[Nivolumab and Gamma Knife Radiosurgery for a Gastric Cancer Patient with Brain Metastasis-A Case Report].

The prognosis of patients with brain metastasis is very poor. Very few cases of combined treatment with nivolumab(240 mg/body, day 1, q2w, a programmed cell death-1[PD-1]inhibitor)and gamma knife radiosurgery(GKR)(27 Gy/3 Fr) for gastric cancer patients with brain metastasis have been reported. Here, we discuss the case of a 55-year-old man with HER2-positive poorly differentiated gastric adenocarcinoma with multiple bone and intra-abdominal lymph node metastases. After 25 courses of SOX(oxaliplatin 100 mg/m2, day 1, q3w plus S-1 120 mg/day, day 1-14, po, q3w)plus trastuzumab( 6 mg/kg, q3w)treatment, brain metastasis was detected. Subsequently, combined treatment with GKR and nivolumab(8 courses, anti-PD-1 monotherapy)was initiated. Both intra-abdominal and brain lesions decreased in response to this treatment, showing that combined therapy with nivolumab and GKR could be effective for treating gastric cancer patients with brain metastasis.

Clinical Significance of Cytotoxic Lesions of the Corpus Callosum in Subarachnoid Hemorrhage Patients: A Retrospective Analysis.

INTRODUCTION: Cytotoxic lesions of the corpus callosum are secondary lesions induced by significant increases in cytokine levels in the brain and are associated with subarachnoid hemorrhage (SAH). However, their clinical significance in SAH patients remains unclear. METHODS: We retrospectively analyzed SAH patients who were treated in our hospital and evaluated between-group differences in the backgrounds, clinical findings, and outcomes between SAH patients who developed cytotoxic lesions of the corpus callosum and those who did not. We further compared patients who achieved good outcomes with those who had poor outcomes. Multivariate logistic regression analysis was used to identify risk factors for poor clinical outcomes. RESULTS: We analyzed 159 SAH patients; 17 patients (10.7%) had cytotoxic lesions of the corpus callosum. Patients with cytotoxic lesions of the corpus callosum were more likely to be in a severe condition (World Federation of Neurosurgical Societies grading IV-V: odds ratio [OR], 4.53; 95% confidence interval [95% CI]: 1.60-12.84; p = 0.0042) and have an intraventricular (OR, 5.98; 95% CI: 1.32-27.13; p = 0.0054) or an intraparenchymal hematoma (OR, 3.62; 95% CI: 1.25-10.45; p = 0.023). Patients with cytotoxic lesions of the corpus callosum had a greater propensity of a poor outcome 3 months after onset (modified Rankin Scale score 0-2: OR, 0.22; 95% CI: 0.07-0.66; p = 0.0043). Multivariate analysis confirmed that cytotoxic lesions of the corpus callosum increased the risk of a poor outcome (OR, 4.39; 95% CI: 1.06-18.1; p = 0.037). DISCUSSION/CONCLUSIONS: The development of cytotoxic lesions of the corpus callosum may be related to the extent of hematomas in SAH patients. Although they are usually reversible lesions, the development of cytotoxic lesions of the corpus callosum may be a predictor of poor outcomes in SAH patients.

Genetically encoding thyronine for fluorescent detection of peroxynitrite.

Peroxynitrite is a highly reactive oxidant effecting cell signaling and cell death. Here we report a fluorescent protein probe to selectively detect peroxynitrite. A novel unnatural amino acid, thyronine (Thy), was genetically encoded in E. coli and mammalian cells by evolving an orthogonal tRNAPyl/ThyRS pair. Incorporation of Thy into the chromophore of sfGFP or cpsGFP afforded a virtually non-fluorescent reporter. Upon treatment with peroxynitrite, Thy was converted into tyrosine via O-dearylation, regenerating GFP fluorescence in a time- and concentration-dependent manner. Genetically encoded thyronine may also be valuable for other redox applications.

[A Case of Intraosseous Meningioma Suspected of Osteosarcoma Based on Preoperative Image Examinations].

Intraosseous meningioma(IM)is one of the less frequent tumors of the skull, which often cannot be distinguished from other common skull tumors based on preoperative radiological findings. Herein, we describe a case of IM suspected to be an osteosarcoma based on preoperative image examinations. A 72-year-old woman experienced an impact to her left parietal area, and a subcutaneous tumor appeared in the same area. It had enlarged gradually over seven months, although she exhibited no obvious symptoms. On preoperative diagnostic imaging, the tumor was mainly found in the skull, extending from the subcutaneous area to the intradural space, and was primarily suspected to be an osteosarcoma. After surgery, the pathological diagnosis was atypical meningioma, and there has been no recurrence for 1 year after the surgery. It is necessary for IM to be considered as a differential diagnosis for skull tumors exhibiting characteristics of osteosarcoma.

Allogeneic adipose-derived mesenchymal stem cell sheet that produces neurological improvement with angiogenesis and neurogenesis in a rat stroke model.

OBJECTIVE: Stem cell therapy is a promising strategy for the treatment of severe cerebral ischemia. However, targeting sufficient grafted cells to the affected area remains challenging. Choosing an adequate transplantation method for the CNS appears crucial for this therapy to become a clinical reality. The authors used a scaffold-free cell sheet as a translational intervention. This method involves the use of cell sheet layers and allows the transplantation of a large number of cells, locally and noninvasively. The authors evaluated the effectiveness of allogeneic adipose tissue-derived mesenchymal stem cell sheets in a rat model of stroke. METHODS: The animals, subjected to middle cerebral artery occlusion, were randomly divided in two groups: one in which a cell sheet was transplanted and the other in which a vehicle was used (n = 10/group). Over a period of 14 days after transplantation, the animals' behavior was evaluated, after which brain tissue samples were removed and fixed, and the extent of angiogenesis and infarct areas was evaluated histologically. RESULTS: Compared to the vehicle group, in the cell sheet group functional angiogenesis and neurogenesis were significantly increased, which resulted in behavioral improvement. Transplanted cells were identified within newly formed perivascular walls as pericytes, a proportion of which were functional. Newly formed blood vessels were found within the cell sheet that had anastomosed to the cerebral blood vessels in the host. CONCLUSIONS: The transplantation approach described here is expected to provide not only a paracrine effect but also a direct cell effect resulting in cell replacement that protects the damaged neurovascular unit. The behavioral improvement seen with this transplantation approach provides the basis for further research on cell sheet-based regenerative treatment as a translational treatment for patients with stroke.

Langerhans Cell Histiocytosis of the Skull in a Burr Hole Site Covered with Hydroxyapatite Material.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare dendritic histiocytic disorder that affects the bones, especially the skull. Langerhans cell histiocytosis (LCH) developing in a burr hole site for chronic subdural hematoma is extremely rare. CASE DESCRIPTION: A 53-year-old man underwent a burr hole irrigation for chronic subdural hematoma, and the burr hole was covered with a burr hole button made of hydroxyapatite. Seven months after the first surgery, the connective tissue rapidly proliferated around the burr hole button, and the pathologic diagnosis was LCH. LCH recurred at 13 and 19 months after the first operation, with curettage performed each time. At 3 months after the final operation, no recurrence was identified on magnetic resonance imaging. CONCLUSIONS: If there is rapid proliferation of connective tissue at an operative site where artificial material has been used, LCH should be considered.

Genetically Encoding Quinoline Reverses Chromophore Charge and Enables Fluorescent Protein Brightening in Acidic Vesicles.

Acidic vesicles and organelles play fundamental roles in a broad range of cellular events such as endocytosis, lysosomal degradation, synaptic transmission, pathogen fate, and drug delivery. Fluorescent reporters will be invaluable for studying these complex and multifunctional systems with spatiotemporal resolution, yet common fluorescent proteins are generally nonfluorescent at acidic conditions due to the decrease of anionic chromophores upon protonation, but are fluorescent at physiological pH, creating interfering fluorescence from nonvesicle regions. Here we developed a novel acid-brightening fluorescent protein (abFP) that fluoresces strongly at acidic pH but is nonfluorescent at or above neutral pH, boasting a pH profile opposite to that of common fluorescent proteins. Through expansion of the genetic code, we incorporated a quinoline-containing amino acid Qui into the chromophore of EGFP to reverse the chromophore charge. Protonation of Qui rendered a cationic chromophore, which resulted in unique fluorescence increase only at acidic pH in vitro, in E. coli cells, and on the mammalian cell surface. We further demonstrated that abFP-tagged δ opioid receptors were fluorescently imaged in lysosome showing distinct features and without background fluorescence from other cellular regions, whereas EGFP-tagged receptors were invisible in lysosome. This Qui-rendered cationic chromophore strategy may be generally applied to other fluorescent proteins to generate a palette of colors for acidic imaging with minimal background, and these abFPs should facilitate the study of molecules in association with various acidic vesicles and organelles in different cells and model organisms.

Retinal artery occlusion during carotid artery stenting with distal embolic protection device.

Retinal artery occlusion associated with carotid artery stenosis is well known. Although it can also occur at the time of carotid artery stenting, retinal artery occlusion via the collateral circulation of the external carotid artery is rare. We encountered two cases of retinal artery occlusion that were thought to be caused by an embolus from the external carotid artery during carotid artery stenting with a distal embolic protection device for the internal carotid artery. A 71-year-old man presented with central retinal artery occlusion after carotid artery stenting using the Carotid Guardwire PS and a 77-year-old man presented with branch retinal artery occlusion after carotid artery stenting using the FilterWire EZ. Because additional new cerebral ischaemic lesions were not detected in either case by postoperative diffusion-weighted magnetic resonance imaging, it was highly likely that the debris that caused retinal artery occlusion passed through not the internal carotid artery but collaterals to retinal arteries from the external carotid artery, which was not protected by a distal embolic protection device. It is suggested that a distal protection device for the internal carotid artery alone cannot prevent retinal artery embolisation during carotid artery stenting and protection of the external carotid artery is important to avoid retinal artery occlusion.

Chronic subdural hematoma of the posterior fossa treated by suboccipital craniotomy.

BACKGROUND: Chronic subdural hematoma (CSDH) of the posterior fossa is uncommon in adults. Only a few cases have been reported, and most of these were secondary to head injury or anticoagulant therapy. We herein describe a case of successful surgical treatment of CSDH in the posterior fossa after surgical removal of a large supratentorial and infratentorial dermoid cyst. CASE DESCRIPTION: A 71-year-old woman underwent removal of a left supratentorial and infratentorial dermoid cyst via a left transzygomatic approach. Three years, 6 months after surgery, screening computed tomography revealed CSDH in the supratentorial and infratentorial regions. Four months later, the patient was transferred to the emergency department with cerebellar ataxia, vomiting, and deterioration of consciousness. Two hematomas, one in the supratentorial region and one in the infratentorial region, were greatly compressing the brain, and seemed to be separate lesions. It was difficult to judge on computed tomography whether there was communication between these two hematoma cavities. The patient underwent hematoma removal via suboccipital craniotomy for the posterior fossa CSDH to resolve brain stem compression. Burr-hole irrigation was used for the supratentorial CSDH to avoid upper herniation. The patient recovered uneventfully and was discharged with no neurological deficits. CONCLUSION: Although the optimal treatment for CSDH of the posterior fossa remains unclear because of the limited number of previous reports, direct decompression of the posterior fossa via suboccipital craniotomy should be considered, especially when CSDH exists primarily at the cerebellopontine angle and strongly compresses the brain stem.

Genetically Encoding Fluorosulfate-l-tyrosine To React with Lysine, Histidine, and Tyrosine via SuFEx in Proteins in Vivo.

Introducing new chemical reactivity into proteins in living cells would endow innovative covalent bonding ability to proteins for research and engineering in vivo. Latent bioreactive unnatural amino acids (Uaas) can be incorporated into proteins to react with target natural amino acid residues via proximity-enabled reactivity. To expand the diversity of proteins amenable to such reactivity in vivo, a chemical functionality that is biocompatible and able to react with multiple natural residues under physiological conditions is highly desirable. Here we report the genetic encoding of fluorosulfate-l-tyrosine (FSY), the first latent bioreactive Uaa that undergoes sulfur-fluoride exchange (SuFEx) on proteins in vivo. FSY was found nontoxic to Escherichia coli and mammalian cells; after being incorporated into proteins, it selectively reacted with proximal lysine, histidine, and tyrosine via SuFEx, generating covalent intraprotein bridge and interprotein cross-link of interacting proteins directly in living cells. The proximity-activatable reactivity, multitargeting ability, and excellent biocompatibility of FSY will be invaluable for covalent manipulation of proteins in vivo. Moreover, genetically encoded FSY hereby empowers general proteins with the next generation of click chemistry, SuFEx, which will afford broad utilities in chemical biology, drug discovery, and biotherapeutics.

Vascular ADAM17 (a Disintegrin and Metalloproteinase Domain 17) Is Required for Angiotensin II/ß-Aminopropionitrile-Induced Abdominal Aortic Aneurysm.

Angiotensin II (AngII)-activated epidermal growth factor receptor has been implicated in abdominal aortic aneurysm (AAA) development. In vascular smooth muscle cells (VSMCs), AngII activates epidermal growth factor receptor via a metalloproteinase, ADAM17 (a disintegrin and metalloproteinase domain 17). We hypothesized that AngII-dependent AAA development would be prevented in mice lacking ADAM17 in VSMCs. To test this concept, control and VSMC ADAM17-deficient mice were cotreated with AngII and a lysyl oxidase inhibitor, ß-aminopropionitrile, to induce AAA. We found that 52.4% of control mice did not survive because of aortic rupture. All other surviving control mice developed AAA and demonstrated enhanced expression of ADAM17 in the AAA lesions. In contrast, all AngII and ß-aminopropionitrile-treated VSMC ADAM17-deficient mice survived and showed reduction in external/internal diameters (51%/28%, respectively). VSMC ADAM17 deficiency was associated with lack of epidermal growth factor receptor activation, interleukin-6 induction, endoplasmic reticulum/oxidative stress, and matrix deposition in the abdominal aorta of treated mice. However, both VSMC ADAM17-deficient and control mice treated with AngII and ß-aminopropionitrile developed comparable levels of hypertension. Treatment of C57Bl/6 mice with an ADAM17 inhibitory antibody but not with control IgG also prevented AAA development. In conclusion, VSMC ADAM17 silencing or systemic ADAM17 inhibition seems to protect mice from AAA formation. The mechanism seems to involve suppression of epidermal growth factor receptor activation.

Using Protein-Confined Proximity To Determine Chemical Reactivity.

Chemical reactivity is essential for functional modification of biomolecules with small molecules and the development of covalent drugs. The reactivity between a chemical functional group of a small molecule and that of a large biomolecule cannot be reliably predicted from the reactivity of the corresponding functional groups separately installed on two small molecules, because the proximity effect on reactivity resulting from the binding of the small molecule to the biomolecule is challenging to achieve by mixing two small molecules. Here we present a new strategy to determine the chemical reactivity of two functional groups in the context of close proximity afforded by proteins. The functional groups to be tested were separately installed at the interface of two interacting proteins in the format of amino acid side chains via the expansion of the genetic code. Reaction of the two functional groups resulted in covalent cross-linking of interacting proteins, readily detectable by gel electrophoresis. Using this strategy, we evolved new synthetases to genetically encode Nε-fluoroacetyllysine (FAcK), an isosteric fluorine analogue of acetyllysine. We demonstrated that fluoroacetamide installed on FAcK, previously thought inert to biological functional groups, actually reacted with the thiol group of cysteine when in proximity. This strategy should be valuable for accurately evaluating chemical reactivity of small molecules toward large biomolecules, which will help avoid undesired side reactions of drugs and expand the repertoire of functional groups to covalently target biomolecules.

Farnesoid X receptor knockdown provides significant growth inhibition in hepatocellular carcinoma cells while it does not interfere with the proliferation of primary human hepatocyte-derived cells.

Identification of substances with specific toxicity for carcinoma cells promises to facilitate the development of cancer chemotherapeutics that cause minimal side effects. Here, we show that knockdown of the farnesoid X receptor (FXR) effectively suppresses the proliferation of human hepatocellular carcinoma cell lines HepG2 and HLE accompanied by elevated expression of cyclin-dependent kinase (CDK) inhibitor p16/INK4a and p21/Cip1 proteins. On the other hand, the growth of the primary human hepatocyte-derived cell line Fa2N-4 is not affected by the treatment with FXR siRNA irrespective of marked increases in the mRNAs of p16/INK4a and p21/Cip1. Surprisingly, the expression levels of p16/INK and p21/Cip1 proteins are left unchanged in Fa2N-4 cells that are subjected to the FXR siRNA treatment. Since the expression levels of these CDK inhibitor proteins in FXR-knockdown Fa2N-4 cells were elevated in the presence of proteasomal inhibitor MG132, these CDK inhibitors may be subjected to the proteasomal degradation, thereby counteracting the increased expression of their cognate mRNAs, therefore similar levels of p16 and p21 proteins were observed in control and FXR-knockdown Fa2N-4 cells. These results suggest that FXR-knockdown is effective for inhibiting the proliferation of hepatocellular carcinoma cells, not interfering with the regulatory mechanism of normal hepatocyte growth.

Discovery of 3H-imidazo[4,5-c]quinolin-4(5H)-ones as potent and selective dipeptidyl peptidase IV (DPP-4) inhibitors: use of a carboxylate prodrug to improve bioavailability.

We have previously reported a novel series of 3H-imidazo[4,5-c]quinolin-4(5H)-ones with potent dipeptidyl peptidase IV (DPP-4) inhibitory activity. However, these compounds showed poor oral absorption. We attempted in this study esterification of the carboxylic acid moiety to improve the compounds 1-4 plasma concentrations. Our efforts yielded 10h with a 5-methyl-2-oxo-1,3-dioxol-4-yl methyl ester as an S9/plasma-cleavable functionality. Compound 10h showed significantly high oral absorption and potent DPP-4 inhibition in vivo and decreased Zucker fatty rats glucose levels in the oral glucose tolerance test. Optimization of the ester moiety revealed that rapid conversion to the carboxyl form in both liver S9 fractions and serum was important for prodrugs not to be detected in the plasma after oral administration. In particular, lability in the serum was found to be an important characteristic. Through our investigation, we were able to develop a novel efficient synthetic method for construction of 3H-imidazo[4,5-c]quinolin-4(5H)-ones using intramolecular radical cyclization.

Epidermal growth factor receptor inhibitor protects against abdominal aortic aneurysm in a mouse model.

Angiotensin II (Ang II) has been implicated in the development of abdominal aortic aneurysm (AAA). In vascular smooth muscle cells (VSMC), Ang II activates epidermal growth factor receptor (EGFR) mediating growth promotion. We hypothesized that inhibition of EGFR prevents Ang II-dependent AAA. C57BL/6 mice were co-treated with Ang II and ß-aminopropionitrile (BAPN) to induce AAA with or without treatment with EGFR inhibitor, erlotinib. Without erlotinib, 64.3% of mice were dead due to aortic rupture. All surviving mice had AAA associated with EGFR activation. Erlotinib-treated mice did not die and developed far fewer AAA. The maximum diameters of abdominal aortas were significantly shorter with erlotinib treatment. In contrast, both erlotinib-treated and non-treated mice developed hypertension. The erlotinib treatment of abdominal aorta was associated with lack of EGFR activation, endoplasmic reticulum (ER) stress, oxidative stress, interleukin-6 induction and matrix deposition. EGFR activation in AAA was also observed in humans. In conclusion, EGFR inhibition appears to protect mice from AAA formation induced by Ang II plus BAPN. The mechanism seems to involve suppression of vascular EGFR and ER stress.

Vascular induction of a disintegrin and metalloprotease 17 by angiotensin II through hypoxia inducible factor 1α.

BACKGROUND: A disintegrin and metalloprotease 17 (ADAM17) is a membrane-spanning metalloprotease overexpressed in various cardiovascular diseases such as hypertension and atherosclerosis. However, little is known regarding the regulation of ADAM17 expression in the cardiovascular system. Here, we test our hypothesis that angiotensin II induces ADAM17 expression in the vasculature. METHODS: Cultured vascular smooth muscle cells were stimulated with 100 nM angiotensin II. Mice were infused with 1 µg/kg/minute angiotensin II for 2 weeks. ADAM17 expression was evaluated by a promoter-reporter construct, quantitative polymerase chain reaction, immunoblotting, and immunohistochemistry. RESULTS: In vascular smooth muscle cells, angiotensin II increased ADAM17 protein expression, mRNA, and promoter activity. We determined that the angiotensin II response involves hypoxia inducible factor 1α and a hypoxia responsive element. In angiotensin II-infused mice, marked induction of ADAM17 and hypoxia inducible factor 1α was seen in vasculatures in heart and kidney, as well as in aortae, by immunohistochemistry. CONCLUSIONS: Angiotensin II induces ADAM17 expression in the vasculatures through a hypoxia inducible factor 1α-dependent transcriptional upregulation, potentially contributing to end-organ damage in the cardiovascular system.

Antidepressant fluvoxamine reduces cerebral infarct volume and ameliorates sensorimotor dysfunction in experimental stroke.

The sigma-1 receptor has been reported to be associated with diverse biological activities including cellular differentiation, neuroplasticity, neuroprotection, and cognitive functioning of the brain. Fluvoxamine, one of the currently known antidepressants, is a sigma-1 receptor agonist; its effectiveness in treating acute cerebral ischemia has not been reported. We studied the in-vivo effects of this compound using an animal model of focal cerebral ischemia. Forty male Sprague-Dawley rats were subjected to right middle cerebral artery occlusion and assigned to five treatment groups (n=8 each). Postischemic neurological deficits and infarct volume were determined 24 h after stroke-inducing surgery. Significant reductions in infarct volume (total and cortical) were found in group 2 (fluvoxamine 20 mg/kg given 6 h before and immediately after ischemic onset) and group 3 (fluvoxamine given immediately after ischemic onset and 2 h later) compared with controls. Fluvoxamine induced significant amelioration of sensorimotor dysfunction, as indicated by the scores of forelimb and hindlimb placing tests. Moreover, NE-100, a selective sigma-1 receptor antagonist, completely blocked the neuroprotective effect of fluvoxamine. The present findings suggest that the sigma-1 receptor agonist fluvoxamine reduces infarct volume and ameliorates neurological impairment even on postischemic treatment. From the clinical viewpoint, fluvoxamine may be a promising new therapeutic approach for cerebral infarction.

Caveolin 1 is critical for abdominal aortic aneurysm formation induced by angiotensin II and inhibition of lysyl oxidase.

Although AngII (angiotensin II) and its receptor AT1R (AngII type 1 receptor) have been implicated in AAA (abdominal aortic aneurysm) formation, the proximal signalling events primarily responsible for AAA formation remain uncertain. Caveolae are cholesterol-rich membrane microdomains that serve as a signalling platform to facilitate the temporal and spatial localization of signal transduction events, including those stimulated by AngII. Cav1 (caveolin 1)-enriched caveolae in vascular smooth muscle cells mediate ADAM17 (a disintegrin and metalloproteinase 17)-dependent EGFR (epidermal growth factor receptor) transactivation, which is linked to vascular remodelling induced by AngII. In the present study, we have tested our hypothesis that Cav1 plays a critical role for the development of AAA at least in part via its specific alteration of AngII signalling within caveolae. Cav1-/- mice and the control wild-type mice were co-infused with AngII and ß-aminopropionitrile to induce AAA. We found that Cav1-/- mice with the co-infusion did not develop AAA compared with control mice in spite of hypertension. We found an increased expression of ADAM17 and enhanced phosphorylation of EGFR in AAA. These events were markedly attenuated in Cav1-/- aortas with the co-infusion. Furthermore, aortas from Cav1-/- mice with the co-infusion showed less endoplasmic reticulum stress, oxidative stress and inflammatory responses compared with aortas from control mice. Cav1 silencing in cultured vascular smooth muscle cells prevented AngII-induced ADAM17 induction and activation. In conclusion, Cav1 appears to play a critical role in the formation of AAA and associated endoplasmic reticulum/oxidative stress, presumably through the regulation of caveolae compartmentalized signals induced by AngII.

Design and synthesis of an aminopiperidine series of γ-secretase modulators.

The design, synthesis, and SAR of cyclic diamines as novel γ secretase modulators (GSMs) are presented in this Letter. Starting from information in the literature and in-house cyclic diamines library, we have found a 3(S)-aminopiperidine as a potent structure for lowering Aß42 production both in vitro and in vivo.