Novel analgesics targeting brain-derived neurotrophic factor for neuropathic pain

　Brain-derived neurotrophic factor (BDNF) is necessary for the development, growth, and maintenance of nerve cells. BDNF is expressed in the dorsal root ganglion (DRG); binds to the Tropomyosin receptor kinasa B (TrkB) receptor, which has a tyrosine kinase domain, in the spinal cord; and plays an important role as a pain modulator. BDNF expression is increased in various types of pain, including acute pain, neuropathic pain, and cancer pain. Activation of the BDNF–TrkB pathway transmits pain information. In order to inhibit the BDNF–TrkB pathway, by sequestering BDNF, we constructed a cDNA expression plasmid encoding the extracellular region of rat TrkB fused to enhanced green fluorescent protein (EGFP). When the expression plasmid vector was administered to rat models of neuropathic pain, induced by spinal nerve ligation, statistically significant relief of pain was observed in terms of a 50% paw-withdrawal threshold using the von Frey test. The expression of TrkB-EGFP mRNA was detected in L5 lumbar vertebral nerves by quantitative reverse transcriptase polymerase chain reaction. To verify the pain-suppressive effect of the expression vector, truncated TrkB protein, without EGFP, was purified, and administered to pain model rats. A statistically significant suppressive effect of the truncated TrkB protein on neuropathic pain was observed 2 days after administration. The pain-suppressive effect of the truncated TrkB protein was more effective than that of the TrkB-Fc chimera protein and lasted longer than that of the TrkB antagonist ANA-12. Our results suggested that the truncated TrkB cDNA expression vector and truncated TrkB protein could be used as molecular targeted drugs in patients with neuropathic pain

Development of a novel analgesic for cancer pain targeting brain-derived neurotrophic factor

Brain-derived neurotrophic factor (BDNF) is necessary for nerve growth. BDNF is expressed in the dorsal root ganglion (DRG) and modulates pain transduction from peripheral nociceptors. TrkB, which is a BDNF receptor with a tyrosine kinase domain, acts as a pain modulator on the cell membrane of second neuron. If an exogenous truncated TrkB lacking a tyrosine kinase domain can competitively block the binding of BDNF to endogenous TrkB, inhibitory effects on pain are expected. We constructed two expression vectors coding truncated TrkB-GFP fusion proteins, lacking intracellular tyrosine kinase domain, with and without the transmembrane domain. By transfection of the vectors to HEK293 cells, the expression and localization of the modified receptor proteins were confirmed. The truncated TrkB with the transmembrane domain, TM (+), was localized on cell membrane surface of the transfected cells, and capable of BDNF binding on cell surface. TM (-) without the transmembrane domain was secreted from the transfected cells, and the secreted TrkB protein was confirmed the capability for binding with BDNF by pull-down assay. Furthermore, we developed a rat model of cancerous osteocopic pain for evaluating an analgesic effect of the modified TrkB vectors on cancer pain. Pain-related behavior, as assessed by von Frey tests, indicated hyperalgesia after cancer cell administration. BDNF expression was higher on the affected side of the DRG at the third lumbar vertebra L3 than on the unaffected side. When the modified TrkB vectors were administrated to the cancer pain model rats, both the TM (+) and TM (-) vector administration groups exhibited an analgesic effect. These results suggest that the modified TrkB receptors and their vectors are applicable as molecular targeted drugs for pain control in cancer patients

Efficient Artificial Fertilization and Ovulated Egg Preservation in Kawakawa <i>Euthynnus affinis</i>

Artificial fertilization of cultured fish is essential for seed production using breeding techniques. However, in tuna species, the success rate of artificial fertilization is tremendously low. In this study, it was reported that the adequate procedure for ovulated egg collection and storage for artificial fertilization in kawakawa Euthynnus affinis. The collection of ovulated eggs was attempted using new techniques that disrupt only spawning activity without discontinuing ovulation. The available time to use ovulated eggs was also examined by assessing the optimal preservation process and temperature. As a result, artificial fertilization was effectively executed by assessing spawning time and thoroughly extracting ovulated eggs immediately after ovulation, with a success rate of 70% and an ovulation rate of 51.7%. Ovulated eggs could be stored with small quantities of ovarian fluid to sustain fertility. However, fertility was better preserved with Hanks’ solution. Ovulated eggs with high productivity were achieved 3 h after egg extraction when maintained in Hanks’ solution at 20 °C, leading to a supply of one-cell stage embryo for microinjection treatment constantly by continuously executing artificial fertilization. This systematic procedure permitted selective breeding by 1:1 mating between top-quality parental fish and applying several developmental engineering techniques to kawakawa breeding

Efficient Artificial Fertilization and Ovulated Egg Preservation in Kawakawa Euthynnus affinis

Artificial fertilization of cultured fish is essential for seed production using breeding techniques. However, in tuna species, the success rate of artificial fertilization is tremendously low. In this study, it was reported that the adequate procedure for ovulated egg collection and storage for artificial fertilization in kawakawa Euthynnus affinis. The collection of ovulated eggs was attempted using new techniques that disrupt only spawning activity without discontinuing ovulation. The available time to use ovulated eggs was also examined by assessing the optimal preservation process and temperature. As a result, artificial fertilization was effectively executed by assessing spawning time and thoroughly extracting ovulated eggs immediately after ovulation, with a success rate of 70% and an ovulation rate of 51.7%. Ovulated eggs could be stored with small quantities of ovarian fluid to sustain fertility. However, fertility was better preserved with Hanks&rsquo; solution. Ovulated eggs with high productivity were achieved 3 h after egg extraction when maintained in Hanks&rsquo; solution at 20 &deg;C, leading to a supply of one-cell stage embryo for microinjection treatment constantly by continuously executing artificial fertilization. This systematic procedure permitted selective breeding by 1:1 mating between top-quality parental fish and applying several developmental engineering techniques to kawakawa breeding

Effectiveness and Safety of Immune Checkpoint Inhibitors Alone or in Combination With Chemotherapy in Pulmonary Sarcomatoid Carcinoma

Introduction: Pulmonary sarcomatoid carcinoma (PSC) is a rare subtype of lung cancer associated with poor prognosis and resistance to conventional chemotherapy. Immune checkpoint inhibitors (ICIs), alone or in combination with chemotherapy, were found to have clinical benefits in PSC in recent studies. Nevertheless, because these studies included a small number of patients owing to disease rarity, larger studies are needed to evaluate the effectiveness and safety of ICI-based therapy for PSC. Methods: This multicenter retrospective study evaluated patients with ICI-naive advanced or metastatic PSC who were treated with ICI-based therapy at 25 hospitals in Japan. Results: A total of 124 patients were evaluated. The overall response rate, median progression-free survival (PFS), and median overall survival (OS) were 59.0%, 10.5 months, and 32.8 months, respectively. The PFS and OS rates at 24 months were 35.3% and 51.5%, respectively. Programmed death-ligand 1 expression, concomitant chemotherapy, and the treatment line were not significantly associated with PFS or OS. Immune-related adverse events (irAEs) were observed in 70 patients (56.5%), including 30 (24.2%) with grade 3 to 5 events. Patients with mild irAEs (grades 1–2) had longer PFS and OS than did those with severe (grades 3–5) or no irAEs. In a multivariate analysis, any-grade irAEs and the absence of liver metastases were independently associated with PFS, whereas any-grade irAEs and Eastern Cooperative Oncology Group performance status less than or equal to 1 were independently associated with OS. Conclusions: ICI-based therapy was found to have promising effectiveness in patients with advanced or metastatic PSC, regardless of programmed death-ligand 1 expression, concomitant chemotherapy, or treatment line