Background: Tumor PD-L1 levels have predictive value in PD-1/PD-L1 checkpoint blockade therapies, yet biopsies can only provide baseline information. Whether PD-L1 expression on circulating tumor cells (CTCs) could serve as an alternative biomarker is of great interest.Design: We established an immunofluorescence assay for semi-quantitative assessment of the PD-L1 expression levels on CTCs with four categories (PD-L1negative, PD-L1low, PD-L1medium and PD-L1high). 35 patients with different advanced gastrointestinal tumors were enrolled in a phase 1 trial of a PD-1 inhibitor, IBI308. The CTC numeration and the PD-L1 expression levels were analyzed.Results: Prior the treatment of IBI308, 97% (34/35) patients had CTCs, ranging from1 to 70 (median 7). 74% (26/35) had PD-L1positive CTCs, and 60% (21/35) had at least one PD-L1high CTCs. The disease control (DC) rate in PD-L1high patients (48%) is much higher than the others (14%). The group with at least 20% abundance of PD-L1high CTCs had even higher DC rate of 64% (9/14), with only 14% DC rate for the rest (3/21). We also observed that the count changes of total CTC, PD-L1postive CTC and PD-L1high CTC correlate quite well with disease outcome (P<0.001, P = 0.002 and 0.007, respectively). In addition, the abundance of PD-L1high CTCs at baseline had predicative significance for progression free survival (PFS).Conclusions: We revealed that the abundance of PD-L1high CTCs at baseline might serve as a predictor to screen patients for PD-1/PD-L1 blockade therapies and measuring the dynamic changes of CTC could indicate the therapeutic response at early time.

In this review we discuss the contribution of NO, prostacyclin and endothelium-derived relaxing factor--endothelium-derived hyperpolarizing factor, or EDHF, to vascular function. We also explore the hypotheses (1): that tissues can store NO as nitrosothiols (RSNOs) and (2) that such RSNO stores can be modulated by physiological and pathophysiological processes. Notably in the microcirculation, EDHF appears to play an important role in the regulation of vascular tone. Leading candidates for EDHF include extracellular potassium (K+), an epoxygenase product, hydrogen peroxide and/or a contribution from myoendothelial gap junctions. Data from our laboratory indicate that in mouse vessels, different endothelium-dependent vasodilators, such as acetylcholine and protease-activated receptor (PAR) agonists, release different endothelium-derived relaxing factors. The combination of two K-channel toxins, apamin and charybdotoxin, inhibits EDHF activity in most protocols. Endothelial dysfunction is considered as the major risk factor and a very early indicator of cardiovascular disease including the cardiovascular complications of type I & types II diabetes. Impaired endothelium-dependent vasodilatation results primarily from a decreased synthesis of endothelium-derived nitric oxide (NO) and/or an increase in the production of reactive oxygen species such as superoxide. We have shown that the administration of tetrahydrobiopterin, an important co-factor for nitric oxide synthase (NOS) partially restores endothelial function (1) in leptin-deficient mice (db/db) with spontaneous type II diabetes, as well as (2) in human vascular tissue harvested for coronary artery bypass grafting (CABG). These data suggest that a deficiency in the availability of tetrahydrobiopterin plays an important role in vascular dysfunction associated with Type II diabetes. In addition, changes in the contribution of EDHF occur in vascular tissue from the db/db mice suggesting a compensatory increase in EDHF production; whether this alteration in EDHF production is physiological or pathophysiological remains controversial.

Background & Aims-Achalasia esophagus is characterized by loss of peristalsis and incomplete esophago-gastric junction (EGJ) relaxation. We studied mechanisms of esophageal emptying in patients with achalasia using simultaneous high-resolution manometery (HRM), multiple intraluminal impedance (MII), and high frequency intra-luminal ultrasound (HFIUS) image recordings.

Programmed death-1 receptor (PD-1) and its ligand (PD-L1) play an integral role in regulating the immune response against cancer. This study investigated the prognostic significance of PD-L1 expression on tumor cells and tumor-infiltrating immune cells (TILs) in the tumor microenvironment in Chinese patients with esophageal squamous cell carcinoma (ESCC). Archival formalin-fixed, paraffin-embedded ESCC samples from treatment-naïve patients with ESCC after surgery or by diagnostic endoscopic biopsy were collected between 2004 and 2014. Expression of PD-L1 in ESCC tumor specimens was assessed by immunohistochemistry (IHC), and the degree of TIL infiltration was evaluated by examining hematoxylin and eosin-stained (H&E) specimens. PD-L1+ as defined as ≥1% of tumor cell membranes showing ≥1+ intensity. In 428 patients, specimens from 341 (79.7%) were PD-L1+. In the definitive treatment group (patients who received curative esophagectomy or definitive [chemo-]radiation therapy), PD-L1 positivity was associated with a significantly shorter DFS and OS. In the palliative chemotherapy group exhibited, neither PFS nor OS correlated significantly with PD-L1 expression. PD-L1 expression was positively associated with TIL density. In 17 paired tumor tissues collected before and after treatment, an increase in PD-L1 expression was associated with disease progression, whereas a decrease in PD-L1 expression was associated with response to chemotherapy or disease control. So, PD-L1 expression was associated with a significantly worse prognosis in patients with ESCC. These observations suggest that PD-L1 may play a critical role in ESCC cancer progression and provide a rationale for developing PD-L1 inhibitors for treatment of a subset of ESCC patients.

Rajasekaran MR, Jiang Y, Bhargava V, Littlefield R, Lee A, Lieber RL, Mittal RK. Length-tension relationship of the external anal sphincter muscle: implications for the anal canal function. Am J Physiol Gastrointest Liver Physiol 295: 367-373, 2008. First published July 3, 2008 doi:10.1152/ajpgi.00033.2008The length at which a muscle operates in vivo (operational length) and the length at which it generates maximal force (optimal length) may be quite different. We studied active and passive length-tension characteristics of external anal sphincter (EAS) in vivo and in vitro to determine the optimal and operational length of rabbit EAS. For the in vitro studies, rings of EAS (n ϭ 4) were prepared and studied in a muscle bath under isometric conditions. For in vivo studies, female rabbits (n ϭ 19) were anesthetized and anal canal pressure was recorded by use of a sleeve sensor placed in the custom-designed catheter holders of 4.5-, 6-, and 9-mm diameters. Measurements were obtained at rest and during EAS electrical stimulation. Sarcomere length of EAS muscle was measured by laser diffraction technique with no probe and three probes in the anal canal. In vitro studies revealed 2,054 mN/cm 2 active tension at optimal length. In vivo studies revealed a probe size-dependent increase in anal canal pressure and tension. Maximal increase in anal canal tension with stimulation was recorded with the 9-mm probe. Increases in anal canal tension with increase in probe size were completely abolished by pancuronium bromide. EAS muscle sarcomere length without and with 9-mm probe in the anal canal were 2.11 Ϯ 0.08 and 2.99 Ϯ 0.07 m, respectively. Optimal sarcomere length, based on the thin filament length measured by thin filament analysis, is 2.44 Ϯ 0.10 m. These data show that the operational length of EAS is significantly shorter than its optimal length. Our findings provide insight into EAS function and we propose the possibility of increasing anal canal pressure by surgical manipulation of the EAS sarcomere length. anal continence; fecal incontinence; sarcomere length SMOOTH MUSCLE OF THE internal anal sphincter (IAS) and striated muscle of the external anal sphincter (EAS) and puborectalis muscle play essential roles in the fecal continence mechanism. Current understanding is that the resting anal canal pressure is mostly related to the IAS and the increase in anal canal pressure with voluntary squeeze is related to contraction of striated EAS muscle (5,8,25). Pressure recording of the anal canal using side-hole manometry and station pull-through technique show that, both at rest and during voluntary squeeze, the highest pressure is located in the part of the anal canal that is surrounded by the EAS, suggesting that EAS is most likely the strongest component of the anal continence mechanism (22). The EAS is also the most commonly affected muscle in women with fecal incontinence because it frequently gets damaged during childbirth-related trauma and episiotomy (29).The EAS muscle in humans shows both resting or sustained s...

We recently found that an orally directed stretch of the esophagus activates a neurally mediated relaxation of the lower esophageal sphincter (LES). Goals of our study were to characterize the neural mechanisms responsible for axial and transverse stretch-activated responses in the LES. LES pressure was monitored in anesthetized and artificially ventilated mice. Sutures were placed in the esophagus to exert graded stretch in the longitudinal and transverse directions. Effects of bilateral vagotomy and pharmacological agents on the stretch-activated LES responses were investigated. The relationship between vagally stimulated axial stretch and LES relaxation was also studied. Stretch in the longitudinal and transverse directions caused a dose-dependent LES relaxation and contraction, respectively, that were not affected by bilateral vagotomy and sympathectomy but were blocked by tetrodotoxin. In bilateral vagotomized animals, hexamethonium, atropine, pyridoxalphosphate-6-azophenyl-2',4' disulfonic acid (PPADS), and ondansetron did not block the stretch-activated LES relaxation and contraction. Axial stretch-activated LES relaxation was blocked by nitric oxide inhibitor and transverse stretch-activated LES contraction was blocked by a combination of atropine and substance P antagonist. Electrical stimulation of the vagus nerve induced LES relaxation and axial stretch on the LES, both of which were blocked by rocuronium. Axial and transverse stretch-activated LES relaxation and contraction were present in the W/W(v) mice that lack interstitial cells of Cajal (ICC). Stretch-activated LES relaxation and contraction are mediated through mechanosensitive neurons located in the myenteric plexus, which involves neither synaptic transmission nor ICC.

1 In the presence of L-NNA (100 mM), indomethacin (10 mM) and ODQ (10 mM), acetylcholine induced a concentration-dependent vasorelaxation of guinea-pig mesenteric and middle cerebral arteries precontracted with cirazoline or histamine, but not with high K + , indicating the contribution of an endothelium-derived hyperpolarizing factor (EDHF). 2 In cerebral arteries, charybdotoxin (ChTX; 0.1 mM) completely inhibited the indomethacin, L-NNA and ODQ-insensitive relaxation; iberiotoxin (IbTX, 0.1 mM), 4-aminopyridine (4-AP, 1 mM), or barium (30 mM) signi®cantly reduced the response; in the mesenteric artery, ChTX and IbTX also reduced this relaxation. Glibenclamide (10 mM) had no a ect in either the mesenteric or cerebral artery. 3 Neither clotrimazole (1 mM) nor 7-ethoxyresoru®n (3 mM) a ected EDHF-mediated relaxation in the mesenteric artery, but abolished or attenuated EDHF-mediated relaxations in the cerebral artery. AM404 (30 mM), a selective anandamide transport inhibitor, did not a ect the vasorelaxation response to acetylcholine in the cerebral artery, but in the mesenteric artery potentiated the vasorelaxation response to acetylcholine in an IbTX, and apamin-sensitive, but SR 141816A-insensitive manner. Ouabain (100 mM) almost abolished EDHF-mediated relaxation in the mesenteric artery, but enhanced the relaxation in the cerebral artery whereas the addition of K + (5 ± 20 mM) to precontracted guinea-pig cerebral or mesenteric artery induced further vasoconstriction. 4 These data suggest that in the guinea-pig mesenteric and cerebral arteries di erent EDHFs mediate acetylcholine-induced relaxation, however, EDHF is unlikely to be mediated by K + .

] cyt homeostasis (8, 23, 49, 66 -68). Two families of plasma membrane Na ϩ /Ca 2ϩ exchange proteins have been described in mammalian tissues, one in which Ca 2ϩ movement is dependent only on sodium (NCX family, including NCX1-3) and one in which Ca 2ϩ movement is also dependent on potassium (NCKX family, including NCKX1-6) (8,12,32,39,41,59). Of note, both NCX and NCKX exchangers can operate either in a forward (Ca 2ϩ exit) mode or in a reverse (Ca 2ϩ entry) mode, depending on the Na ϩ , Ca 2ϩ (and K ϩ ) gradients and the potential across the plasma membrane (8, 41). NCX1 is abundantly expressed in heart, brain, kidney, and smooth muscle (45). However, NCX2 and NCX3 are expressed only in brain and skeletal muscle (40,46). NCKX1 is expressed only in retinal rod photoreceptors, whereas NCKX2 expression is restricted to brain neurons and cone photoreceptors (41). NCKX3 and NCKX4 are expressed not only in brain but also in many other tissues, including aorta, uterus, and intestine, which are rich in smooth muscle cells (SMC) (12,32,39). NCKX5 has recently been demonstrated to be expressed in skin and retinal pigmented epithelium, where it is thought to present on the melanosome membrane and not the plasma membrane (33). NCKX6 has also been characterized; however, the physiological function of this protein remains controversial (12,48). Studies examining the physiological role(s) for NCKX proteins have been restricted to NCKX1 in retinal rod photoreceptors and some recent reports describing NCKX function in brain, spermatozoa, mast cells, and platelet (1, 28 -31, 37, 56, 58). Indeed, the latter studies could not discriminate among NCKX isoforms, and thus the function of NCKX3 and NCKX4 proteins has only been determined in recombinant systems (32, 39) and never in an endogenous setting.Many functional studies have demonstrated the involvement of plasma membrane Na ϩ /Ca 2ϩ exchange in Ca 2ϩ homeostasis of blood vessels (3,4,8,50). The evidence for plasma membrane Na ϩ /Ca 2ϩ exchange in vessels is also supported by direct demonstration that NCX mRNA and protein are expressed in vascular smooth muscle cells (VSMC) and endothelial cells (22,55). Immunocytochemical studies revealed that the exchanger in gastric SMC and VSMC appears to be restricted primarily to plasma membrane regions that are adjacent to junctional SR (22, 42), implying that a major role of