Satoshi Imai

Hypoxia-induced sensitisation of TRPA1 in painful dysesthesia evoked by transient hindlimb ischemia/reperfusion in mice

Dysesthesia is an unpleasant abnormal sensation, which is often accompanied by peripheral neuropathy or vascular impairment. Here, we examined the roles of transient receptor potential ankyrin 1 (TRPA1) in dysesthesia-like behaviours elicited by transient hindlimb ischemia (15–60u2009min) by tightly compressing the hindlimb, and reperfusion by releasing the ligature. The paw-withdrawal responses to tactile stimulation were reduced during ischemia and lasted for a while after reperfusion. Hindlimb ischemia/reperfusion elicited spontaneous licking of the ischemic hindpaw that peaked within 10u2009min. The licking was inhibited by reactive oxygen species (ROS) scavengers, a TRPA1 antagonist, or TRPA1 deficiency, but not by TRPV1 deficiency. In human TRPA1-expressing cells as well as cultured mouse dorsal root ganglion neurons, the H2O2-evoked TRPA1 response was significantly increased by pretreatment with hypoxia (80u2009mmHg) for 30u2009min. This hypoxia-induced TRPA1 sensitisation to H2O2 was inhibited by overexpressing a catalytically-inactive mutant of prolyl hydroxylase (PHD) 2 or in a TRPA1 proline mutant resistant to PHDs. Consistent with these results, a PHD inhibitor increased H2O2-evoked nocifensive behaviours through TRPA1 activation. Our results suggest that transient hindlimb ischemia/reperfusion-evoked spontaneous licking, i.e. painful dysesthesia, is caused by ROS-evoked activation of TRPA1 sensitised by hypoxia through inhibiting PHD-mediated hydroxylation of a proline residue in TRPA1.

Disruption of Slc52a3 gene causes neonatal lethality with riboflavin deficiency in mice

Homeostasis of riboflavin should be maintained by transporters. Previous in vitro studies have elucidated basic information about riboflavin transporter RFVT3 encoded by SLC52A3 gene. However, the contribution of RFVT3 to the maintenance of riboflavin homeostasis and the significance in vivo remain unclear. Here, we investigated the physiological role of RFVT3 using Slc52a3 knockout (Slc52a3−/−) mice. Most Slc52a3−/− mice died with hyperlipidemia and hypoglycemia within 48u2009hr after birth. The plasma and tissue riboflavin concentrations in Slc52a3−/− mice at postnatal day 0 were dramatically lower than those in wild-type (WT) littermates. Slc52a3−/− fetuses showed a lower capacity of placental riboflavin transport compared with WT fetuses. Riboflavin supplement during pregnancy and after birth reduced neonatal death and metabolic disorders. To our knowledge, this is the first report to indicate that Rfvt3 contributes to placental riboflavin transport, and that disruption of Slc52a3 gene caused neonatal mortality with hyperlipidemia and hypoglycemia owing to riboflavin deficiency.

Effects of Cryotherapy on Objective and Subjective Symptoms of Paclitaxel-Induced Neuropathy: Prospective Self-Controlled Trial

Abstract Background Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting and disabling side effect of taxane anticancer agents. We prospectively evaluated the efficacy of cryotherapy for CIPN prevention. Methods Breast cancer patients treated weekly with paclitaxel (80u2009mg/m2 for one hour) wore frozen gloves and socks on the dominant side for 90u2009minutes, including the entire duration of drug infusion. Symptoms on the treated sides were compared with those on the untreated (nondominant) sides. The primary end point was CIPN incidence assessed by changes in tactile sensitivity from pretreatment baseline in a monofilament test at a cumulative dose of 960u2009mg/m2. We also assessed thermosensory deficits, subjective symptoms (Patient Neuropathy Questionnaire [PNQ]), manipulative dexterity, and the time to events and hazard ratio by PNQ. All statistical tests were two-sided. Results Among the 40 patients, four did not reach the cumulative dose (due to the occurrence of pneumonia, severe fatigue, severe liver dysfunction, and macular edema), leaving 36 patients for analysis. None dropped out due to cold intolerance. The incidence of objective and subjective CIPN signs was clinically and statistically significantly lower on the intervention side than on the control (hand: tactile sensitivity = 27.8% vs 80.6%, odds ratio [OR] = 20.00, 95% confidence interval [CI] = 3.20 to 828.96, P < .001; foot: tacile sensitivity = 25.0% vs 63.9%, OR = infinite, 95% CIu2009=u20093.32 to infinite, P < .001; hand: warm sense = 8.8% vs 32.4%, ORu2009=u20099.00, 95% CIu2009=u20091.25 to 394.48, P = .02; foot: warm sense: 33.4% vs 57.6%, ORu2009=u20095.00, 95% CIu2009=u20091.07 to 46.93, P = .04; hand: PNQ = 2.8% vs 41.7%, OR = infinite, 95% CIu2009=u20093.32 to infinite, P < .001; foot: PNQ = 2.8% vs 36.1%, OR = infinite, 95% CIu2009=u20092.78 to infinite, P < .001; hand: hazard ratio [HR] = 0.13, 95% CIu2009=u20090.05 to 0.34; foot: HRu2009=u20090.13, 95% CIu2009=u20090.04 to 0.38, dexterity mean delayu2009=u2009−2.5u2009seconds, SDu2009=u200912.0u2009seconds, vsu2009+u20098.6u2009seconds, SDu2009=u200925.8u2009seconds, P = .005). Conclusions Cryotherapy is useful for preventing both the objective and subjective symptoms of CIPN and resultant dysfunction.

Taxanes and platinum derivatives impair Schwann cells via distinct mechanisms

Impairment of peripheral neurons by anti-cancer agents, including taxanes and platinum derivatives, has been considered to be a major cause of chemotherapy-induced peripheral neuropathy (CIPN), however, the precise underlying mechanisms are not fully understood. Here, we examined the direct effects of anti-cancer agents on Schwann cells. Exposure of primary cultured rat Schwann cells to paclitaxel (0.01u2009μM), cisplatin (1u2009μM), or oxaliplatin (3u2009μM) for 48u2009h induced cytotoxicity and reduced myelin basic protein expression at concentrations lower than those required to induce neurotoxicity in cultured rat dorsal root ganglion (DRG) neurons. Similarly, these anti-cancer drugs disrupted myelin formation in Schwann cell/DRG neuron co-cultures without affecting nerve axons. Cisplatin and oxaliplatin, but not paclitaxel, caused mitochondrial dysfunction in cultured Schwann cells. By contrast, paclitaxel led to dedifferentiation of Schwann cells into an immature state, characterized by increased expression of p75 and galectin-3. Consistent with in vitro findings, repeated injection of paclitaxel increased expression of p75 and galectin-3 in Schwann cells within the mouse sciatic nerve. These results suggest that taxanes and platinum derivatives impair Schwan cells by inducing dedifferentiation and mitochondrial dysfunction, respectively, which may be important in the development of CIPN in conjunction with their direct impairment in peripheral neurons.

Gefitinib and Erlotinib Lead to Phosphorylation of Eukaryotic Initiation Factor 2 Alpha Independent of Epidermal Growth Factor Receptor in A549 Cells.

Gefitinib and erlotinib are anticancer agents, which inhibit epidermal growth factor receptor (EGFR) tyrosine kinase. Interstitial lung disease (ILD) occurs in patients with non-small cell lung cancer receiving EGFR inhibitors. In the present study, we examined whether gefitinib- and erlotinib-induced lung injury related to ILD through endoplasmic reticulum (ER) stress, which is a causative intracellular mechanism in cytotoxicity caused by various chemicals in adenocarcinomic human alveolar basal epithelial cells. These two EGFR inhibitors increased Parkinson juvenile disease protein 2 and C/EBP homologous protein mRNA expressions, and activated the eukaryotic initiation factor (eIF) 2α/activating transcription factor 4 pathway without protein kinase R-like ER kinase activation in A549 cells. Gefitinib and erlotinib caused neither ER stress nor cell death; however, these agents inhibited cell growth via the reduction of cyclin-D1 expression. Tauroursodeoxycholic acid, which is known to suppress eIF2α phosphorylation, cancelled the effects of EGFR inhibitors on cyclin-D1 expression and cell proliferation in a concentration-dependent manner. The results of an EGFR-silencing study using siRNA showed that gefitinib and erlotinib affected eIF2α phosphorylation and cyclin-D1 expression independent of EGFR inhibition. Therefore, the inhibition of cell growth by these EGFR inhibitors might equate to impairment of the alveolar epithelial cell repair system via eIF2α phosphorylation and reduced cyclin-D1 expression.

TRPA1 sensitization during diabetic vascular impairment contributes to cold hypersensitivity in a mouse model of painful diabetic peripheral neuropathy

Background Diabetic peripheral neuropathy is a common long-term complication of diabetes. Accumulating evidence suggests that vascular impairment plays important roles in the pathogenesis of diabetic peripheral neuropathy, while the mechanism remains unclear. We recently reported that transient receptor potential ankyrin 1 (TRPA1) is sensitized by hypoxia, which can contribute to cold hypersensitivity. In this study, we investigated the involvement of TRPA1 and vascular impairment in painful diabetic peripheral neuropathy using streptozotocin-induced diabetic model mice. Results Streptozotocin-induced diabetic model mice showed mechanical and cold hypersensitivity with a peak at two weeks after the streptozotocin administration, which were likely to be paralleled with the decrease in the skin blood flow of the hindpaw. Streptozotocin-induced cold hypersensitivity was significantly inhibited by an antagonist HC-030031 (100 mg/kg) or deficiency for TRPA1, whereas mechanical hypersensitivity was unaltered. Consistent with these results, the nocifensive behaviors evoked by an intraplantar injection of the TRPA1 agonist allyl isothiocyanate (AITC) were enhanced two weeks after the streptozotocin administration. Both streptozotocin-induced cold hypersensitivity and the enhanced AITC-evoked nocifensive behaviors were significantly inhibited by a vasodilator, tadalafil (10 mg/kg), with recovery of the decreased skin blood flow. Similarly, in a mouse model of hindlimb ischemia induced by the ligation of the external iliac artery, AITC-evoked nocifensive behaviors were significantly enhanced three and seven days after the ischemic operation, whereas mechanical hypersensitivity was unaltered in TRPA1-knockout mice. However, no difference was observed between wild-type and TRPA1-knockout mice in the hyposensitivity for current or mechanical stimulation or the deceased density of intraepidermal nerve fibers eight weeks after the streptozotocin administration. Conclusion These results suggest that TRPA1 sensitization during diabetic vascular impairment causes cold, but not mechanical, hypersensitivity in the early painful phase of diabetic peripheral neuropathy. However, TRPA1 may play little or no role in the progression of diabetic peripheral neuropathy.

The impact of mouse strain-specific spatial and temporal immune responses on the progression of neuropathic pain

The present study was designed to investigate the correlation between the spatial and temporal aspects of immune responses and genetic heterogeneity in the progression of peripheral neuropathic pain. To address this issue, we first screened four inbred mouse strains (C57BL/6J, C3H/He, DBA/2, and A/J mice) to identify high- and low-responder strains to mechanical hypersensitivity induced by partial sciatic nerve ligation (pSNL). Among these strains, the C57BL/6J strain showed the highest vulnerability to pSNL-induced mechanical hypersensitivity, whereas the C3H/HeSlc strain was most resistant. C3H/HeSlc mice exhibited a significant increase in CD206-immunoreactivity (anti-inflammatory macrophages) in the dorsal root ganglia (DRG) at 3 and 7u202fdays, and lower Iba1-immunoreactivity (microglia) in the spinal cord from 3 to 14u202fdays after pSNL than C57BL/6J mice. These phenomena might be associated with a decrease in the production of inflammatory factors (interleukin-1β, interleukin-6, and CX3CL1) in the DRG and the poor responsiveness of spinal microglia (i.e. microglial production of IL1β, CCL2, and TNFα) against CX3CL1 in C3H/HeSlc mice. Behavioral experiments using bone marrow (BM) chimeric mice derived by crossing C3H/HeSlc and C57BL/6J strains showed that the strength of mechanical hypersensitivity 3u202fdays following pSNL was inversely correlated with the increase in the ratio of anti-inflammatory/pro-inflammatory DRG macrophages, which was based on the BM-derived hematopoietic cells from donor mice. By contrast, the intensity of Iba1-immunoreactivity (microglia) in the spinal cord was dependent on the phenotypes of recipient mice, but not affected by the phenotypes of BM-derived donor hematopoietic cells. These findings suggest that the strain-specific aspects of DRG macrophages and spinal microglia might be related to the early and late phases of pSNL-induced mechanical hypersensitivity, respectively. This study presents a greater understanding of the differences in neuropathic pain among genetically heterogeneous inbred mouse strains, and provides further insights into the spatial and temporal roles of the immune system in the pathogenesis of neuropathic pain.

Analysis of Glycoforms and Amino Acids in Infliximab and a Biosimilar Product using New Method with LC/TOF-MS

Biosimilar products of therapeutic antibodies have been launched all over the world. They can relieve some of the economic burden of medicines. Although clinical trials have demonstrated the equivalency of biosimilar products with their reference product, biosimilar products are not commonly used in clinical practice. One reason is that the structural difference between the reference product and a biosimilar one remains unclear. We analyzed glycoforms and amino acids of an infliximab biosimilar product approved in Japan compared to that of the reference product (Remicade®). By combination of papain digestion and LC/ time-of-flight (TOF)-MS, we established a valuable method to analyze these therapeutic antibodies. Nine glycoforms were detected in infliximab, and a difference in amino acids was observed. In the glycoforms of MMF, MGnF/GnMF, GnGn, GnGnF, AGnF/GnAF, and AAF, the relative intensities were significantly different between the reference and biosimilar product. Furthermore, we elucidated that the content rate of the C-terminal lysine was different among glycoforms. In conclusion, our analytical method can analyze not only amino acids but also carbohydrate chains of therapeutic antibodies, and will provide a useful strategy to evaluate bio-medicines including biosimilar antibodies.

Oxicam-derived non-steroidal anti-inflammatory drugs suppress 1-methyl-4-phenyl pyridinium-induced cell death via repression of endoplasmic reticulum stress response and mitochondrial dysfunction in SH-SY5Y cells

We have previously reported that oxicam-derived non-steroidal anti-inflammatory drugs (oxicam-NSAIDs), including meloxicam, piroxicam and tenoxicam, elicit protective effects against 1-methyl-4-phenyl pyridinium (MPP+)-induced cell death in a fashion independent of cyclooxygenase (COX) inhibition. We have also demonstrated that oxicam-NSAIDs suppress the decrease in phosphorylation of Akt caused by MPP+. The molecular mechanism through which oxicam-NSAIDs provide cytoprotection remains unclear. In this study, we speculated a possibility that endoplasmic reticulum (ER) stress and/or mitochondrial dysfunction, which are both causative factors of Parkinsons disease (PD), may be involved in the neuroprotective mechanism of oxicam-NSAIDs. We demonstrated here that oxicam-NSAIDs suppressed the activation of caspase-3 and cell death caused by MPP+ or ER stress-inducer, tunicamycin, in SH-SY5Y cells. Furthermore, oxicam-NSAIDs suppressed the increases in the ER stress marker CHOP (apoptosis mediator) caused by MPP+ or tunicamycin, beside suppressing eukaryotic initiation factor 2α (eIF2α) phosphorylation and the increase in ATF4 caused by MPP+. Taken together, these results suggest that oxicam-NSAIDs suppress the eIF2α-ATF4-CHOP pathway, one of the three signaling pathways in the ER stress response. Oxicam-NSAIDs suppressed the decrease in mitochondrial membrane potential depolarization caused by MPP+, indicating they also rescue cells from mitochondrial dysfunction. Akt phosphorylation levels were suppressed after the incubation with MPP+, whereas phosphorylation of eIF2α was enhanced. These results suggest that oxicam-NSAIDs prevented eIF2α phosphorylation and mitochondrial dysfunction by maintaining Akt phosphorylation (reduced by MPP+), thereby preventing cell death.

Riboflavin Transporters RFVT/SLC52A Mediate Translocation of Riboflavin, Rather than FMN or FAD, across Plasma Membrane

Riboflavin (vitamin B2) plays a role in various biochemical oxidation-reduction reactions. Flavin mononucleotide (FMN) and FAD, the biologically active forms, are made from riboflavin. Riboflavin transporters (RFVTs), RFVT1-3/Slc52a1-3, have been identified. However, the roles of human (h)RFVTs in FMN and FAD homeostasis have not yet been fully clarified. In this study, we assessed the contribution of each hRFVT to riboflavin, FMN and FAD uptake and efflux using in vitro studies. The transfection of hRFVTs increased cellular riboflavin concentrations. The uptake of riboflavin by human embryonic kidney cells transfected with hRFVTs was significantly increased, and the efflux was accelerated in a time-dependent manner. However, the uptake and efflux of FMN and FAD hardly changed. These results strongly suggest that riboflavin, rather than FMN or FAD, passes through plasma membranes via hRFVTs. Our findings could suggest that hRFVTs are involved in riboflavin homeostasis in the cells, and that FMN and FAD concentrations are regulated by riboflavin kinase and FAD synthase.