Fluid Hypersecretion Research Articles

Choroid plexus CCL2‒CCR2 signaling orchestrates macrophage recruitment and cerebrospinal fluid hypersecretion in hydrocephalus

The choroid plexus (ChP) serves as the principal origin of cerebrospinal fluid (CSF). CSF hypersecretion due to ChP inflammation has emerged as an important pathogenesis of hydrocephalus recently. Nevertheless, the precise mechanisms of ChP inflammation and the ensuing CSF hypersecretion in hydrocephalus remain ill-defined. In the present study, we elucidate the critical role of macrophages in the pathogenesis of ChP inflammation. Specifically, we identify the chemokine CCL2, released by ChP epithelial cells, recruits CCR2+ monocytes to the ChP thereby inciting hydrocephalus pathogenesis. The accumulated ChP macrophages increase the inflammation in ChP epithelial cells through TNF-α/TNFR1/NF-κB signaling cascade, thereby leading to CSF hypersecretion. Strikingly, augmentation of ChP‒CCL2 using an adeno-associated viral approach (AAV) exacerbates macrophage recruitment, activation, and ventriculomegaly in rat PHH models. Systemic application of Bindarit, a specific CCL2 inhibitor, significantly inhibits ChP macrophage infiltration and activation and reduces CSF secretion rate. Furthermore, the administration of CCR2 antagonist (INCB 3284) reduces ChP macrophage accumulation and ventriculomegaly. This study not only unveils the ChP CCL2‒CCR2 signaling in the pathophysiology of hydrocephalus but also unveils Bindarit as a promising therapeutic choice for the management of posthemorrhagic hydrocephalus.

NLRP3-dependent lipid droplet formation contributes to posthemorrhagic hydrocephalus by increasing the permeability of the blood–cerebrospinal fluid barrier in the choroid plexus

Hydrocephalus is a severe complication that can result from intracerebral hemorrhage, especially if this hemorrhage extends into the ventricles. Our previous study indicated that the NLRP3 inflammasome mediates cerebrospinal fluid hypersecretion in the choroid plexus epithelium. However, the pathogenesis of posthemorrhagic hydrocephalus remains unclear, and therapeutic strategies for prevention and treatment are lacking. In this study, an Nlrp3−/− rat model of intracerebral hemorrhage with ventricular extension and primary choroid plexus epithelial cell culture were used to investigate the potential effects of NLRP3-dependent lipid droplet formation and its role in the pathogenesis of posthemorrhagic hydrocephalus. The data indicated that NLRP3-mediated dysfunction of the blood–cerebrospinal fluid barrier (B-CSFB) accelerated neurological deficits and hydrocephalus, at least in part, through the formation of lipid droplets in the choroid plexus; these lipid droplets interacted with mitochondria and increased the release of mitochondrial reactive oxygen species that destroyed tight junctions in the choroid plexus after intracerebral hemorrhage with ventricular extension. This study broadens the current understanding of the relationship among NLRP3, lipid droplets and the B-CSFB and provides a new therapeutic target for the treatment of posthemorrhagic hydrocephalus. Strategies to protect the B-CSFB may be effective therapeutic approaches for posthemorrhagic hydrocephalus.

Regulation of Ncbe in the Choroid Plexus of Mice after Hemorrhage‐Induced Hydrocephalus

The choroid plexus (CP) is a small tissue located inside brain ventricles. It is responsible for the production of most of the cerebrospinal fluid (CSF); approximately 500 mL of CSF per day in the adult human [1, 2]. CSF production occurs as a net result of transcellular movement of salt and water, carried out by various cellular transporters.Ncbe is a sodium‐coupled bicarbonate transporter located in the basolateral membrane of the choroid plexus epithelial (CPE) cells. It transports Na+ and HCO3‐ into the cell in exchange for Cl‐. Ncbe is the main sodium loader of the cell and CSF production is probably dependent on this transporter [3].Posthemorrhagic hydrocephalus (PHH) is a pathological state, caused by an accumulation of CSF in the ventricular system following intraventricular hemorrhage. CSF buildup is caused by a disproportion in CSF production and reabsorption. This leads to an expansion of the brain ventricles. Intraventricular hemorrhage (IVH) is known to cause inflammation in the CP, which is coupled to an over‐production of CSF [4‐6].Initially we hypothesized that IVH would stimulate Ncbe in the CPE through increased protein abundance as well as transporter activity. This would lead to increased CSF formation, similar to the response found after treatment with e.g. the cyclic‐AMP agonist forskolin [7]. Our preliminary data, however, suggest that Ncbe abundance is reduced by 24% (n=4, p=0.0329) in mice in the initial stages of PHH and then normalizes.The aim of this study is to investigate which role Ncbe plays in PHH and how Ncbe knock down affects CSF production in both healthy mice and a disease model.An adeno‐associated virus (AAV) shRNA knockdown vector system has been developed to knock down Ncbe gene expression by intracerebroventricular injections. Our preliminary data shows that the CPE cells incorporate the vector when tagged with GFP.We have established an IVH model and recording of CSF secretion rates in mice. Increased CSF formation rate was validated with ventriculo‐cisternal perfusion (data shown in figure). The resulting changes in ventricular volume will be investigated using magnetic resonance imaging (MRI).We propose that Ncbe knock down inhibits CSF production and could potentially target inflammation‐dependent hypersecretion by reducing the availability of the sodium supplying the apical transporters in the CP.

In vitro Investigation of the Role of NCBE in Inflammation Induced Cerebrospinal Fluid Hypersecretion by the Choroid Plexus

Post‐hemorrhagic hydrocephalus (PHH) can be the result of inflammation or deposition of FeCl3 in the choroid plexus (CP) after intraventricular hemorrhage (IVH). The hemorrhage leads to increased CSF secretion from the CP also known as the blood‐CSF‐barrier (BCSFB). The precise molecular mechanism is unknown, but the Na+ dependent Cl‐/HCO3‐ exchanger, NCBE, located in the basolateral membrane of the CP epithelia has been suggested as a possible facilitator of CSF hypersecretion.The aim of this study is to investigate the role of the NCBE located in the CP epithelia after inflammation or deposition of FeCl3in relation to the development of PHH. An in vitro model of the BCSFB was established and validated. The model was generated from mouse pup CP epithelial cells. The cells were treated with either TNF‐□, IL‐1□, LPS or FeCl3. NCBE protein expression of the CP epithelial cells was determined by immunoblotting.The CP epithelial cell cultures showed typical cobblestone‐like morphology, polarization and formed an intermediate tight barrier demonstrated by TEER values of ~ 100 Ω*cm2and expression of tight junctions. Compared with controls TNF‐□ decreased NCBE expression by 23.8 % after 24 h (p=0.0048, n=6/group). IL‐1□ had same effect with 28.3 % decrease after 24 h (p=0.0018, n= 6/group) persisting with a decrease of 88.5 % after 3 and 82.4 % after 7 days (p=0.0004 and p=0.0035, n=6/group). LPS had no effect on NCBE expression(n=5/group). FeCl3 increased NCBE expression by 52 % after 24 h (p=0.024, n=7/group) followed by a decrease of 57.8 % after 7 days (p=0.043, n=3/group).We hypothesized that IVH‐induced CP inflammation or FeCl3deposition leads to hydrocephalus by increasing the expression of the basolateral NCBE in the CP cells which leads to an increased rate of CSF secretion. However, the preliminary results suggest a decrease in expression of NCBE after inflammation whereas only an increase in NCBE expression was seen after FeCl3 deposition.In conclusion, NCBE expression of the CP epithelium in cultures decreases under pro‐inflammatory conditions and increases after FeCl3 deposition. Further studies are needed to define the pathways that lead to regulation of NCBE following hemorrhage.

Bumetanide lowers acute hydrocephalus in a rat model of subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) can lead to acute hydrocephalus (AH). AH pathophysiology is classically attributed to an obstruction of the arachnoid granulations by blood. Recent findings in rodents suggest that after intraventricular hemorrhage, AH is related to cerebrospinal fluid (CSF) hypersecretion by the choroid plexus (CP), as it can be reduced by intracerebroventricular (ICV) injection of bumetanide. Here, we investigated if and how CSF hypersecretion and/or CSF outflow disorders contribute to post-SAH hydrocephalus. Ninety-four Wistar rats were used. SAH was induced by the endovascular perforation technique. The presence of AH was confirmed by magnetic resonance imaging (MRI), and rats with AH were randomly assigned to 4 groups: control group, superior sagittal sinus (SSS) thrombosis to block CSF reabsorption, ICV injection of saline, and ICV injection of bumetanide to decrease CSF secretion. Clinical outcome was evaluated with a neuroscore. A second MRI was performed 24h later to evaluate the ventricular volume. Fifty percent of rats that survived SAH induction had AH. Their ventricular volume correlated well to the functional outcome after 24h (r = 0.803). In rats with AH, 24h later, ventricular volume remained equally increased in the absence of any further procedure. Similarly, ICV injection of saline or SSS thrombosis had no impact on the ventricular volume. However, ICV injection of bumetanide reduced AH by 35.9% (p = 0.002). In rodents, post-SAH hydrocephalus is may be due to hypersecretion of CSF by the CP, as it is limited by ICV injection of bumetanide. However, we cannot exclude other mechanisms involved in post-SAH acute hydrocephalus.

Lipopolysaccharides induce a RAGE-mediated sensitization\xa0of sensory neurons and fluid hypersecretion in the upper airways

Thoracic dorsal root ganglia (tDRG) contribute to fluid secretion in the upper airways. Inflammation potentiates DRG responses, but the mechanisms remain under investigation. The receptor for advanced glycation end-products (RAGE) underlies potentiation of DRG responses in pain pathologies; however, its role in other sensory modalities is less understood. We hypothesize that RAGE contributes to electrophysiological and biochemical changes in tDRGs during inflammation. We used tDRGs and tracheas from wild types (WT), RAGE knock-out (RAGE-KO), and with the RAGE antagonist FPS-ZM1, and exposed them to lipopolysaccharides (LPS). We studied: capsaicin (CAP)-evoked currents and action potentials (AP), tracheal submucosal gland secretion, RAGE expression and downstream pathways. In WT neurons, LPS increased CAP-evoked currents and AP generation, and it caused submucosal gland hypersecretion in tracheas from WT mice exposed to LPS. In contrast, LPS had no effect on tDRG excitability or gland secretion in RAGE-KO mice or mice treated with FPS-ZM1. LPS upregulated full-length RAGE (encoded by Tv1-RAGE) and downregulated a soluble (sRAGE) splice variant (encoded by MmusRAGEv4) in tDRG neurons. These data suggest that sensitization of tDRG neurons contributes to hypersecretion in the upper airways during inflammation. And at least two RAGE variants may be involved in these effects of LPS.

Luteolin reduces fluid hypersecretion by inhibiting TMEM16A in interleukin-4 treated Calu-3 airway epithelial cells.

Rhinorrhea in allergic rhinitis (AR) is characterized by the secretion of electrolytes in the nasal discharge. The secretion of Cl– and HCO3– is mainly regulated by cystic fibrosis transmembrane conductance regulator (CFTR) or via the calcium-activated Cl– channel anoctamin-1 (ANO1) in nasal gland serous cells. Interleukin-4 (IL-4), which is crucial in the development of allergic inflammation, increases the expression and activity of ANO1 by stimulating histamine receptors. In this study, we investigated ANO1 as a potential therapeutic target for rhinorrhea in AR using an ANO1 inhibitor derived from a natural herb. Ethanolic extracts (30%) of Spirodela polyrhiza (SPEtOH) and its five major flavonoids constituents were prepared. To elucidate whether the activity of human ANO1 (hANO1) was modulated by SPEtOH and its chemical constituents, a patch clamp experiment was performed in hANO1-HEK293T cells. Luteolin, one of the major chemical constituents in SPEtOH, significantly inhibited hANO1 activity in hANO1-HEK293T cells. Further, SPEtOH and luteolin specifically inhibited the calcium-activated chloride current, but not CFTR current in human airway epithelial Calu-3 cells. Calu-3 cells were cultured to confluency on transwell inserts in the presence of IL-4 to measure the electrolyte transport by Ussing chamber. Luteolin also significantly inhibited the ATP-induced increase in electrolyte transport, which was increased in IL-4 sensitized Calu-3 cells. Our findings indicate that SPEtOH and luteolin may be suitable candidates for the prevention and treatment of allergic rhinitis. SPEtOH- and luteolin-mediated ANO1 regulation provides a basis for the development of novel approaches for the treatment of allergic rhinitis-induced rhinorrhea.

MON-342 Severe Hypocalcemia and Psuedotumor Cerebri: Old, Still Not so Well Known Relationship

Introduction:Severe headaches with nonspecific characteristics raise concern for idiopathic intracranial hypertension (IIH) in a young adult female patient. Clinical features of severe headache, vision loss, and papilledema; normal neuroimaging and elevated opening pressure on lumbar puncture are diagnostic of IIH or pseudotumor cerebri. Etiology is often attributed to obesity in young female patients. We present a rare case of Pseudotumor cerebri in a non-obese female patient with severe hypocalcemia.Case:A 26 year old Hispanic female patient with past medical history of hypocalcemia, but off her medications, presented to ER for worsening headache for over a month. Without any specific triggers, she began having headaches in her bitemporal region. The pain was continuous, pressure-type, and 9/10 intensity at its worst. It was associated with nausea and blurring of vision and was refractory to ibuprofen. Patient endorsed severe muscle cramps on review of systems. Physical examination demonstrated mild distress due to the headache, positive Trousseau’s sign, and bilateral papilledema. Laboratory studies revealed serum total calcium level of 4.8 mg/dL, albumin 3.5 g/dL, ionized calcium 0.71 mmol/L, serum phosphate 5.3 mg/dL, intact PTH 300.1 pg/mL, 25-OH Vitamin D 14.3 ng/mL and 1, 25-Dihydoxy Vitamin D 14.1 pg/mL, alkaline phosphatase 131 IU/L, 24-hour urinary calcium 48mg/d and 24 hour urinary calcium/creatinine ratio 50 mg/g. Her MRI brain and MR venogram were unremarkable. Lumbar puncture had elevated opening pressure of 46 cm of H2O. The patient was started on Acetazolamide, oral calcium, and calcitriol without resolution of headaches and hypocalcemia. Acetazolamide was discontinued when she developed severe hypokalemia. With diagnosis of pseudohypoparathyroidism, the dose of elemental calcium was doubled and calcitriol dose increased to 1 mcg BID with improvement of serum calcium, symptoms, and papilledema.Discussion:While IIH could be associated with certain medications and systemic conditions, obesity or recent weight gain are the most commonly cited causes of IIH. Severe hypocalcemia is a rare and less known etiology of IIH. Cases of adult patients are reported with a variety of etiologies of severe hypocalcemia. Severe hypocalcemia leading to hypersecretion of cerebrospinal fluid is postulated as a possible mechanism but evidence is lacking in literature. Chronic, rather than acute, severe hypocalcemia is likely to precipitate IIH. Visual symptoms, headache and papilledema are difficult to resolve with conventional medical therapy without correction of underlying severe hypocalcemia. We conclude that before considering surgical interventions for cases which are refractory to medical therapy, rare underlying conditions like severe hypocalcemia must be investigated and treated optimally.

Modulation of brain cation-Cl\u2212 cotransport via the SPAK kinase inhibitor ZT-1a

The SLC12A cation-Cl− cotransporters (CCC), including NKCC1 and the KCCs, are important determinants of brain ionic homeostasis. SPAK kinase (STK39) is the CCC master regulator, which stimulates NKCC1 ionic influx and inhibits KCC-mediated efflux via phosphorylation at conserved, shared motifs. Upregulation of SPAK-dependent CCC phosphorylation has been implicated in several neurological diseases. Using a scaffold-hybrid strategy, we develop a novel potent and selective SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano)methyl)-2-methylphenyl)-2-hydroxybenzamide (“ZT-1a”). ZT-1a inhibits NKCC1 and stimulates KCCs by decreasing their SPAK-dependent phosphorylation. Intracerebroventricular delivery of ZT-1a decreases inflammation-induced CCC phosphorylation in the choroid plexus and reduces cerebrospinal fluid (CSF) hypersecretion in a model of post-hemorrhagic hydrocephalus. Systemically administered ZT-1a reduces ischemia-induced CCC phosphorylation, attenuates cerebral edema, protects against brain damage, and improves outcomes in a model of stroke. These results suggest ZT-1a or related compounds may be effective CCC modulators with therapeutic potential for brain disorders associated with impaired ionic homeostasis.

Activation of the Calcium Sensing Receptor Decreases Secretagogue-Induced Fluid Secretion in the Rat Small Intestine.

BackgroundThe calcium-sensing receptor (CaSR) has been localized and characterized in numerous tissues throughout the body. In the mammalian gastrointestinal tract, the CaSR is known to act as a nutrient sensor and has recently been found to play a role in intestinal fluid and electrolyte balance. This study aims to demonstrate the functionality of the CaSR as a modulator of fluid secretion and absorption along the small intestine.MethodsSmall intestine regions (proximal, middle, and distal) were isolated from Sprague Dawley rats and loaded into an ex vivo intestinal perfusion device that provides independent intraluminal and extraluminal (serosa/basolateral) perfusion. The regions were perfused with 5 and 7 mM of Ca2+, both in the presence and absence of forskolin (FSK), a potent secretagogue. Control experiments were conducted with intraluminal perfusate containing standard Ringer-HEPES buffer with a physiological concentration of Ca2+ (1 mM). A second set of comparison experiments was performed with intraluminal perfusates containing AC-265347, a CaSR activator and agonist, in the presence of FSK. In all experimental conditions, the intraluminal perfusate contained fluorescein isothiocyanate (FITC)-inulin, a nonabsorbable fluorescent marker of secretion and/or absorption. Intraluminal fluorescence signal was utilized as a measure of water movement at the start of the experiment and every 15 min for 90 min.ResultsUnder physiological conditions, increasing the concentration of Ca2+ in the luminal perfusate reduced intestinal fluid secretion in all regions. At a Ca2+ concentration of 7 mM, net fluid absorption was observed in all regions. In the presence of FSK, 5 mM Ca2+ significantly decreased fluid secretion and 7 mM Ca2+ abolished FSK-induced fluid secretion. Intraluminal perfusion with 5 mM Ca2+ was as effective as AC-265347, in reducing secretagogue-induced fluid hypersecretion in the proximal and middle regions.ConclusionThis study concludes that apical CaSR is active along the small intestine. Its activation by Ca2+ and/or calcimimetics reduces fluid secretion in a dose-dependent manner, with higher Ca2+ concentrations, or application of a calcimimetic, leading to fluid absorption. We furthermore show that, in the presence of FSK, receptor activation abates FSK secretagogue-induced fluid secretion. This presents a new therapeutic target to address secretory diarrheal illnesses.

The WNK-SPAK/OSR1 Kinases and the Cation-Chloride Cotransporters as Therapeutic Targets for Neurological Diseases

In recent years, cation-chloride cotransporters (CCCs) have drawn attention in the medical neuroscience research. CCCs include the family of Na+-coupled Cl- importers (NCC, NKCC1, and NKCC2), K+-coupled Cl- exporters (KCCs), and possibly polyamine transporters (CCC9) and CCC interacting protein (CIP1). For decades, CCCs have been the targets of several commonly used diuretic drugs, including hydrochlorothiazide, furosemide, and bumetanide. Genetic mutations of NCC and NKCC2 cause congenital renal tubular disorders and lead to renal salt-losing hypotension, secondary hyperreninemia, and hypokalemic metabolic alkalosis. New studies reveal that CCCs along with their regulatory WNK (Kinase with no lysine (K)), and SPAK (Ste20-related proline-alanine-rich kinase)/OSR1(oxidative stress-responsive kinase-1) are essential for regulating cell volume and maintaining ionic homeostasis in the nervous system, especially roles of the WNK-SPAK-NKCC1 signaling pathway in ischemic brain injury and hypersecretion of cerebrospinal fluid in post-hemorrhagic hydrocephalus. In addition, disruption of Cl- exporter KCC2 has an effect on synaptic inhibition, which may be involved in developing pain, epilepsy, and possibly some neuropsychiatric disorders. Interference with KCC3 leads to peripheral nervous system neuropathy as well as axon and nerve fiber swelling and psychosis. The WNK-SPAK/OSR1-CCCs complex emerges as therapeutic targets for multiple neurological diseases. This review will highlight these new findings.

Influence of chemical compositions of anterior chamber aqueous humour and blood serum on the secretion of intraocular fluid

to identify the standard chemical composition of anterior chamber aqueous humour (AH) using energy dispersive spectrometer (EDS) integrated in scanning electron microscope (SEM); to study the influence of concentration gradient of chemical elements between aqueous humor and blood serum (BS) on the secretion of intraocular fluid (IOF) at normal and increased intraocular pressure (IOP). The study involved two groups of patients consisting of 33 people (33 eyes) each. The 1st group included patients with cataract and normal IOP (control), the 2nd group included patients with POAG and decompensated IOP. The samples (AH and BS) were taken during surgeries and studied using Oxford X-Max50 EDS integrated in EVO LS10 SEM. The concentration ratios (mmol/l) of AH/BS for Na at normal IOP was 1.472/1.278, for K - 0.106/0.035. In cases with POAG with decompensated IOP, the concentration ratio of Na was 1.424/1.248, K - 0.114/0.036. The concentration ratio of N between AH in cases with decompensated IOP and AH of normotonic eyes was 1.151/0.960, for S - 0.020/0.012. Consistent excess of Na and K concentrations in AH compared to BS indicates active participation of these osmotically active elements in the normal process of intraocular fluid secretion. The absence of significant differences in the ratios of Na and K in AH and BS at normal and decompensated IOP indicates low significance of these elements for pathological hypersecretion of intraocular fluid. Increased concentration of N in AH eyes with decompensated IOP compared with AH at normal IOP indicates possible involvement of nitrogen-containing osmotically active substances in the abnormal hypersecretion of intraocular fluid.

Inflammation-dependent cerebrospinal fluid hypersecretion by the choroid plexus epithelium in posthemorrhagic hydrocephalus.

The choroid plexus epithelium (CPE) secretes higher volumes of fluid (cerebrospinal fluid, CSF) than any other epithelium and simultaneously functions as the blood-CSF barrier to gate immune cell entry into the central nervous system. Posthemorrhagic hydrocephalus (PHH), an expansion of the cerebral ventricles due to CSF accumulation following intraventricular hemorrhage (IVH), is a common disease usually treated by suboptimal CSF shunting techniques. PHH is classically attributed to primary impairments in CSF reabsorption, but little experimental evidence supports this concept. In contrast, the potential contribution of CSF secretion to PHH has received little attention. In a rat model of PHH, we demonstrate that IVH causes a Toll-like receptor 4 (TLR4)- and NF-κB-dependent inflammatory response in the CPE that is associated with a ∼3-fold increase in bumetanide-sensitive CSF secretion. IVH-induced hypersecretion of CSF is mediated by TLR4-dependent activation of the Ste20-type stress kinase SPAK, which binds, phosphorylates, and stimulates the NKCC1 co-transporter at the CPE apical membrane. Genetic depletion of TLR4 or SPAK normalizes hyperactive CSF secretion rates and reduces PHH symptoms, as does treatment with drugs that antagonize TLR4-NF-κB signaling or the SPAK-NKCC1 co-transporter complex. These data uncover a previously unrecognized contribution of CSF hypersecretion to the pathogenesis of PHH, demonstrate a new role for TLRs in regulation of the internal brain milieu, and identify a kinase-regulated mechanism of CSF secretion that could be targeted by repurposed US Food and Drug Administration (FDA)-approved drugs to treat hydrocephalus.

Synergistic mucus secretion by histamine and IL-4 through TMEM16A in airway epithelium.

Histamine is an important mediator of allergic reactions, and mucus hypersecretion is a major allergic symptom. However, the direct effect of histamine on mucus secretion from airway mucosal epithelia has not been clearly demonstrated. TMEM16A is a Ca2+-activated chloride channel, and it is closely related to fluid secretion in airway mucosal epithelia. We investigated whether histamine directly induces fluid secretion from epithelial cells or submucosal glands (SMG) and mechanisms related, therewith, in allergic airway diseases. In pig airway tissues from the nose or trachea, histamine was a potent secretagogue that directly induced strong responses. However, gland secretion from human nasal tissue was not induced by histamine, even in allergic rhinitis patients. Histamine type 1 receptor (H1R) and histamine type 2 receptor (H2R) were not noted in SMG by in situ hybridization. Cultured primary human nasal epithelial (NHE) cells were used for the measurement of short-circuit current changes with the Ussing chamber. Histamine-induced slight responses of anion secretions under normal conditions. The response was enhanced by IL-4 stimulation through TMEM16A, which might be related to fluid hypersecretion in allergic rhinitis. Pretreatment with IL-4 augmented the histamine response that was suppressed by a TMEM16A inhibitor. TMEM16A expression was enhanced by 24-h treatment of IL-4 in human nasal epithelial cells. The expression of TMEM16A was significantly elevated in an allergic rhinitis group, compared with a control group. We elucidated histamine-induced fluid secretions in synergy with IL-4 through TMEM16A in the human airway epithelium. In addition, we observed species differences between pigs and humans in terms of gland secretion of histamine.

Caffeine Inhibits Fluid Secretion by Interlobular Ducts From Guinea Pig Pancreas.

Caffeine is contained in coffee, tea, and numerous beverages and foods. We examined the direct effects of caffeine on the physiological function of pancreatic duct cells by using interlobular duct segments isolated from guinea pig pancreas. The rate of fluid secretion was continuously measured by monitoring the luminal volume of isolated duct segments. Changes in intracellular Ca concentration ([Ca]i) were estimated by microfluorometry in ducts loaded with Fura-2. Both secretin-stimulated and acetylcholine (ACh)-stimulated fluid secretions were substantially and reversibly inhibited by relatively low concentrations of caffeine as low as 0.03 mM relevant to blood levels after ingestion of caffeine-containing beverages. Caffeine inhibited ACh-induced elevation of [Ca]i and secretin-induced fluctuation of [Ca]i. Caffeine abolished thapsigargin-induced intracellular Ca release but did not affect the entry of extracellular Ca. Caffeine (0.05 mM) abolished ethanol (1 mM)-induced fluid hypersecretion in secretin-stimulated pancreatic duct. Low concentrations of caffeine directly inhibit pancreatic ductal fluid secretion stimulated by secretin or ACh and also ethanol-induced fluid hypersecretion. The inhibition by caffeine seems to be mediated by the blockade of intracellular Ca mobilization. Daily intake of caffeine may reduce the volume of pancreatic juice secretion.

Antibodies derived from a toxoid MEFA (multiepitope fusion antigen) show neutralizing activities against heat-labile toxin (LT), heat-stable toxins (STa, STb), and Shiga toxin 2e (Stx2e) of porcine enterotoxigenic Escherichia coli (ETEC)

Enterotoxigenic Escherichia coli (ETEC) strains are the main cause of diarrhea in pigs. Pig diarrhea especially post-weaning diarrhea remains one of the most important swine diseases. ETEC bacterial fimbriae including K88, F18, 987P, K99 and F41 promote bacterial attachment to intestinal epithelial cells and facilitate ETEC colonization in pig small intestine. ETEC enterotoxins including heat-labile toxin (LT) and heat-stable toxins type Ia (porcine-type STa) and type II (STb) stimulate fluid hyper-secretion, leading to watery diarrhea. Blocking bacteria colonization and/or neutralizing enterotoxicity of ETEC toxins are considered effective prevention against ETEC diarrhea. In this study, we applied the MEFA (multiepitope fusion antigen) strategy to create toxoid MEFAs that carried antigenic elements of ETEC toxins, and examined for broad antitoxin immunogenicity in a murine model. By embedding STa toxoid STaP12F (NTFYCCELCCNFACAGCY), a STb epitope (KKDLCEHY), and an epitope of Stx2e A subunit (QSYVSSLN) into the A1 peptide of a monomeric LT toxoid (LTR192G), two toxoid MEFAs, ‘LTR192G-STb-Stx2e-STaP12F’ and ‘LTR192G-STb-Stx2e-3xSTaP12F’ which carried three copies of STaP12F, were constructed. Mice intraperitoneally immunized with each toxoid MEFA developed IgG antibodies to all four toxins. Induced antibodies showed in vitro neutralizing activities against LT, STa, STb and Stx2e toxins. Moreover, suckling piglets born by a gilt immunized with ‘LTR192G-STb-Stx2e-3xSTaP12F’ were protected when challenged with ETEC strains, whereas piglets born by a control gilt developed diarrhea. Results from this study showed that the toxoid MEFA induced broadly antitoxin antibodies, and suggested potential application of the toxoid MEFA for developing a broad-spectrum vaccine against ETEC diarrhea in pigs.

Toxicity and Immunogenicity of Enterotoxigenic Escherichia coli Heat-Labile and Heat-Stable Toxoid Fusion 3xSTaA14Q-LTS63K/R192G/L211A in a Murine Model

Diarrhea is the second leading cause of death to young children. Enterotoxigenic Escherichia coli (ETEC) are the most common bacteria causing diarrhea. Adhesins and enterotoxins are the virulence determinants in ETEC diarrhea. Adhesins mediate bacterial attachment and colonization, and enterotoxins including heat-labile (LT) and heat-stable type Ib toxin (STa) disrupt fluid homeostasis in host cells that leads to fluid hyper-secretion and diarrhea. Thus, adhesins and enterotoxins have been primarily targeted in ETEC vaccine development. A recent study reported toxoid fusions with STa toxoid (STaP13F) fused at the N- or C-terminus, or inside the A subunit of LTR192G elicited neutralizing antitoxin antibodies, and suggested application of toxoid fusions in ETEC vaccine development (Liu et al., Infect. Immun. 79:4002-4009, 2011). In this study, we generated a different STa toxoid (STaA14Q) and a triple-mutant LT toxoid (LTS63K/R192G/L211A, tmLT), constructed a toxoid fusion (3xSTaA14Q-tmLT) that carried 3 copies of STaA14Q for further facilitation of anti-STa immunogenicity, and assessed antigen safety and immunogenicity in a murine model to explore its potential for ETEC vaccine development. Mice immunized with this fusion antigen showed no adverse effects, and developed antitoxin antibodies particularly through the IP route. Anti-LT antibodies were detected and were shown neutralizing against CT in vitro. Anti-STa antibodies were also detected in the immunized mice, and serum from the IP immunized mice neutralized STa toxin in vitro. Data from this study indicated that toxoid fusion 3xSTaA14Q-tmLT is safe and can induce neutralizing antitoxin antibodies, and provided helpful information for vaccine development against ETEC diarrhea.

エタノールが膵導管細胞機能に及ぼす影響

慢性膵炎は，膵腺房細胞の脱落壊死と小葉間の線維化を主体とする進行性の炎症性疾患であり，慢性膵炎の原因として最多のものがアルコールの多飲である．アルコールによる膵障害に関する細胞レベルの研究は従来から単離膵腺房細胞を用いて高濃度のエタノールに対する作用が検討されてきた．しかし，飲酒後の血中エタノール濃度は通常30mM以下であり（約20mMで酩酊．1 pintのビールの飲酒では，アルコールの血中濃度は5mM程度），飲酒が膵障害を起こすメカニズムとは言えない．著者らは，膵導管細胞を用いて，通常の飲酒後の血中濃度であるエタノール1mMでも膵導管細胞からの水の過分泌を認めること，および，その細胞生理学的メカニズムについてもn-アルコールを用いて検討した．また，膵導管細胞におけるエタノールの水の過分泌が，なぜ，慢性膵炎と結び付くのか，他の研究を交えて述べる．

Diagnosis and neurosurgical treatment of glossopharyngeal neuralgia: clinical findings and 3-D visualization of neurovascular compression in 19 consecutive patients.

Glossopharyngeal neuralgia is a rare condition with neuralgic sharp pain in the pharyngeal and auricular region. Classical glossopharyngeal neuralgia is caused by neurovascular compression at the root entry zone of the nerve. Regarding the rare occurrence of glossopharyngeal neuralgia, we report clinical data and magnetic resonance imaging (MRI) findings in a case series of 19 patients, of whom 18 underwent surgery. Two patients additionally suffered from trigeminal neuralgia and three from additional symptomatic vagal nerve compression. In all patients, ipsilateral neurovascular compression syndrome of the IX cranial nerve could be shown by high-resolution MRI and image processing, which was confirmed intraoperatively. Additional neurovascular compression of the V cranial nerve was shown in patients suffering from trigeminal neuralgia. Vagal nerve neurovascular compression could be seen in all patients during surgery. Sixteen patients were completely pain free after surgery without need of anticonvulsant treatment. As a consequence of the operation, two patients suffered from transient cerebrospinal fluid hypersecretion as a reaction to Teflon implants. One patient suffered postoperatively from deep vein thrombosis and pulmonary embolism. Six patients showed transient cranial nerve dysfunctions (difficulties in swallowing, vocal cord paresis), but all recovered within 1 week. One patient complained of a gnawing and burning pain in the cervical area. Microvascular decompression is a second-line treatment after failure of standard medical treatment with high success in glossopharyngeal neuralgia. High-resolution MRI and 3D visualization of the brainstem and accompanying vessels as well as the cranial nerves is helpful in identifying neurovascular compression before microvascular decompression procedure.

House dust mite extract activates apical Cl− channels through protease‐activated receptor 2 in human airway epithelia

Adequate fluid secretion from airway mucosa is essential for maintaining mucociliary clearance, and fluid hypersecretion is a prominent feature of inflammatory airway diseases such as allergic rhinitis. House dust mite extract (HDM) has been reported to activate protease-activated receptors (PARs), which play various roles in airway epithelia. However, the role of HDM in regulating ion transporters and fluid secretion has not been investigated. We examined the effect of HDM on ion transport in human primary nasal epithelial cells. The Ca(2+)-sensitive dye Fura2-AM was used to determine intracellular Ca(2+) concentration ([Ca(2+)](i)) by means of spectrofluorometry in human normal nasal epithelial cells (NHNE). Short-circuit current (Isc) was measured using Ussing chambers. Fluid secretion from porcine airway mucosa was observed by optical measurement. HDM extract (10 microg/Ml) effectively cleaved the PAR-2 peptide and induced an increase of [Ca(2+)](i) that was abolished by desensitization with trypsin, but not with thrombin. Apical application of HDM-induced Isc sensitive to both a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and a Ca(2+)-activated Cl(-) channel (CaCC) inhibitor. HDM extract also stimulated fluid secretion from porcine airway mucosa. HDM extract activated PAR-2 and apical Cl(-) secretion via CaCC and CFTR, and HDM-induced fluid secretion in porcine airway mucosa. Our results suggest a role for PAR-2 in mucociliary clearance and fluid hypersecretion of airway mucosa in response to air-borne allergens such as HDM.