High-sensitivity whole-mount in situ Hybridization of Mouse Oocytes and Embryos Visualizes the Super-resolution Structures and Distributions of mRNA Molecules.

Mammalian oocytes accumulate more than ten thousand mRNAs, of which three to four thousand mRNAs are translationally repressed. The timings and sites of translational activation of these dormant mRNAs are crucial for promoting oocyte maturation and embryonic development. How these mRNAs are accumulated and distributed in oocytes is therefore a fundamental issue to be explored. A method that enables visualization of mRNA molecules with high resolution in a simple manner would be valuable for understanding how oocytes accumulate and regulate the dormant mRNAs. We have developed a highly sensitive whole-mount in situ hybridization method using in vitro-synthesized RNA probes and the tyramide signal amplification (TSA) system optimized for mouse oocytes and embryos. By using this method, Pou5f1/Oct4, Emi2, and cyclin B1 mRNAs were detected in immature oocytes and 2-cell stage embryos. Confocal microscopy showed that these mRNAs formed granular structures in the oocyte cytoplasm. The structures of Pou5f1/Oct4 and cyclin B1 mRNAs persisted in 2-cell stage embryos. Pou5f1/Oct4 RNA granules exhibited a solid-like property in immature oocytes and became liquid-like droplets in 2-cell stage embryos. Double-staining of cyclin B1 mRNA with Emi2 or Pou5f1/Oct4 mRNA revealed that these mRNAs were distributed as different RNA granules without overlapping each other and that the size of cyclin B1 RNA granules tended to be larger than that of Emi2 RNA granules. The structures and distribution patterns of these mRNAs were further analyzed by N-SIM super-resolution microscopy. This analysis revealed that the large-sized RNA granules consist of many small-sized granules, suggesting the accumulation and regulation of dormant mRNAs as basal-sized RNA granules. The method established in this study can easily visualize the structure and distribution of mRNAs accumulated in mammalian oocytes and embryos with high sensitivity and super-resolution. This method is useful for investigating the cellular and molecular mechanisms of translational control of mRNAs by which maturation and early developmental processes are promoted.

[A Case of Early-Stage Cecal Cancer with Mesenteric Phlebosclerosis Requiring Laparoscopic Right Hemicolectomy of the Colon].

The patient was a 71-year-old woman diagnosed with mesenteric phlebosclerosis(MP)2 years earlier. CT performed to investigate her abdominal pain revealed an ascending colon obstruction. Colonoscopy(CS)revealed MP extending to the ascending colon hepatic flexure with stenosis and a cecal tumor(biopsy tub1). Although the cancerous lesion itself was potentially curable by endoscopic treatment, it was surgically resected because of the ascending colon stenosis caused by the MP that had also caused intestinal obstruction. Intraoperative findings revealed wall thickening and stiffening from the cecum to the ascending colon hepatic flexure. Postoperative pathological examination revealed cecal carcinoma pTis, N0, M0, pStage 0. The background mucosal tissue was consistent with MP, but no findings suggested a relationship between the MP and tumor. Although the relationship between MP and carcinogenesis is unknown, and no such relationship was identified in this case, we report this case because a further accumulation of cases of MP and carcinoma is necessary, considering the rarity of MP itself and the non-negligible number of cases with carcinoma.

Attenuated humoral response against SARS-CoV-2 mRNA vaccination in allogeneic stem cell transplantation recipients.

Antibody persistence several months after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccination in allogeneic stem cell transplantation recipients remains largely unknown. We sequentially evaluated the humoral response to two doses of mRNA vaccines in 128 adult recipients and identified the risk factors involved in a poor response. The median interval between stem cell transplantation and vaccination was 2.7 years. The SARS-CoV-2 S1 Ab became positive after the second vaccination dose in 87.6% of the recipients, and the median titer was 1235.4 arbitrary units (AU)/ml. In patients on corticosteroid treatment, the corticosteroid dose inversely correlated with Ab titer. Multivariate analysis identified risk factors for poor peak response such as an interval from stem cell transplantation ≤1 year, history of clinically significant CMV infection, and use of >5 mg/day prednisolone at vaccination. Six months after vaccination, the median titer decreased to 185.15 AU/ml, and use of >5 mg/day prednisolone at vaccination was significantly associated with a poor response. These results indicate that early vaccination after stem cell transplantation (<12 months) and CMV infection are risk factors for poor peak response, while steroid use is important for a peak as well as a persistent response. In conclusion, although humoral response is observed in many stem cell transplantation recipients after two doses of vaccination, Ab titers diminish with time, and factors associated with persistence and a peak immunity should be considered separately.

Qualitative MR features to identify non-enhancing tumors within glioblastoma's T2-FLAIR hyperintense lesions.

PURPOSE: To identify qualitative MRI features of non-(contrast)-enhancing tumor (nCET) in glioblastoma's T2-FLAIR hyperintense lesion. METHODS: Thirty-three histologically confirmed glioblastoma patients whose T1-, T2- and contrast-enhanced T1-weighted MRI and 11C-methionine positron emission tomography (Met-PET) were available were included in this study. Met-PET was utilized as a surrogate for tumor burden. Imaging features for identifying nCET were searched by qualitative examination of 156 targets. A new scoring system to identify nCET was established and validated by two independent observers. RESULTS: Three imaging features were found helpful for identifying nCET; "Bulky gray matter involvement", "Around the rim of contrast-enhancement (Around-rim)," and "High-intensity on T1WI and low-intensity on T2WI (HighT1LowT2)" resulting in an nCET score = 2 × Bulky gray matter involvement - 2 × Around-rim + HighT1LowT2 + 2. The nCET score's classification performances of two independent observers measured by AUC were 0.78 and 0.80, with sensitivities and specificities using a threshold of four being 0.443 and 0.771, and 0.916 and 0.768, respectively. The weighted kappa coefficient for the nCET score was 0.946. CONCLUSION: The current investigation demonstrated that qualitative assessments of glioblastoma's MRI might help identify nCET in T2/FLAIR high-intensity lesions. The novel nCET score is expected to aid in expanding treatment targets within the T2/FLAIR high-intensity lesions.

Intranasal therapeutic vaccine containing HBsAg and HBcAg for patients with chronic hepatitis B; 18 months follow-up results of phase IIa clinical study.

AIMS: HBsAg loss with anti-HBs acquisition is considered a functional cure and ideal treatment goal for patients with CHB. Our group have reported the efficacy of therapeutic vaccine with HBsAg and HBcAg (NASVAC) by intranasal and subcutaneous injection. In this study, we investigated the safety and efficacy of newly developed CVP-NASVAC, which contained NASVAC with mucoadhesive carboxyl vinyl polymer (CVP) in the dedicated device. METHODS: A single dose, open-label, phase IIa clinical trial of CVP-NASVAC was conducted. Patients with CHB treated with nucleoside/nucleotide analogs (NAs) and HBV carriers not undergoing anti-HBV treatment were enrolled. CVP-NASVAC was injected through the nose for, in total, 10 times. Participants were followed-up for 18 months, and their HBsAg reduction and anti-HBs induction assessed as endpoints. RESULTS: Among the patients with CHB treated with NAs (n = 27) and HBV carriers without NAs (n = 36), 74.1% and 75.0% exhibited reductions in their baseline HBsAg, and the mean reductions were -0.1454 log10  IU/ml (p < 0.05) and -0.2677 log10  IU/ml (p < 0.05), respectively. Anti-HBs antibody was detected in 40.7% and 58.3% of patients treated with and without NAs, respectively. Six of 71 (9.5%) patients were functionally cured after the CVP-NASVAC treatment. CONCLUSIONS: Anti-HBs induction and HBsAg reduction was observed after CVP-NASVAC treatment in some patients with CHB. The CVP-NASVAC is a safe treatment, which might expect to achieve functional cure for patients with CHB.

Antibody response to third and fourth BNT162b2 mRNA booster vaccinations in healthcare workers in Tokyo, Japan.

BACKGROUND: Booster vaccinations against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are being promoted worldwide to counter the coronavirus disease 2019 (COVID-19) pandemic. In this study, we analyzed the longitudinal effect of the third BNT162b2 mRNA vaccination on antibody responses in healthcare workers. Additionally, antibody responses induced by the fourth vaccination were analyzed. METHODS: The levels of anti-spike (S) IgG and neutralizing antibody against SARS-CoV-2 were measured at 7 months after the second vaccination (n = 1138), and at 4 (n = 701) and 7 (n = 417) months after the third vaccination using an iFlash 3000 chemiluminescence immunoassay analyzer. Among the 417 participants surveyed at 7 months after the third vaccination, 40 had received the fourth vaccination. A multiple linear regression analysis was performed to clarify which factors were associated with the anti-S IgG and neutralizing antibody. Variables assessed included sex, age, number of days after the second or third vaccination, diagnostic history of COVID-19, and anti-nucleocapsid (N) IgG level. RESULTS: At 7 months after the third vaccination, antibody responses were significantly higher than those at the same time after the second vaccination. Unlike the second vaccination, age had no effect on the antibody responses induced by the third vaccination. Furthermore, the fourth vaccination resulted in a further increase in antibody responses. The multiple linear regression analysis identified anti-N IgG level, presumably associated with infection, as a factor associated with antibody responses. CONCLUSIONS: Our findings showed that BNT162b2 booster vaccinations increased and sustained the antibody responses against SARS-CoV-2.

Serologic Survey of IgG Against SARS-CoV-2 Among Hospital Visitors Without a History of SARS-CoV-2 Infection in Tokyo, 2020-2021.

BACKGROUND: Tokyo, the capital of Japan, is a densely populated city of >13 million people, so the population is at high risk of epidemic severe acute respiratory coronavirus 2 (SARS-CoV-2) infection. A serologic survey of anti-SARS-CoV-2 IgG would provide valuable data for assessing the city's SARS-CoV-2 infection status. Therefore, this cross-sectional study estimated the anti-SARS-CoV-2 IgG seroprevalence in Tokyo. METHODS: Leftover serum of 23,234 hospital visitors was tested for antibodies against SARS-CoV-2 using an iFlash 3000 chemiluminescence immunoassay analyzer (Shenzhen YHLO Biotech, Shenzhen, China) with an iFlash-SARS-CoV-2 IgG kit (YHLO) and iFlash-SARS-CoV-2 IgG-S1 kit (YHLO). Serum samples with a positive result (≥10 AU/mL) in either of these assays were considered seropositive for anti-SARS-CoV-2 IgG. Participants were randomly selected from patients visiting 14 Tokyo hospitals between September 1, 2020 and March 31, 2021. No participants were diagnosed with coronavirus disease 2019 (COVID-19), and none exhibited COVID-19-related symptoms at the time of blood collection. RESULTS: The overall anti-SARS-CoV-2 IgG seroprevalence among all participants was 1.83% (95% confidence interval [CI], 1.66-2.01%). The seroprevalence in March 2021, the most recent month of this study, was 2.70% (95% CI, 2.16-3.34%). After adjusting for population age, sex, and region, the estimated seroprevalence in Tokyo was 3.40%, indicating that 470,778 individuals had a history of SARS-CoV-2 infection. CONCLUSIONS: The estimated number of individuals in Tokyo with a history of SARS-CoV-2 infection was 3.9-fold higher than the number of confirmed cases. Our study enhances understanding of the SARS-CoV-2 epidemic in Tokyo.

[Two Cases of Neuroendocrine Carcinoma of Colon and Rectum].

We report 2 cases of neuroendocrine carcinoma(NEC)of colon and rectum with distant metastasis. The treatment of NEC with distant metastasis is based on the treatment of small cell lung cancer, but that is controversial because NEC is relatively rare. Case 1: A 75-year-old man who was admitted for anal pain. Physical examination showed the anal tumor and swelling inguinal lymph nodes. CT showed rectum tumor and multiple lymph node metastases to the pelvis and inguinal region. Colonoscopy showed a Type 3 tumor in the rectum. He was diagnosed with NEC based on biopsy and immunostaining. Colostomy was performed for pain relief and etoposide/cisplatin(EP)therapy was introduced. After 6 courses of the chemotherapy, CT showed progression of the tumor, then we made the shift to palliative treatment. Case 2: A 69-year-old man who was admitted for abdominal pain and back pain. CT showed transverse colon tumor with multiple metastases to the liver, lung, and lymph nodes. Colonoscopy showed a circumferential tumor in the transverse colon. He was diagnosed with NEC based on biopsy and immunostaining. He refused chemotherapy and died 2 months later.

[A Case of BRAF-Mutated Obstructive Colon Cancer with Liver Metastases Treated with Multidisciplinary Therapy].

A 39-year-old woman visited our hospital with complaints of nausea, vomiting, and lower abdominal pain for 2 weeks. Abdominal CT revealed thickening of the transverse colonic wall, dilated bowel, and a metastatic ischemic tumor in the liver (S7). We diagnosed her with obstructive colon cancer, clinical Stage Ⅳa(T, type 2, cT3, N0, M1a[liver]). At first, we placed a self-expanding metallic stent(SEMS)to decompress bowel obstructions. We planned a surgical resection of the primary tumor followed by partial resection of the liver. We performed a laparoscopic right hemicolectomy(D3)24 days after the stenting. Pathologically, we diagnosed her with BRAF-mutated colon cancer, pStage Ⅳa(pT4a, N1b[2/43], M1a[liver]). On completion of 4 courses of mFOLFOXIRI and bevacizumab, we confirmed a reduction of the S7 tumor but found a new tumor in S6. Since the tumors were potentially resectable, we performed partial liver resection(S6, S7)1 month later. A month following the hepatectomy, CT revealed a new tumor in S4. The patient has been receiving general chemotherapy (CapeOX and bevacizumab)without disease progression for 6 months. We experienced a challenging case of BRAF- mutated obstructive colon cancer with liver metastases.

Intranasal vaccination with HBs and HBc protein combined with carboxyl vinyl polymer induces strong neutralizing antibody, anti-HBs IgA, and IFNG response.

Hepatitis B virus (HBV) infection causes acute and chronic hepatitis, which is a major public health concern worldwide. Immunization methods incorporating hepatitis B surface-small (HBs-S) antigen and hepatitis B core antigen (HBc) have been proposed as candidate therapeutic vaccines, but the elimination of existing HBV infection remains a challenge. To enhance the efficacy of HBs and HBc vaccination, we investigated HBs-large (HBs-L) as an immunogen, and carboxyl vinyl polymer (CVP) as an excipient. HBs-S or HBs-L, in combination with HBc antigen, was administered subcutaneously (without CVP) or intranasally (with or without CVP) for the evaluation of immune response in the tree shrew, which is considered to be a suitable small animal model of HBV infection. Immunization with HBs-L antigen by either route induced a rapid IgG response. Intranasal immunization with HBs-S or HBs-L and HBc formulated with CVP strongly induced neutralizing antibody activity, IgA response, and HBc-specific expression of the interferon gamma-encoding gene. These data indicated the potential of HBs-L and HBc intranasal immunization with CVP, not only as a therapeutic vaccine, but also as a prophylactic vaccine candidate.

[A Case of Long-Term Survival of a Postoperative Recurrence of Duodenal Papilla Adenosquamous Carcinoma Associated with the Annular Pancreas].

A woman in her mid-50's presented to our hospital with jaundice, fatigue, and fever. Jaundice, elevated tumor markers, and lower bile duct stricture suggested malignancy, for which subtotal stomach-preserving pancreaticoduodenectomy was performed. The patient also had annular pancreas as the second part of the duodenum was surrounded by pancreatic parenchyma. The histopathological diagnosis was adenosquamous carcinoma of the duodenal papilla associated with annular pancreas. Adjuvant chemotherapy with TS-1 was administered for 1 year. Although para-aortic lymph node metastasis was detected radiographically 3 years 9 months after surgery, the recurrence remains under control and she is alive at 5 years 9 months after surgery due to multidisciplinary therapy.

[A Successful Case of Multimodal Therapy for Multiple Hypervascular Tumor of Unknown Primary Origin in the Liver].

We experienced a successful case in which the patient was treated with a combination of operation and transcatheter arterial chemoembolization for a hypervascular tumor of unknown primary origin in the liver; we report this case with a literature review. A 76-year-old man noticed an upper abdominal tumor and he visited our department for detailed examination. Enhanced CT scan showed multiple hypervascular tumors scattered across both lobes of the liver, and the pattern of contrast effect implied that the tumors were primary neuroendocrine tumors or endocrine metastases. Gastroscopy and colonoscopy showed no tumor in the intestine. TACE was performed mainly for the tumors in the right lobe. One month later, we performed left lateral segmentectomy and partial hepatectomy for segment 6. Histopathological findings showed that the tumors in the left lateral segment had neuroendocrine properties, and the tumor in segment 6 was necrosed. One year has passed since the surgery without recurrence of neuroendocrine tumors in the liver or other organs.

[A Case of Advanced Gastric Cancer with Right Gastroepiploic Vein Tumor Thrombus Treated by Preoperative S-1 plus CDDP That Resulted in Pathological Complete Response].

We report a case of advanced gastric cancer with right gastroepiploic vein tumor thrombus treated using preoperative S-1 plus cisplatin(CDDP)in which pathological complete response was achieved. A 78-year-old man was diagnosed with type 2 gastric cancer located at the greater curvature of the antrum, accompanied by right gastroepiploic vein tumor thrombus. Four courses of S-1 plus CDDP were administered as neoadjuvant chemotherapy. After 2 courses, computed tomography(CT) revealed the disappearance of the tumor in the right gastroepiploic vein thrombus. Distal gastrectomy with D2 lymphadenec- tomy was performed, and the diagnosis was pathological complete response(CR). Eight courses of S-1(100mg/day on days 1-28, followed by 2 weeks of rest)were administered as adjuvant chemotherapy. During the 1-year postoperative follow up, the patient showed no recurrence. An S-1 plus CDDP regimen can be a useful preoperative chemotherapy option for advanced gastric cancer with tumor vein thrombus.

[Intrahepatic Bile Duct Metastasis from Rectal Adenocarcinoma Eight Years after Curative Operation].

A 60-year-old male undergoing pelvic evisceration with D3 lymph node dissection for pR0 in 2006f or carcinoma of the rectum was diagnosed pathologically with moderately differentiated adenocarcinoma, fStage ⅡA(UICC pT3, pN0, M0). Follow-up CT revealed hypovascular liver tumors with intrahepatic bile duct dilation in the right lobe 8 years after the pelvic evisceration. We conducted right lobe hepatectomy. The resected specimens showed that the tumor extended predominantly along the right intrahepatic bile duct. Histological findings and an immunohistochemical examination of CK7, CK20, and CDX2 revealed metastasis of the rectum cancer. We finally diagnosed him with intrahepatic bile duct metastasis from rectal adenocarcinoma.

[A Case of Cecal Cancer with Simultaneous Liver Metastases Successfully Treated with Multidisciplinary Therapy Including Two-Stage Hepatectomy].

We report a case of colon cancer with simultaneous liver metastases successfully treated with multidisciplinary therapy including two-stage hepatectomy. This is a case of a 72-year-old woman diagnosed with type 1 cecal cancer with 2 simulta- neous liver metastases(S4/8: 98mm, S3: 63mm). At first, laparoscopic resection of the primary site was performed to prevent colon ileus. Liver metastases rapidly grew and became unresectable; fortunately, 11 courses of mFOLFOX6 and bevacizu- mab were effective and liver metastases shrunk(S4/8: 85mm, S3: 67mm). At last, two-stage hepatectomy was performed as conversion surgery, and the patient survived without recurrence for more than 2 years.

[Two Cases of Aneurysm Rupture in Advanced Colorectal Cancer Successfully Treated with Selective Trans-Catheter Arterial Embolization].

We report 2 successful cases treated with trans-catheter arterial embolization(TAE)for hemorrhagic shock due to pseudoaneurysm rupture at the primary tumor site in advanced colorectal cancer. Case 1: A 73-year-old female underwent Hartmann surgery at another hospital for rectal perforation and peritonitis. Six months later, she had a massive bloody stool and was transferred to our hospital. She was diagnosed with bleeding from advanced rectal cancer that remained following the first surgery. Contrast computed tomography(CT)showed that a branch of the superior rectal artery(SRA)developed an aneurysm at the primary tumor site, and TAE was performed to stop bleeding on hospital day 3. Case 2: A 79-year-old male with type 2 sigmoid colon cancer and simultaneous multiple liver metastases developed copious blood in the stool before primary tumor resection and was transferred to our hospital. Contrast CT showed that a branch of the SRA developed an aneurysm at the primary tumor site, and TAE was immediately performed to stop bleeding.

[A Case of Perforated Gastric MALT Lymphoma with Total Gastrectomy].

A 62-year-old man presented with complaints of acute abdominal pain. Abdominal enhanced CT showed free air in the abdominal cavity, a thickened stomach wall accompanied by deep ulcer, and enlarged lymph nodes around the stomach. Emergent surgery was conducted, and a perforation in the upper part of the stomach was found. Total gastrectomy was performed. On pathological examination, a mucosa-associated lymphoid tissue lymphoma was diagnosed. Histologic examination revealed the absence of Helicobacter pylori in the gastric mucosa. After the operation, adjuvant chemotherapy was not administered. However, he has been well for 11 months without any recurrence. Gastric MALT lymphoma with spontaneous perforation is considered extremely rare. A brief review of the literature is presented.

[A Case of Extrarenal Retroperitoneal Angiomyolipoma].

We report a rare case of a extrarenal retroperitoneal angiomyolipoma. A 64-year-old female visited our hospital because of an abdominal mass in her lower right abdomen. Enhanced CT scan revealed a 67×52mm tumor in lower right retroperitoneal space. MRI revealed adipose component in the tumor on T1 weighted images and T2 weighted images. Our preoperative diagnosis was retroperitoneal liposarcoma, and we performed retroperitoneal tumor resection. At operation the tumor was on the fossa iliaca, which was covered with capsule. It did not invade surrounding tissues. The resected tumor measured 90× 60×45 mm, and tiny hemorrhage was seen in the tumor. Histopathological findings showed that the tumor was composed of a mixture of adipose tissue, vessels, and smooth muscle. Immunohistochemistry demonstrated the tumor was stained with asmooth muscle actin(aSMA). We diagnosed the tumor as extrarenal retroperitoneal angiomyolipoma.

[A Case of Non-Functioning Pancreatic Neuroendocrine Tumor Accompanied by Stenosis of the Main Pancreatic Duct].

We experienced a rare case of pancreatic neuroendocrine tumor(pNET)in which stenosis of the main pancreatic duct occurred despite the tumor diameter being less than 1 cm; therefore, here, we report this case with some literature review.A 41-year-old woman showed dilation of the main pancreatic duct on an abdominal ultrasound as part of a health examination, and she visited our department for a more detailed examination.Enhanced CT scan showed a 7mm subtle enhancing mass in the head of the pancreas and the upstream main pancreatic duct was dilated up to 7 mm.ERCP and MRCP images also showed a dilated main pancreas duct.We suspected that her tumor was a pNET.We decided not to perform surgery immediately. After 6 months of follow-up, the tumor size increased to 10 mm; therefore, we performed subtotal stomach-preserving pancreaticoduodenectomy considering the possibility of pancreatic cancer.Histopathological findings showed that the pancreatic tumor had the property of a neuroendocrine tumor and the proliferated fibrous stroma around it caused the pancreas duct stenosis.