Interleukin-1beta upregulates Na+-K+-2Cl- cotransporter in human middle ear epithelia

Disruption of periciliary fluid homeostasis is the main pathogenesis of otitis media with effusion (OME), one of the most common childhood diseases. Although the underlying molecular mechanisms are unclear, it has been suggested that the altered functions of ion channels and transporters are involved in the fluid collection of middle ear cavity of OME patients. In the present study, we analyzed the effects of a major cytokine interleukin (IL)-1beta, which was known to be involved in the pathogenesis of OME, on Na(+)-K(+)-2Cl(-) cotransporter (NKCC) in human middle ear cells. Intracellular pH (pH(i)) was measured in primary cultures of normal human middle ear epithelial (NHMEE) cells using a double perfusion chamber, which enabled us to analyze the membrane-specific transporter activities. NKCC activities were estimated by the pH(i) reduction due to bumetanide-sensitive intracellular uptake of NH(4) (+). In NHMEE cells, NKCC activities were observed only in the basolateral membrane, and immunoblotting using specific antibodies revealed the expression of NKCC1. Interestingly, IL-1beta treatments augmented the basolateral NKCC activities and increased NKCC1 expression. In addition, IL-1beta treatments stimulated bumetanide-sensitive fluid transport across the NHMEE cell monolayers. Furthermore, an elevated NKCC1 expression was observed in middle ear cells from OME patients when compared to those from control individuals. The above results provide in vitro and in vivo evidence that the inflammatory cytokine IL-1beta upregulates NKCC1 in middle ear epithelial cells, which would be one of the important underlying mechanisms of excess fluid collection in OME patients.     

Role of the pro-inflammatory cytokines tumor necrosis factor-alpha,interleukin-1 beta,interleukin-6 and interleukin-8 in the pathogenesis of the otitis media with effusion

Inflammation in the middle ear mucosa, caused usually by bacterial and viral pathogens, is the primary event in the middle ear predisposing the development of otitis media with effusion (OME). Numerous inflammatory mediators have been identified in OME. However, cytokines play a central role as initiators, mediators and regulators of middle ear inflammation and subsequent molecular-pathological processes in middle ear tissues, leading to histopathological changes in the middle ear cavity and the pathogenesis of OME. In this article, we aim to present an overview of current research developments in the pro-inflammatory cytokine involvement in the aetiology of otitis media with effusion.     

The immunoregulatory and allergy-associated cytokines in the aetiology of the otitis media with effusion

Inflammation in the middle ear mucosa, which can be provoked by different primary factors such as bacterial and viral infection, local allergic reactions and reflux, is the crucial event in the pathogenesis of otitis media with effusion (OME). Unresolved acute inflammatory responses or defective immunoregulation of middle inflammation can promote chronic inflammatory processes and stimulate the chronic condition of OME. Cytokines are the central molecular regulators of middle ear inflammation and can switch the acute phase of inflammation in the chronic stage and induce molecular-pathological processes leading to the histopathological changes accompanying OME. In this review we present cytokines identified in otitis media, immunoregulatory [interleukin (IL)-2, IL-10, transforming growth factor-beta]) and allergy associated (IL-4, IL-5, granulocyte-macrophage colony-stimulating factor), as crucial molecular regulators, responsible for chronic inflammation in the middle ear and the chronic condition of OME.     

Inhibiting hypoxia-inducible factor 1 for cancer therapy

Hypoxia has long been recognized as a common feature of solid tumors and a negative prognostic factor for response to treatment and survival of cancer patients. The discovery of hypoxia-inducible factor 1 (HIF-1), a molecular determinant of the response of mammalian cells to hypoxia, has led to the identification of a "molecular target" of hypoxia suitable for the development of cancer therapeutics. Early controversy about whether or not HIF-1 is a good target for therapy has not discouraged academic groups and pharmaceutical companies from actively engaging in the discovery of small-molecule inhibitors of HIF. However, what is the best strategy to inhibit HIF and how HIF inhibitors should be developed for treatment of human cancers is still poorly defined. In this review, aspects related to the identification and early development of novel HIF inhibitors are discussed. Identification and validation of pharmacodynamic end points relevant to the HIF-1 pathway is essential for a rational development of HIF inhibitors. Integration of these biomarkers in early clinical trials may provide valuable information to determine the contribution of HIF inhibitors to response to therapy. Finally, HIF inhibitors should be incorporated in combination strategies to effectively target multiple cellular components of the tumor microenvironment and redundant signaling pathways frequently deregulated in human cancer.     

分泌性中耳炎大鼠咽鼓管鼓室β-防御素的表达

目的检测大鼠β防御素(ratβ-defensin,rBD)在分泌性中耳炎大鼠咽鼓管鼓室的表达,探讨β防御素在分泌性中耳炎发病机制中的作用。方法排除中耳感染的清洁级SD大鼠48只,随机分为4组,前3组36只行颈部切口经右侧听泡注入脂多糖(lipopolysaccharide,LPS)溶液(1mg/mL)30μL制作分泌性中耳炎动物模型,造模后分别于第1、3、7天断头取咽鼓管鼓室黏膜;对照组12只右侧听泡注入生理盐水30μL,左侧听泡作为正常组,3d后断头取咽鼓管鼓室黏膜。逆转录聚合酶链反应(RT-PCR)检测咽鼓管鼓室rBD-1mRNA和rBD-2mRNA的表达。结果正常大鼠咽鼓管鼓室存在rBD-1和rBD-2的表达,且rBD-1的表达较rBD-2强,差异有统计学意义;造模后第1、3、7天,rBD-1表达变化不明显;rBD-2则在造模后第1、3天明显增加,差异有统计学意义,第7天渐回复到正常水平。结论在大鼠,rBD-1可能参与正常咽鼓管鼓室的防御功能,造模后rBD-2的表达增加可能与病原体入侵后的清除相关。     

The human otitis media with effusion: a numerical-based study

Otitis media is a group of inflammatory diseases of the middle ear. Acute otitis media and otitis media with effusion (OME) are its two main types of manifestation. Otitis media is common in children and can result in structural alterations in the middle ear which will lead to hearing losses. This work studies the effects of an OME on the sound transmission from the external auditory meatus to the inner ear. The finite element method was applied on the present biomechanical study. The numerical model used in this work was built based on the geometrical information obtained from The visible ear project. The present work explains the mechanisms by which the presence of fluid in the middle ear affects hearing by calculating the magnitude, phase and reduction of the normalized umbo velocity and also the magnitude and phase of the normalized stapes velocity. A sound pressure level of 90 dB SPL was applied at the tympanic membrane. The harmonic analysis was performed with the auditory frequency varying from 100 Hz to 10 kHz. A decrease in the response of the normalized umbo and stapes velocity as the tympanic cavity was filled with fluid was obtained. The decrease was more accentuated at the umbo.     

Bacterial etiology of otitis media with effusion; focusing on the high positivity of Alloiococcus otitidis

The etiology of otitis media with effusion (OME) is unclear. The bacterial analyses of middle ear effusion (MEE) in OME may reveal important information regarding its etiology. Alloiococcus otitidis, Heamophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis were investigated by using microbiologic culture and a multiplex PCR method in the middle ear fluid of 32 children (54 samples) with chronic OME. PCR yielded positive results in 18 (33.3%) middle ear effusions while culture resulted positive for 3 (5.6%). The PCR method detected A. otitidis in 10 (18.5%) specimens, H. influenzae in 7 (13%), M. catarrhalis in 4 (7.4%) and S. pneumoniae in 2 (3.7%) specimens. The multiplex PCR method enhances the detection rate significantly compared to that of the conventional culture method. A. otitidis is the most common detected pathogen in the MEE of the OME.     

The role of DNA sensing and innate immune receptor TLR9 in otitis media

Otitis media (OM), a common infectious disease in children, is associated with bacterial middle ear (ME) infection. Toll-like receptors (TLRs) are important mediators of innate immune responses, and TLR9 specifically recognizes the unmethylated cytidine-phosphate-guanosine (CpG) motifs in bacterial DNA. Additional sensors of foreign DNA have recently been identified. The role of DNA sensing and TLR9 was investigated in a murine model of OM induced by non-typeable Haemophilus influenzae (NTHi). Expression of genes related to DNA-sensing pathways involved in innate immunity was assessed via DNA microarray, qPCR and immunohistochemistry. Middle ear responses to NTHi were examined in wild-type and TLR9(-/-) mice by histopathology and bacterial culture. Expression of TLR9 signaling genes was modestly up-regulated during OM, as was TLR9 protein in both ME mucosal cells and infiltrating leukocytes. However, genes known to be regulated by CpG DNA were dramatically up-regulated, as were genes involved in DNA sensing by DIA, Pol-III and AIM2. Toll-like receptor 9 deletion significantly prolonged the inflammatory response induced by NTHi in the ME and delayed bacterial clearance. The results suggest that DNA sensing via TLR9 plays a role in OM pathogenesis and recovery. Alternative forms of DNA sensing may also contribute to OM.     

Blocking autocrine VEGF signaling by sunitinib,an anti-cancer drug,promotes embryonic stem cell self-renewal and somatic cell reprogramming

Maintaining the self-renewal of embryonic stem cells (ESCs) could be achieved by activating the extrinsic signaling, i.e., the use of leukemia inhibitory factor (LIF), or blocking the intrinsic differentiation pathways, i.e., the use of GSK3 and MEK inhibitors (2i). Here we found that even in medium supplemented with LIF, mESCs still tend to differentiate toward meso-endoderm lineages after long-term culture and the culture spontaneously secretes vascular endothelial growth factors (VEGFs). Blocking VEGF signaling with sunitinib, an anti-cancer drug and a receptor tyrosine kinase (RTK) inhibitor mainly targeting VEGF receptors (VEGFRs), is capable of maintaining the mESCs in the undifferentiated state without the need for feeder cells or LIF. Sunitinib facilitates the derivation of mESCs from blastocysts, and the mESCs maintained in sunitinib-containing medium remain pluripotent and are able to contribute to chimeric mice. Sunitinib also promotes iPSC generation from MEFs with only Oct4. Knocking down VEGFR2 or blocking it with neutralizing antibody mimicks the effect of sunitinib, indicating that blocking VEGF/VEGFR signaling is indeed beneficial to the self-renewal of mESCs. We also found that hypoxia-inducible factor alpha (HIF1α) and endoplasmic reticulum (ER) stress are involved in the production of VEGF in mESCs. Blocking both pathways inhibits the expression of VEGF and prevents spontaneous differentiation of mESCs. Interestingly, LIF may also exert its effect by downregulating HIF1α and ER stress pathways and subsequent VEGF expression. These results indicate the existence of an intrinsic differentiation pathway in mESCs by activating the autocrine VEGF signaling. Blocking VEGF signaling with sunitinib or other small molecules help to maintain the mESCs in the ground state of pluripotency.     

Myocardial Hypertrophy Overrides the Angiogenic Response to Hypoxia

Background

Cyanosis and myocardial hypertrophy frequently occur in combination. Hypoxia or cyanosis can be potent inducers of angiogenesis, regulating the expression of hypoxia-inducible factors (HIF), vascular endothelial growth factors (VEGF), and VEGF receptors (VEGFR-1 and 2); in contrast, pressure overload hypertrophy is often associated with impaired pro-angiogenic signaling and decreased myocardial capillary density. We hypothesized that the physiological pro-angiogenic response to cyanosis in the hypertrophied myocardium is blunted through differential HIF and VEGF-associated signaling.

Methods and Results

Newborn rabbits underwent aortic banding and, together with sham-operated littermates, were transferred into a hypoxic chamber (FiO₂ = 0.12) at 3 weeks of age. Control banded or sham-operated rabbits were housed in normoxia. Systemic cyanosis was confirmed (hematocrit, arterial oxygen saturation, and serum erythropoietin). Myocardial tissue was assayed for low oxygen concentrations using a pimonidazole adduct. At 4 weeks of age, HIF-1α and HIF-2α protein levels, HIF-1α DNA-binding activity, and expression of VEGFR-1, VEGFR-2, and VEGF were determined in hypoxic and normoxic rabbits. At 6 weeks of age, left-ventricular capillary density was assessed by immunohistochemistry. Under normoxia, capillary density was decreased in the banded rabbits compared to non-banded littermates. As expected, non-hypertrophied hearts responded to hypoxia with increased capillary density; however, banded hypoxic rabbits demonstrated no increase in angiogenesis. This blunted pro-angiogenic response to hypoxia in the hypertrophied myocardium was associated with lower HIF-2α and VEGFR-2 levels and increased HIF-1α activity and VEGFR-1 expression. In contrast, non-hypertrophied hearts responded to hypoxia with increased HIF-2α and VEGFR-2 expression with lower VEGFR-1 expression.

Conclusion

The participation of HIF-2α and VEGFR-2 appear to be required for hypoxia-stimulated myocardial angiogenesis. In infant rabbit hearts with pressure overload hypertrophy, this pro-angiogenic response to hypoxia is effectively uncoupled, apparently in part due to altered HIF-mediated signaling and VEGFR subtype expression.