Exosomes in Sepsis.

Sepsis is a severe state of infection with high mortality. Pathogen-associated molecular patterns and damage-associated molecular patterns (DAMPs) initiate dysregulated systemic inflammation upon binding to pattern recognition receptors. Exosomes are endosome-derived vesicles, which carry proteins, lipids and nucleic acids, and facilitate intercellular communications. Studies have shown altered contents and function of exosomes during sepsis. In sepsis, exosomes carry increased levels of cytokines and DAMPs to induce inflammation. Exosomal DAMPs include, but are not limited to, high mobility group box 1, heat shock proteins, histones, adenosine triphosphate, and extracellular RNA. Exosomes released during sepsis have impact on multiple organs, including the lungs, kidneys, liver, cardiovascular system, and central nervous system. Here, we review the mechanisms of inflammation caused by exosomes, and their contribution to multiple organ dysfunction in sepsis.

Cyclin-dependent kinase inhibitors plus aromatase inhibitor in first-line treatment hormone-receptor-positive/HER2-negative advanced breast cancer women with or without visceral disease: time to turn page?

Breast cancer is the most common female tumour type and accounts for the leading cancer mortality in women worldwide. Up to 75% of breast cancers express the oestrogen receptor or progesterone receptor (hormone-receptor-positive). Aromatase inhibitors were the preferred first-line treatment option. New and acquired resistance to hormonal blockade has led to the development of targeted treatments. Cyclin-dependent kinases (CDKs) are a large family of serine-threonine kinases that play an important role in regulating cell cycle progression: palbociclib, ribociclib, and abemaciclib. We conducted a study to evaluate the efficacy of CDK inhibitors (CDKi) plus aromatase inhibitor in hormone-receptor-positive/HER2-negative ABC patients with visceral disease, postponing the use of chemotherapeutic agents and strengthening the power of endocrine agents. We enrolled 22 patients treated with CDKi (palbocilib) plus aromatase inhibitor (group A) and 38 patients treated with chemotherapy (group B). Our small study confirms the effectiveness of treatment with CDKi plus aromatase inhibitor, even in patients with visceral metastases, when compared with chemotherapy.

Acute Toxic Injuries of Rat's Visceral Tissues Induced by Different Oximes.

Certain AChE reactivators, asoxime, obidoxime, K027, K048, and K075, when taken in overdoses and sometimes even when introduced within therapeutic ranges, may injure the different organs. As a continuation of previously published data, in this study, Wistar rats have sacrificed 24 hrs and 7 days after single im application of 0.1LD50, 0.5LD50 and 1.0LD50 of each reactivator, and examinated tissue samples were obtained for pathohistological and semiquantitative analysis. A severity of tissue alteration, expressed as different tissue damage scores were evaluated. Morphological structure of examinated tissues treated with of 0.1LD50 of all reactivators was comparable with the control group of rats. Moderate injuries were seen in visceral tissues treated with 0.5LD50 of asoxime, obidoxime and K027. Acute damages were enlarged after treatment with 0.5LD50 and 1.0LD50 of all reactivators during the next 7 days. The most prominent changes were seen in rats treated with 1.0LD50 of K048 and K075 (P < 0.001 vs. control and asoxime-treated group). All reactivators given by a single, high, unitary dose regimen, have an adverse effect not only on the main visceral tissue, but on the whole rat as well, but the exact mechanism of cellular injury remains to be confirmed in further investigation.

Histological characterisation of visceral changes in a patient with type 2 Gaucher disease treated with enzyme replacement therapy.

Gaucher disease is a lysosomal storage disease caused by deficiency of glucocerebrosidase and accumulation of glucocerebroside. Three major sub-types have been described, type 2 is an acute neurological form that exhibits serious general symptoms and poor prognosis, compared with the other types. This case was a girl diagnosed with type 2 Gaucher disease at 12months of age who presented with poor weight gain from infancy, stridor, hypertonia, hepatosplenomegaly, trismus and an eye movement disorder. Enzyme replacement therapy (ERT) was administered, but she had frequent myoclonus and developmental regression. She needed artificial ventilation because of respiratory failure. She died at 11years of age. An autopsy demonstrated infiltrating CD68-positive large cells containing abundant lipids in alveoli, while in the liver, kidney and bone marrow CD68-positive cells were small and round. In the bone marrow, myelodysplastic changes were present without Gaucher cells. The infiltration of Gaucher cells in alveoli was marked, suggesting that ERT was relatively ineffective in pulmonary involvement, particularly intra-alveolar. Additional treatments are necessary to improve the neurological and pulmonary prognosis of type 2Gaucher disease.

Sulfated Cyclocarya paliurus polysaccharides markedly attenuates inflammation and oxidative damage in lipopolysaccharide-treated macrophage cells and mice.

Natural polysaccharides and their modified derivatives are crucial supplements to the prevention of inflammation. This study aimed to evaluate the effect of sulfated modification on the anti-inflammatory and anti-oxidative activities of Cyclocarya paliurus polysaccharides (CP). A sulfated CP, S-CP1-4 was obtained using chlorosulfonic acid-pyridine method. The chemical components and FT-IR spectrum confirmed that sulfated group was synthesized to the polysaccharide chains successfully. S-CP1-4 was found to inhibit nitric oxide production, phagocytic activity and the release of interleukin (IL)-6 and IL-1ß in lipopolysaccharide-treated macrophage cells, RAW 264.7. S-CP1-4 significantly decreased the secretion of IL-6 and TNF-α and the thymus and spleen indexes, and increased the production of IL-10 in lipopolysaccharide-treated mice. S-CP1-4 could better protect the liver by inhibiting the activities of alanine aminotransferase and aspartate aminotransferase, and malondialdehyde level while increasing the superoxide dismutase activity and total anti-oxidative capacity. These results suggested that S-CP1-4 may be an effective anti-inflammatory agent, and sulfated modification may be a reliable method for the development of food supplements.

Assessment of metabolism-dependent drug efficacy and toxicity on a multilayer organs-on-a-chip.

Pharmaceutical development is greatly hindered by the poor predictive power of existing in vitro models for drug efficacy and toxicity testing. In this work, we present a new and multilayer organs-on-a-chip device that allows for the assessment of drug metabolism, and its resultant drug efficacy and cytotoxicity in different organ-specific cells simultaneously. Four cell lines representing the liver, tumor (breast cancer and lung cancer), and normal tissue (gastric cells) were cultured in the compartmentalized micro-chambers of the multilayer microdevice. We adopted the prodrug capecitabine (CAP) as a model drug. The intermediate metabolites 5'-deoxy-5-fluorocytidine (DFUR) of CAP that were metabolized from liver and its active metabolite 5-fluorouracil (5-FU) from the targeted cancer cells and normal tissue cells were identified using mass spectrometry. CAP exhibited strong cytoxicity on breast cancer and lung cancer cells, but not in normal gastric cells. Moreover, the drug-induced cytotoxicity on cells varied in various target tissues, suggesting the metabolism-dependent drug efficacy in different tissues as exisits in vivo. This in vitro model can not only allow for characterizing the dynamic metabolism of anti-cancer drugs in different tissues simultaneously, but also facilitate the assessment of drug bioactivity on various target tissues in a simple way, indicating the utility of this organs-on-chip for applications in pharmacodynamics/pharmacokinetics studies, drug efficacy and toxicity testing.

Tonsil-Derived Mesenchymal Stem Cells Promote Bone Mineralization and Reduce Marrow and Visceral Adiposity in a Mouse Model of Senile Osteoporosis.

Osteoporosis is a disease that affects 35% women and 20% men aged more than 65 years. Reduction in bone formation and increased bone resorption are known factors that drive osteoporosis, but recent studies suggest a positive correlation between bone marrow adipose tissue (MAT) and osteoporosis. Previously, we have observed that tonsil-derived mesenchymal stem cells (T-MSCs) reduce MAT in a mouse model of bone marrow depletion. That prompted us to investigate on the senile osteoporosis to characterize the bone-forming effect, as well as MAT-reducing effect of T-MSCs. In a mouse model of senescence-accelerated mouse prone 6 (SAMP6), we injected T-MSCs or T-MSC conditioned medium (CM) through tail vein and examined changes in bone microstructure using micro-CT scan and hematoxylin & eosin (H&E) staining. Biochemical markers of osteoporosis, deoxypyridinoline (DPD) and osteocalcin, were examined by ELISA. Results demonstrated attenuation in the progression of osteoporosis, in part, by sustaining osteocalcin production and by blocking MAT accumulation. Increase in matrix mineralization was determined using in vitro culture of murine preosteoblast cell line by treatment of T-MSC CM. Interestingly, T-MSC CM induced continuous weight loss and selectively reduced visceral adipose tissue mass. Finally, antiadipogenic effects of T-MSC CM were determined in vitro. In conclusion, regulation of bone together with MAT could be considered as a new therapeutic option for the treatment of senile osteoporosis and this report may provide a framework for future cell therapy using T-MSCs.

Crizotinib-induced toxicity in an experimental rat model.

AIM: The aim of the present study was to evaluate the effect of crizotinib on visceral organs in an experimental rat model. METHODS: Eighteen Wistar albino rats were divided into three groups: experimental toxicity was induced with crizotinib (10 mg/kg) administered for 28 days (Group 1), 42 days (Group 2) orally by gavage. Control group received only distilled water. Rats in Group 1 and Group 2 were sacrificed after the collection of blood and tissue samples on the 28th and 42nd days, respectively. RESULTS: Subjects in Group 1 and Group 2 had abnormal histology mainly in lung and liver. There were intraalveolar hemorrhage in lungs; mild portal inflammation, perivenular focal and confluent necrosis in liver; inflammatory reaction in renal pelvis and periureteral areas, and focal pancreatitis in pancreas. CONCLUSION: This study is the first to evaluate the histopathological features of toxicity of crizotinib in a rat model.

Antitoxic Activity of Extract from Salix Viminalis Leaves under Conditions of 5-Fluorouracil Treatment.

Injection of 5-fluorouracil to animals caused a pronounced toxic effect. Therapeutic and preventive treatment with Salix viminalis leaf extract significantly reduced the negative effects of the antitumor drug: promoted recovery of the bone marrow, peripheral blood, and visceral parameters and prevented ulceration. Combined use of the cytostatic and Salix viminalis extract increased the efficiency of antitumor therapy.

Plasma cholesterol-lowering activity of dietary dihydrocholesterol in hypercholesterolemia hamsters.

OBJECTIVE: Cholesterol analogs have been used to treat hypercholesterolemia. The present study was to examine the effect of dihydrocholesterol (DC) on plasma total cholesterol (TC) compared with that of ß-sitosterol (SI) in hamsters fed a high cholesterol diet. METHODS AND RESULTS: Forty-five male hamsters were randomly divided into 6 groups, fed either a non-cholesterol diet (NCD) or one of five high-cholesterol diets without addition of DC and SI (HCD) or with addition of 0.2% DC (DA), 0.3% DC (DB), 0.2% SI (SA), and 0.3% SI (SB), respectively, for 6 weeks. Results showed that DC added into diet at a dose of 0.2% could reduce plasma TC by 21%, comparable to that of SI (19%). At a higher dose of 0.3%, DC reduced plasma TC by 15%, less effective than SI (32%). Both DC and SI could increase the excretion of fecal sterols, however, DC was more effective in increasing the excretion of neutral sterols but it was less effective in increasing the excretion of acidic sterols compared with SI. Results on the incorporation of sterols in micellar solutions clearly demonstrated both DC and SI could displace the cholesterol from micelles with the former being more effective than the latter. CONCLUSION: DC was equally effective in reducing plasma cholesterol as SI at a low dose. Plasma TC-lowering activity of DC was mediated by inhibiting the cholesterol absorption and increasing the fecal sterol excretion.

The Quinovic Acid Glycosides Purified Fraction from Uncaria tomentosa Protects against Hemorrhagic Cystitis Induced by Cyclophosphamide in Mice.

Uncaria tomentosa is widely used in folk medicine for the treatment of numerous diseases, such as urinary tract disease. Hemorrhagic cystitis (HE) is an inflammatory condition of the bladder associated with the use of anticancer drugs such as cyclophosphamide (CYP). Sodium 2-mercaptoethanesulfonate (Mesna) has been used to prevent the occurrence of HE, although this compound is not effective in established lesions. It has been demonstrated that the purinergic system is involved in several pathophysiological events. Among purinergic receptors, P2X7 deserves attention because it is involved in HE induced by CYP and, therefore, can be considered a therapeutic target. The objective of this study was to investigate the potential therapeutic effect of the quinovic acid glycosides purified fraction (QAPF) from U. tomentosa in the mouse model of CYP-induced HE. Pretreatment with QAPF not only had a protective effect on HE-induced urothelial damage (edema, hemorrhage and bladder wet weight) but was also able to control visceral pain, decrease IL-1ß levels and down-regulates P2X7 receptors, most likely by inhibit the neutrophils migration to the bladder. This research clearly demonstrates the promising anti-inflammatory properties of QAPF, supporting its use as complementary therapy. QAPF represents a promising therapeutic option for this pathological condition.

Epigenetic upregulation of metabotropic glutamate receptor 2 in the spinal cord attenuates oestrogen-induced visceral hypersensitivity.

OBJECTIVE: Epigenetic mechanisms are potential targets to relieve somatic pain. However, little is known whether epigenetic regulation interferes with visceral pain. Previous studies show that oestrogen facilitates visceral pain. This study aimed to determine whether histone hyperacetylation in the spinal cord could attenuate oestrogen-facilitated visceral pain. DESIGN: The effect of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) on the magnitude of the visceromotor response (VMR) to colorectal distention was examined in ovariectomised rats with/without oestrogen replacement. An additional interaction with the metabotropic glutamate receptor 2/3 (mGluR2/3) antagonist LY341495 was tested. The levels of acetylated histone and mGluR2 mRNA and protein were analysed. The binding of acetylated H3 and oestrogen receptor α (ERα) to the GRM2 promoter was measured by chromatin immunoprecipitation coupled with qPCR. RESULTS: In ovariectomised rats, 17ß-estradiol (E2), but not safflower oil, increased the magnitude of the VMR to colorectal distention. SAHA attenuated the E2-facilitated VMR, but had no effect in safflower oil-treated rats. Subsequent spinal administration of LY341495 reversed the antinociceptive effect of SAHA in E2 rats. In addition, SAHA increased mGluR2 mRNA and protein in the spinal dorsal horn following E2, but not vehicle, treatment. In contrast, neither E2 nor SAHA alone altered mGluR2 mRNA. SAHA increased binding of H3K9ac and ERα to the same regions of the GRM2 promoter in E2-SAHA-treated animals. CONCLUSIONS: Histone hyperacetylation in the spinal cord attenuates the pronociceptive effects of oestrogen on visceral sensitivity, suggesting that epigenetic regulation may be a potential approach to relieve visceral pain.

Micellization of antineoplastic agent to significantly upregulate efficacy and security.

The amphiphilic diblock copolymer composed of methoxy poly(ethylene glycol) and racemic oligoleucine was synthesized which formed into micelle with uniform size in aqueous environment. Doxorubicin (DOX) was loaded into micelle aided by noncovalent interactions with high drug loading efficiency. The DOX-loaded micelle (referred as M-DOX) demonstrated the sustained drug release in vitro and excellent antiproliferative capability toward both MG63 and Saos-2 cells. Furthermore, for both MG63 and Saos-2-xenografted BALB/c nude mouse models, M-DOX exhibited enhanced intratumoral distribution, improved antitumor efficacy, and reduced side effects compared with free DOX. Therefore, the polypeptide micelle showed a bright prospect for controlled delivery of antitumor drugs in vivo.

Preparation of curcumin-loaded poly(ester amine) nanoparticles for the treatment of anti-angiogenesis.

The aim of this study was to prepare curcumin loaded poly(ester amine) nanoparticles and enhance their hydrophilicity and treatment efficacy on anti-angiogenesis zebra fish model. Poly(ester amine) (PEA) copolymer was synthesized in this study. The curcumin-loaded PEA nanoparticles were prepared through double emulsion-solvent evaporation technique. The average particle size of obtained nanoparticles was about 100 nm. The zeta potential of prepared nanoparticles was about 35.8+/-2.4 mV. Transmission electron microscopy demonstrated a narrow size distribution with in vitro release profile demonstrating in vitro slow release of curcumin from the PEA nanoparticles. The in vitro cytotoxicity of the curcumin encapsulated PEA nanoparticles nearly had the same tendency of cytotoxic activity in vitro with free curcumin on tumor cells. In vitro cellular uptake of the curcumin-loaded nanoparticles demonstrated in Hela cells demonstrated that this kind of nanoparticles can be a promising candidate as a drug delivery system to cancer cells. The Cur/PEA nanoparticles more efficiently inhibited angiogenesis (in vivo) in transgenic zebra fish model and Alginate-encapsulated tumor cells than free curcumin. No mortality or significant lesions were observed from histopathological study of the major organs. From our results, we can conclude that the prepared PEA nanoparticles are an efficient curcumin drug delivery system for anti-angiogenesis therapy.

Toxicity of imidazolium-based ionic liquids on Physa acuta and the snail antioxidant stress response.

In the present study, the acute and developmental toxicities of imidazolium ionic liquids (ILs) with different alkyl chain lengths, as well as the antioxidant response and lipid peroxidation levels were evaluated in the snail, Physa acuta. Longer alkyl chains corresponded to increased IL toxicity in snails. Long-term IL exposure at lower concentrations inhibited snail growth and reproduction. We also found that IL inhibited the activities of superoxide dismutase (SOD) and glutathione S-transferase (GST), promoted the activity of catalase (CAT), and increased the glutathione content. However, SOD, GST, and CAT activities returned to control levels after 96 h of recovery. In addition, malondialdehyde levels were increased in treatment groups compared with the control and did not return to control levels even after a recovery period, indicating that ILs induced lipid peroxidation in snail viscera. These results suggest that oxidative stress and lipid peroxidation may be involved in the mechanism of toxicity for ILs.

Changes in tissue lipid and fatty acid composition of farmed rainbow trout in response to dietary camelina oil as a replacement of fish oil.

Camelina oil (CO) replaced 50 and 100 % of fish oil (FO) in diets for farmed rainbow trout (initial weight 44 ± 3 g fish(-1)). The oilseed is particularly unique due to its high lipid content (40 %) and high amount of 18:3n-3 (α-linolenic acid, ALA) (30 %). Replacing 100 % of fish oil with camelina oil did not negatively affect growth of rainbow trout after a 12-week feeding trial (FO = 168 ± 32 g fish(-1); CO = 184 ± 35 g fish(-1)). Lipid and fatty acid profiles of muscle, viscera and skin were significantly affected by the addition of CO after 12 weeks of feeding. However, final 22:6n-3 [docosahexaenoic acid (DHA)] and 20:5n-3 [eicosapentaenoic acid (EPA)] amounts (563 mg) in a 75 g fillet (1 serving) were enough to satisfy daily DHA and EPA requirements (250 mg) set by the World Health Organization. Other health benefits include lower SFA and higher MUFA in filets fed CO versus FO. Compound-specific stable isotope analysis (CSIA) confirmed that the δ(13)C isotopic signature of DHA in CO fed trout shifted significantly compared to DHA in FO fed trout. The shift in DHA δ(13)C indicates mixing of a terrestrial isotopic signature compared to the isotopic signature of DHA in fish oil-fed tissue. These results suggest that ~27 % of DHA was synthesized from the terrestrial and isotopically lighter ALA in the CO diet rather than incorporation of DHA from fish meal in the CO diet. This was the first study to use CSIA in a feeding experiment to demonstrate synthesis of DHA in fish.

Environmental concentration of nonylphenol alters the development of urogenital and visceral organs in avian model.

Nonylphenol (NP) is an endocrine disruptor with harmful effects including feminization and carcinogenesis on various organisms. This substance is a degradation product of nonylphenol ethoxylates (NPEO) that is used in several industrial and agricultural processes. In this paper, we examined the assessment of NP exposure on chick embryo development, using a concentration consistent with the environmental concentrations of NP. With this aim, NP (between 0.1 and 50 µg/egg) was injected into the yolk of egg through a small needle hole in the shell. We report the effect of NP on chick reproductive system development although the effect we observed is lower than those observed by exposition to other endocrine disruptors. However, histological analysis highlighted a decrease of intraluminal seminiferous surface area in 64.12% of case (P=0.0086) and an heterogeneous organization of the renal tubules when 10 µg/egg were injected. Moreover, an impairment of liver development with an abnormal bile spillage was observed when higher concentration of NP was injected (50 µg/egg).

BMP4 signaling directs primitive endoderm-derived XEN cells to an extraembryonic visceral endoderm identity.

The visceral endoderm (VE) is an epithelial tissue in the early postimplantation mouse embryo that encapsulates the pluripotent epiblast distally and the extraembryonic ectoderm proximally. In addition to facilitating nutrient exchange before the establishment of a circulation, the VE is critical for patterning the epiblast. Since VE is derived from the primitive endoderm (PrE) of the blastocyst, and PrE-derived eXtraembryonic ENdoderm (XEN) cells can be propagated in vitro, XEN cells should provide an important tool for identifying factors that direct VE differentiation. In this study, we demonstrated that BMP4 signaling induces the formation of a polarized epithelium in XEN cells. This morphological transition was reversible, and was associated with the acquisition of a molecular signature comparable to extraembryonic (ex) VE. Resembling exVE which will form the endoderm of the visceral yolk sac, BMP4-treated XEN cells regulated hematopoiesis by stimulating the expansion of primitive erythroid progenitors. We also observed that LIF exerted an antagonistic effect on BMP4-induced XEN cell differentiation, thereby impacting the extrinsic conditions used for the isolation and maintenance of XEN cells in an undifferentiated state. Taken together, our data suggest that XEN cells can be differentiated towards an exVE identity upon BMP4 stimulation and therefore represent a valuable tool for investigating PrE lineage differentiation.

Apolipoprotein E-dependent inverse regulation of vertebral bone and adipose tissue mass in C57Bl/6 mice: modulation by diet-induced obesity.

The long prevailing view that obesity is generally associated with beneficial effects on the skeleton has recently been challenged. Apolipoprotein E (apoE) is known to influence both adipose tissue and bone. The goal of the current study was to examine the impact of apoE on the development of fat mass and bone mass in mice under conditions of diet-induced obesity (DIO). Four week-old male C57BL/6 (WT) and apoE-deficient (apoE(-/-)) mice received a control or a diabetogenic high-fat diet (HFD) for 16 weeks. The control-fed apoE(-/-) animals displayed less total fat mass and higher lumbar trabecular bone volume (BV/TV) than WT controls. When stressed with HFD to induce obesity, apoE(-/-) mice had a lower body weight, lower serum glucose, insulin and leptin levels and accumulated less white adipose tissue mass at all sites including bone marrow. While WT animals showed no significant change in BV/TV and bone formation rate (BFR), apoE deficiency led to a decrease of BV/TV and BFR when stressed with HFD. Bone resorption parameters were not affected by HFD in either genotype. Taken together, under normal dietary conditions, apoE-deficient mice acquire less fat mass and more bone mass than WT littermates. When stressed with HFD to develop DIO, the difference of total body fat mass becomes larger and the difference of bone mass smaller between the genotypes. We conclude that apoE is involved in an inverse regulation of bone mass and fat mass in growing mice and that this effect is modulated by diet-induced obesity.

Amylin suppresses acetic acid-induced visceral pain and spinal c-fos expression in the mouse.

Amylin is a member of calcitonin or calcitonin gene-related peptide (CGRP) family. Immunohistochemical study revealed a dense network of amylin-immunoreactive (irAMY) cell processes in the superficial dorsal horn of the mice. Numerous dorsal root ganglion (DRG) and trigeminal ganglion cells expressed moderate to strong irAMY. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed amylin receptor mRNA in the mouse spinal cord, brain stem, cortex, hypothalamus and hippocampus. The nociceptive or antinociceptive effects of amylin were evaluated in the acetic acid-induced writhing test. Amylin (0.1, 0.5 and 1 mg/kg, intraperitoneally (i.p.) or 1-10 microg, intrathecally (i.t.)) reduced the number of writhes in a dose-dependent manner. Pretreatment of the mice with the amylin receptor antagonist salmon calcitonin (8-32), either by i.p. or i.t., antagonized the effect of amylin on acetic acid-induced writhing test. Locomotor activity was not significantly modified by amylin injected either i.p. (0.01-1 mg/kg) or i.t. (1-10 microg). Measurement of c-fos mRNA by RT-PCR or proteins by Western blot showed that the levels were upregulated in the spinal cord of mice injected with acetic acid and the increase was attenuated by pretreatment with amylin (10 microg, i.t.). Collectively, our result demonstrates that irAMY is expressed in DRG neurons with their cell processes projecting to the superficial layers of the dorsal horn, and that the peptide by interacting with amylin receptors in the spinal cord may be antinociceptive.