Anti-SARS-CoV-2 gapmer antisense oligonucleotides targeting the main protease region of viral RNA.

Given the worldwide risk for the outbreak of emerging/re-emerging respiratory viruses, establishment of new antiviral strategies is greatly demanded. In this study, we present a scheme to identify gapmer antisense oligonucleotides (ASOs) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA that efficiently inhibit viral replication. We synthesized approximately 300 gapmer ASOs designed to target various SARS-CoV-2 RNA regions and evaluated their activity in cell-based assays. Through a multistep screening in cell culture systems, we identified that ASO#41, targeting the coding region for viral main protease, reduced SARS-CoV-2 RNA levels in infected cells and inhibited virus-induced cytopathic effects. Antiviral effect of ASO#41 was also observed in iPS cell-derived human lung organoids. ASO#41 depleted intracellular viral RNAs during genome replication in an endogenous RNaseH-dependent manner. ASO#41 showed a wide range of antiviral activity against SARS-CoV-2 variants of concern including Alpha, Delta, and Omicron. Intranasal administration to mice exhibited intracellular accumulation of ASO#41 in the lung and significantly reduced the viral infectious titer, with milder body weight loss due to SARS-CoV-2 infection. Further chemical modification with phosphoryl guanidine-containing backbone linkages provided an elevation of anti-SARS-CoV-2 activity, with 23.4 nM of 50% antiviral inhibitory concentration, one of the strongest anti-SARS-CoV-2 ASOs reported so far. Our study presents an approach to identify active ASOs against SARS-CoV-2, which is potentially useful for establishing an antiviral strategy by targeting genome RNA of respiratory viruses.

Does Central Sensitization Influence Outcomes of Lumbar Discectomy Surgery in Patients With Lumbar Disc Herniation? A Multicenter Prospective Study.

STUDY DESIGN: Multicenter prospective study. OBJECTIVE: Patients with central sensitization (CS) are reported to be at high risk of poor outcomes after spinal surgery. However, the influence of CS on surgical outcomes for lumbar disc herniation (LDH) remains unknown. This study aimed to examine the association between preoperative CS and surgical outcomes in LDH patients. METHODS: A total of 100 consecutive patients with LDH (mean age 51.2) who underwent lumbar surgery were included in this study. The extent of CS was evaluated using the central sensitization inventory (CSI), a screening tool for CS-related symptoms. The patients completed the following CSI and clinical outcome assessments (COAs) preoperatively and 12 months postoperatively: the Japanese Orthopaedic Association (JOA) score for back pain, JOA back pain evaluation questionnaire (JOABPEQ), and Oswestry Disability Index (ODI). The association between preoperative CSI scores, and preoperative and postoperative COAs was analyzed, and the postoperative changes were statistically evaluated. RESULTS: The preoperative CSI score significantly decreased 12 months postoperatively. Preoperative CSI scores showed a significant correlation with most COAs; however, a significant correlation was only identified in the social function and mental health domains of JOABPEC postoperatively. Higher preoperative CSI showed worse preoperative COAs; however, all COAs significantly improved regardless of CSI severity. There were no significant differences in any COAs among the CSI severity groups 12 months postoperatively. CONCLUSIONS: The results of this study showed that lumbar surgeries significantly improved the COAs regardless of preoperative severity of CS in patients with LDH.

Central sensitization as a predictive factor for the surgical outcome in patients with lumbar spinal stenosis: a multicenter prospective study.

PURPOSE: The impact of central sensitization (CS) on neurological symptoms and surgical outcomes in patients with lumbar spinal stenosis (LSS) remains unknown. This study aimed to investigate the influence of preoperative CS on the surgical outcomes of patients with LSS. METHODS: A total of 197 consecutive patients with LSS (mean age 69.3) who underwent posterior decompression surgery with or without fusion were included in this study. The participants completed the CS inventory (CSI) scores and the following clinical outcome assessments (COAs) preoperatively and 12 months postoperatively: the Japanese Orthopaedic Association (JOA) score for back pain, JOA back pain evaluation questionnaire, and Oswestry Disability Index (ODI). The association between preoperative CSI scores and preoperative and postoperative COAs was analyzed, and postoperative changes were statistically evaluated. RESULTS: The preoperative CSI score significantly decreased at 12 months postoperatively and was significantly correlated with all COAs preoperatively and 12 months postoperatively. Higher preoperative CSI showed worse postoperative COAs and inferior postoperative improvement rates in the JOA score, VAS score for neurological symptoms, and ODI. Multiple regression analysis demonstrated that preoperative CSI was significantly associated with postoperative low back pain (LBP), mental health, quality of life (QOL), and neurological symptoms at 12 months postoperatively. CONCLUSIONS: Preoperative CS evaluated by CSI had a significantly worse impact on surgical outcomes, including neurological symptoms, disability, and QOL, especially related to LBP and psychological factors. CSI can be used clinically as a patient-reported measure for predicting postoperative outcomes in patients with LSS.

Evaluation of Central Sensitization Inventory in Patients Undergoing Elective Spine Surgery in a Multicenter Study.

STUDY DESIGN: This study is a retrospective review. OBJECTIVE: Central sensitization (CS) is a neurological phenomenon that involves hypersensitivity of the central nervous system. The central sensitization inventory (CSI) was developed as a screening tool to assess CS-related symptoms. The purpose of this study was to evaluate the association of preoperative CSI scores with patient-reported outcome measures (PROMs) including neurological symptoms for patients who underwent spine surgeries in a multicenter study. METHODS: A consecutive 673 patients who underwent spine surgery at 8 different institutions were included in this study. Preoperative CSI scores were assessed for all subjects. The participants completed the following PROMs: the Oswestry Disability Index (ODI), the Japanese Orthopaedic Association (JOA) back pain evaluation questionnaire (JOABPEQ) for lumbar spinal diseases, and the JOA cervical myelopathy evaluation questionnaire (JOACMEQ) for cervical spinal diseases. The association of CSI scores with PROMs was statistically evaluated. RESULTS: The average CSI score for the total subjects was 23.6 ± 13.5. The subjects with CS-related symptoms (CSI ≥ 40) were 13.2% (n = 89). The CSI score showed a significant and weak-to-moderate correlation with the PROMs including neurological symptoms that included all the domains of the JOACMEQ for cervical spinal diseases, and JOABPEQ and ODI for lumbar spinal diseases. Among these, psychological factors had the most influence on the correlation with CSI score. CONCLUSION: Central sensitization evaluated by the CSI is related to neurological symptoms and health-related quality of life in patients undergoing elective spine surgery.

Influence of Central Sensitization on Surgical Outcomes of Patients With Degenerative Cervical Myelopathy After Posterior Decompression Surgery: A Multicenter Prospective Study.

STUDY DESIGN: Multicenter prospective study. OBJECTIVE: The influence of central sensitization (CS) on neurological symptoms and surgical outcomes in patients with degenerative cervical myelopathy (DCM) remains unknown. This study aimed to investigate the effects of preoperative CS on surgical outcomes of patients with DCM following posterior decompression surgery. METHODS: 77 consecutive patients with DCM (mean age 67.1) who received posterior decompression surgery were included in this study. The participants completed CS inventory (CSI) scores and the following patient-reported outcome measures (PROMs) preoperatively and 12 months postoperatively: the Japanese Orthopaedic Association (JOA) score for cervical myelopathy and JOA cervical myelopathy evaluation questionnaire (JOACMEQ) for cervical spinal diseases. The association of preoperative CSI scores with preoperative and postoperative PROMs was analyzed, and their changes were statistically evaluated. RESULTS: The preoperative CSI score was significantly decreased at 12 months postoperatively, and it was significantly associated with the JOA score and JOACMEQ preoperatively and at 12 months postoperatively. However, no significant association was observed between preoperative CSI and the postoperative change of any PROMs at 12 months. The posterior decompression surgery significantly improved the JOA scores and 'lower extremity function' and 'quality of life (QOL)' domains of the JOACMEQ, independent of the severity of preoperative CSI score. Multiple regression analysis demonstrated that preoperative CSI was significantly associated with the 'QOL' domain of JOACMEQ and original JOA score at 12 months postoperatively. CONCLUSION: The CSI score can be an auxiliary indicator of surgical outcomes of patients with DCM following posterior decompression surgery.

Antibacterial and Immunosuppressive Effects of OPS-2071, a Candidate Therapy for Inflammatory Bowel Disease.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the gastrointestinal tract. Although many types of drug are used, clinical outcomes are still unsatisfactory. Previous studies have suggested that intestinal bacteria are involved in the pathogenesis of IBD. Accordingly, in an IBD model we evaluated the therapeutic effects of OPS-2071, a low-absorption quinolone antibacterial agent indicated for intestinal infection, and investigated its mechanism of action. METHODS: The therapeutic effects of OPS-2071 and comparison therapies were evaluated using naive CD4 + T cell-transfer IBD model mice. In vitro inhibition of LPS-induced TNF-α production and inhibitory effects on T cell responses stimulated using anti-CD3/CD28 antibody-loaded beads were evaluated using mouse splenocytes and human peripheral blood mononuclear cells. In addition, in vitro activities against bacteria implicated in IBD pathogenesis were tested. RESULTS: OPS-2071 dose-dependently decreased both colonic weight/length ratio and the colitis histological score as compared with the vehicle group. The therapeutic effect of OPS-2071 was equivalent to that of anti-IL-12/23 (p40) antibody. In vitro, OPS-2071 suppressed TNF-α production induced by LPS stimulation and T cell responses in a dose-dependent manner. At high concentrations, these effects were comparable to those of existing immunosuppressive agents, such as prednisolone, in both mouse and human cells. OPS-2071 also showed antibacterial activity against IBD-related bacteria. CONCLUSIONS: Our results suggest that OPS-2071 had both immunosuppressive and antibacterial effects. This dual effect makes OPS-2071 a unique and promising candidate for IBD.

[Acute respiratory failures caused by post-tracheotomy tracheomalacia].

A 77-year-old man underwent thoracic surgery. He had a history of two previous operations: parapharyngeal tumor removal with temporal tracheotomy 14 years ago and, two years later, a sinus surgery when, according to our anesthesia registry, intubation was extremely difficult due to stricture of the trachea underneath the tracheotomy scar. Pathology was not fully elucidated. Preoperative examinations including chest x-ray, spirogram and CT were not remarkable. The scar above the suprasternal notch was visibly sunken and retracted with respiration. Stridor was auscultated but breathing was not labored. The patient was anesthetized with propofol and intubation was smooth. During surgery anesthesia was maintained with sevoflurane, remifentanil and rocuronium. However, extubation was followed by desperate gasping and severe respiratory distress. The tracheotomy scar caved in and the airway collapsed. Continuous airway pressure via a facemask restored airway patency and improved breathing. After overnight respiratory support with non-invasive positive pressure ventilation (NPPV), patient was weaned from ventilator. Airway collapse and the two episodes of respiratory failures while under general anesthesia were attributed to post-tracheotomy tracheomalacia.

[Comparison of the Parker flex-tip tube with the standard tube for Airtraq intubation].

BACKGROUND: During tracheal intubation with the Airtraq laryngoscope (AL), the tracheal tube tip sometimes impinges on the glottis or other laryngeal structures. We tested a hypothesis that Parker flex-tip tube (PT) makes AL assisted tracheal intubation easier and faster than a conventional tube (Mallinckrod Hi-Lo tube : HT). METHODS: Study 1. Fifty patients were assigned to group PT (n = 25) or group HT (n = 25). After induction of general anesthesia, intubation was performed with AL. We recorded tube passage time, the number of tube impingements, circulatory changes and incidences of postoperative complications. Study 2. Twenty patients were divided into two groups and intubated either by HT or PT. We assessed the tip direction and contact points of each tube. RESULTS: Study 1. The use of PT significantly reduced the number of tube impingements compared to HT. Tube passage time in group PT was shorter than that in group HT, but the difference was not statistically significant. Study 2. The tube tip contact with the vocal cords was more frequent with HT. CONCLUSIONS: PT significantly reduced the number of impingements. The shape of tube tip and the smaller angle of PT when fitted in AL contributed to the relative ease of tube passage.

Effects of slightly acidic electrolysed drinking water on mice.

Slightly acidic electrolysed (SAE) water is a sanitizer with strong bactericidal activity due to hypochlorous acid. We assessed the safety of SAE water as drinking water for mice at a 5 ppm total residual chlorine (TRC) concentration to examine the possibility of SAE water as a labour- and energy-saving alternative to sterile water. We provided SAE water or sterile water to mice for 12 weeks, during which time we recorded changes in body weight and weekly water and food intakes. At the end of the experiment, all of the subject animals were sacrificed to assess serum aspartate aminotransferase, alanine aminotransferase and creatinine levels and to examine the main organs histopathologically under a light microscope. In addition, we investigated the bacteria levels of both types of water. We found no difference in functional and morphological health condition indices between the groups. Compared with sterile water, SAE water had a relatively higher ability to suppress bacterial growth. We suggest that SAE water at 5 ppm TRC is a safe and useful alternative to sterile water for use as drinking water in laboratory animal facilities.

Expression of proteinase-activated receptor-2 in the intervertebral disc.

STUDY DESIGN: Immunohistochemical and biochemical analyses of proteinase-activated receptor-2 (PAR-2) in rat and human intervertebral discs (IVDs). OBJECTIVES: To examine the expression and function of PAR-2 in rat IVD cells, and to determine if PAR-2 is expressed in human IVDs. SUMMARY OF BACKGROUND DATA: PAR-2 is a G protein-coupled receptor that contributes to the regulation of inflammatory reactions and the pathophysiology of inflammatory diseases, including arthritis. The expression of PAR-2 in the IVD has not been determined. METHODS: PAR-2 expression by rat IVD cells and tissues was examined using immunohistochemistry and western blot. Rat anulus fibrosus cells in monolayer culture were used to examine the biologic role of PAR-2 in vitro. The effect of PAR-2-activating peptide (PAR-2AP) on the catabolic cascade was assessed by western blot and real-time PCR. The expression of PAR-2 by human IVD tissues at different stages of degeneration was determined by immunohistochemical analyses. RESULTS: PAR-2 was expressed by rat IVD cells and in both anulus fibrosus and nucleus pulposus tissues, PAR-2 expression was up-regulated by interleukin-1beta (IL-1beta). PAR-2AP significantly increased the release of IL-1beta into the medium. Although PAR-2AP had no direct effect on matrix metalloproteinase-3 (MMP-3) and MMP-13 mRNA levels, treatment with PAR-2AP significantly up-regulated the mRNA levels of a disintegrin and metalloproteinase with thrombospondin motif-4. The simultaneous administration of PAR-2AP and IL-1beta synergistically up-regulated the mRNA levels of a disintegrin and metalloproteinase with thrombospondin motif-4, MMP-3, and MMP-13. The expression of PAR-2 was identified in human IVD tissues. The number of PAR-2-expressing cells was significantly elevated in advanced stages of IVD degeneration compared with those in early stages of degeneration. CONCLUSION: Our results demonstrate for the first time that IVD cells express PAR-2. The expression of PAR-2 is regulated by IL-1beta stimulation. PAR-2 activation accelerates the expression of matrix-degrading enzymes. PAR-2 may play an important role in the cytokine-mediated catabolic cascade and consequently may be involved in IVD degeneration.

[Awake intubation using lightwand technique under conscious sedation with remifentanil].

We experienced two cases of difficult airway due to deformities in the oral cavities. The first patient was a 62-year-old woman with a large benign soft palate tumor and the second was a 64-year-old woman with macroglossia secondary to acromegaly. Both patients were evaluated difficult to ventilate via face mask and presenting serious risks for tracheal intubation under general anesthesia. The tracheal intubation was planned using the lightwand (Trachilight) under conscious sedation with continuous administration of remifentanil. Remifentanil (0.1-0.25 microg x kg(-1) x min(-1)) maintained the patients' spontaneous ventilation and increased their tolerance to the pain and discomfort caused by insertion of the lightwand. In both patients, remifentanil mildly suppressed the coughing reflex as well as the autonomic responses to stimuli to the airway. Tracheal intubation was managed successfully in both cases and the operations were completed under general anesthesia. Although the patients were aware of being intubated, they could not recall the procedures postoperatively. The awake intubation technique using the lightwand under conscious sedation with remifentanil can be safely applied to a patient with difficult airway.

Identification of a novel NY-ESO-1 promiscuous helper epitope presented by multiple MHC class II molecules found frequently in the Japanese population.

NY-ESO-1 is a cancer-testis antigen that elicits strong cellular and humoral immune responses against NY-ESO-1-expressing tumors. Although CD4(+) T cells play a critical role in inducing antitumor immunity, little is known about MHC class II-restricted helper epitopes of the NY-ESO-1 antigen compared with MHC class I-restricted epitopes. Here, we searched for new NY-ESO-1 helper epitopes presented by MHC class II molecules, especially those found frequently in the Japanese population. We established five NY-ESO-1-specific helper T-cell lines from healthy Japanese donors using NY-ESO-1 recombinant protein and peptide. Using MHC class II-specific antibodies and a panel of Epstein-Barr virus-transformed B-cell lines, it was demonstrated that four out of the five T-cell lines recognized a region within NY-ESO-1(119-143) in the context of HLA-DRB1*0802, DRB1*0901, DRB1*1502 or DRB1*0405/*0410. In addition, using a set of overlapping 15-mer synthetic peptides, we found that NY-ESO-1(122-138) was a promiscuous region that bound to four distinct HLA-DR molecules found in the Japanese population. These findings expand the usefulness of NY-ESO-1 as a tool for tumor vaccine therapy in eliciting NY-ESO-1-specific helper T-cell responses, especially in Japanese cancer patients.

[Successful epidural anesthesia using 120 mm epidural needle for cesarean section in a morbidly obese parturient with body mass index 50.2 kg x m(-2)].

In a morbidly obese parturient, epidural anesthesia is occasionally difficult because of great distance from the skin to the epidural space, and difficulty in identification of appropriate landmarks. We successfully managed cesarean section in a morbidly obese parturient with body mass index of 50.2 kg x m(-2) under epidural anesthesia. We used a 17 G custom-made epidural needle 120 mm long (Hakko, Tokyo, Japan), and the depth from the skin to the epidural space was 95 mm. We conclude that an extremely long epidural needle was useful in a morbidly obese parturient for overcoming the difficulties in epidural puncture and avoiding general anesthesia-related complications.

Antitumor activity of chimeric immunoreceptor gene-modified Tc1 and Th1 cells against autologous carcinoembryonic antigen-expressing colon cancer cells.

To generate tumor-specific and interferon (IFN)-gamma-producing Tc1 and Th1 cells applicable for many cancer patients, we previously developed a protocol for generating carcinoembryonic antigen (CEA)-specific Tc1 and Th1 cells from healthy human T cells by transduction with a lentivirus containing a chimeric immunoglobulin T-cell receptor (cIgTCR) gene composed of single-chain variable fragments from an anti-CEA-specific monoclonal antibody fused to an intracellular signaling domain of CD28 and CD3zeta. These cells, designated Tc1-T and Th1-T bodies, respectively, showed strong antitumor activity against CEA-expressing tumor cells in RAG2-/- mice when both of them were transferred. However, it remains unclear whether it is possible to generate Tc1-T and Th1-T bodies from cancer patients with defective T-cell function because of significant immunosuppression. Here, we prepared Tc1-T and Th1-T bodies from T cells of a colon cancer patient, and asked whether these T bodies can exert effective T-cell function against autologous tumor cells. These T bodies showed high cytotoxicity and produced IFN-gamma in response to CEA-expressing autologous tumor cells, even in the presence of soluble CEA. It was also demonstrated that Th1-T bodies supported the survival of Tc1-T bodies through cell-to-cell interactions. Furthermore, our protocol utilized retrovirus for cIgTCR transduction to achieve better induction efficiency compared to lentivirus-mediated transduction. Taken together, our findings here indicate that retrovirally transduced Tc1-T and Th1-T bodies will become a promising strategy for adoptive immunotherapy of human cancer.

Expression of the MAGE-A4 and NY-ESO-1 cancer-testis antigens and T cell infiltration in non-small cell lung carcinoma and their prognostic significance.

Cancer-testis (CT) antigens were identified as a group of highly attractive targets for cancer immunotherapy because of their expression in a variety of malignant tumors but solely in the testis among the normal adult tissues. To evaluate the potential of two members of this family, MAGE-A4 and NY-ESO-1 antigens, for cancer vaccine in non-small cell lung carcinoma (NSCLC), we examined the expression of these antigens and T cell infiltration in tumor tissue, and evaluated their prognostic significance. One hundred fifty-seven patients with NSCLC were studied. Reverse transcription-PCR was performed to evaluate MAGE-A4 and NY-ESO-1 expression. Immunohistochemistry was performed for NY-ESO-1 expression and T cell infiltration into the tumor site. Survival analysis was also performed. MAGE-A4 and NY-ESO-1 were expressed in 40 of 141 (28.4%) and 13 of 157 (8.3%) NSCLC respectively. Both CT antigens were more frequently expressed in squamous cell carcinoma (SCC) than in adenocarcinoma. An inverse correlation was found between MAGE-A4 expression and patient survival in advanced stage cancers. Combined infiltration of both CD4+ and CD8+ T cells into tumor nest predicted better survival. There was no correlation, however, between lymphocyte infiltration and antigen expression in the tumor. MAGE-A4 expression in advanced group and T cell infiltration may provide prognostic information. Lastly, these CT antigens, especially MAGE-A4, may represent potential targets for cancer immunotherapy in patients with NSCLC.

Development of a functional cDNA array for evaluation of the Th1/Th2 balance.

The immune balance controlled by CD4(+) helper T cell subsets (T helper 1 (Th1) and T helper 2 (Th2)) is crucial for immunoregulation and its imbalance causes various immune diseases including infections, allergic disorders and autoimmune diseases. Therefore, it is of great importance to develop a system of diagnosing Th1/Th2 imbalances for curing immune diseases. Here we developed a functional cDNA array filter useful for assessing the Th1/Th2 balance in mice. To overcome the disadvantages of conventional microarrays carrying thousands of genes, we prepared an array filter containing 40 Th1-specific and 32 Th2-specific genes, which were selected from over 8700 genes based on (i) the specificity of expression in Th1 or Th2 cells and (ii) an expression level which is high enough for detection using a DNA array. This array filter provided a prompt and precise evaluation for the skewing of the Th1/Th2 balance combined with our calculation algorithm. The bias toward Th1 or Th2 was evaluated visually at a glance by aligning the genes on the filter. Moreover, we succeeded in evaluating the skewing of the Th1/Th2 balance in vivo during acute graft versus host disease (GVHD). Thus, this array filter will provide a novel tool for evaluation of the Th1/Th2 balance in a variety of immune diseases.

Uptake of gallium-67 by the hepatocytes during liver regeneration in carbon tetrachloride-treated mice.

Gallium-67 (67Ga) has been used as a tumor or inflammation-imaging agent in nuclear medicine. We recently reported that the peak of serum alanine aminotransferase (ALT) level was 1 d after carbon tetrachloride (CCl4) treatment in rats. However, the peak of 67Ga uptake by the liver tissue and hepatocytes was 2 d after the treatment. If 67Ga is taken in the hepatic disorder phase, the pattern of 67Ga uptake by the hepatocytes should be consistent with that of the ALT level. In order to answer why it was not, we carried out a detailed examination. The lipid peroxidation of hepatocytes from CCl4-treated mice was greatly increased 1 d after CCl4 treatment; serum ALT was also increased 1 d after the treatment. The uptake of 67Ga by the liver tissue reached a maximum 1 and 2 d after the treatment, while maximum by the hepatocytes was achieved after 2 d. The incorporation of bromodeoxyuridine into the hepatocytes also reached maximum 2 d after the treatment. These results suggest that the uptake of 67Ga by the hepatocytes is carried out during liver regeneration rather than during hepatic disorder by CCl4 treatment.