesis of lead induced short stature in children. METHODS: 880 short stature children were recruited from clinics and divided into GHD group and idiopathic short stature (ISS) group according to the GH peak in growth hormone stimulation test. The height, body weight, serum IGF-1 level and blood lead level were determined. A rat model of lead poisoning was used to establish and western blot assay was employed to detect the phosphorylation of signaling molecules (MAPK and PI3K/Akt) related to IGF-1 signaling pathway. RESULTS: In GHD group, the height, body weight and serum IGF-1 level were significantly lower, but the blood lead level was significantly higher than those in ISS group (P<0.05). Western blot assay confirmed that the protein expression of phosphorylated ERK1/2, JNK, p38, Akt473 and Akt308 increased significantly (P<0.01) in lead exposure rats. CONCLUSION: Our study suggesting that reduction in IGF-1 in children with GHD is associated with blood lead level. Lead exposure may induce expression of phosphorylated MAPK and Akt signaling molecules. The activation of these molecules may influence binding of IGF-1 and tyrosine kinase receptor IGFIR to regulate cell growth via the MAPK and Akt signaling pathways, which then interfere with growth-promoting effect of IGF-1 in short children.

Reduction of brain barrier tight junctional proteins by lead exposure: role of activation of nonreceptor tyrosine kinase Src via chaperon GRP78.

Lead (Pb) has long been recognized as a neurodevelopmental toxin. Developing blood-brain barrier (BBB) is known to be a target of Pb neurotoxicity; however, the underlying mechanisms are still unclear. Recent evidence suggests that intracellular nonreceptor protein tyrosine kinase Src regulates tight junctional proteins (TJPs). This study was designed to investigate whether Pb acted on the Src-mediated cascade event leading to an altered TJP expression at BBB. Rats aged 20-22 days were exposed to Pb in drinking water (0, 100, 200, and 300 ppm Pb) for eight weeks. Electron microscopic and Western blot analyses revealed a severe leakage of BBB and significantly decreased expressions of TJP occludin and ZO-1. When cultured brain endothelial RBE4 cells were exposed to 10µM Pb for 24 h, expressions of phosphor-Src and an upstream regulator GRP78 were significantly increased by 6.42-fold and 8.29-fold (p < 0.01), respectively. Inactivation of Src pathway by a Src-specific inhibitor reversed Pb-induced downregulation of occludin, but not ZO-1; small interfering RNA knockdown of GRP78 attenuated Pb-induced Src phosphorylation and occludin reduction. Furthermore, Pb exposure caused redistribution of GRP78 from endoplasmic reticulum to cytosol and toward cell member. However, the data from immunoneutralization studies did not show the involvement of cell-surface GRP78 in regulating Src phosphorylation upon Pb exposure, suggesting that the cytosolic GRP78, rather than cell-surface GRP78, was responsible to Pb-induced Src activation and ensuing occludin reduction. Taken together, this study provides the evidence of a novel linkage of GRP78, Src activation to downregulation of occludin, and BBB disruption during Pb exposure.

Involvement of microglia activation in the lead induced long-term potentiation impairment.

Exposure of Lead (Pb), a known neurotoxicant, can impair spatial learning and memory probably via impairing the hippocampal long-term potentiation (LTP) as well as hippocampal neuronal injury. Activation of hippocampal microglia also impairs spatial learning and memory. Thus, we raised the hypothesis that activation of microglia is involved in the Pb exposure induced hippocampal LTP impairment and neuronal injury. To test this hypothesis and clarify its underlying mechanisms, we investigated the Pb-exposure on the microglia activation, cytokine release, hippocampal LTP level as well as neuronal injury in in vivo or in vitro model. The changes of these parameters were also observed after pretreatment with minocycline, a microglia activation inhibitor. Long-term low dose Pb exposure (100 ppm for 8 weeks) caused significant reduction of LTP in acute slice preparations, meanwhile, such treatment also significantly increased hippocampal microglia activation as well as neuronal injury. In vitro Pb-exposure also induced significantly increase of microglia activation, up-regulate the release of cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß) and inducible nitric oxide synthase (iNOS) in microglia culture alone as well as neuronal injury in the co-culture with hippocampal neurons. Inhibiting the microglia activation with minocycline significantly reversed the above-mentioned Pb-exposure induced changes. Our results showed that Pb can cause microglia activation, which can up-regulate the level of IL-1ß, TNF-α and iNOS, these proinflammatory factors may cause hippocampal neuronal injury as well as LTP deficits.