Changes in salivary microbiota due to gastric cancer resection and its relation to gastric fluid microbiota.

Gastric cancer is one of the leading causes of death worldwide, and resections are performed to cure the disease. We have previously reported the changes in the gastric microbiota after gastric cancer resection, which may be associated with the oral microbiota; however, the changes in the oral microbiota remain uncharacterized. This study aimed to characterize the changes in the salivary microbiota caused by gastric cancer resection and to evaluate their association with the gastric fluid microbiota. Saliva and gastric fluid samples were collected from 63 patients who underwent gastrectomy before and after surgery, and a 16S rRNA metagenomic analysis was performed to compare the microbiota composition. The number of bacterial species in the salivary microbiota decreased, and the bacterial composition changed after the resection of gastric cancer. In addition, we identified several bacterial genera that varied significantly in the salivary microbiota, some of which also showed similar changes in the gastric fluid microbiota. These findings indicate that changes in the gastric environment affect the oral microbiota, emphasizing the close association between the oral and gastric fluid microbiota. Our study signifies the importance of focusing on the oral microbiota in the perioperative period of gastrectomy in patients with gastric cancer.

Flap Viability Evaluation Using a Tissue Oximetry Camera as an Alternative to Indocyanine Green Fluorescence Imaging.

Indocyanine green (ICG) fluorescence imaging is useful for assessing flap viability; however, it is associated with a risk of anaphylactic shock, even in patients with no history of drug allergies. SnapshotNIR is a noncontact, camera-type handheld tissue oximeter that can measure the tissue oxygen saturation of the body surface. The device emits red and near infrared light wavelengths and then optimizes the measurement of the differential reflectance from oxygenated and deoxygenated hemoglobin, and StO2 is calculated. A 20 × 15 cm surgical field can be evaluated in less than 3 seconds by holding the camera at a distance of 30 cm. We applied this device at zone II in a deep inferior epigastric perforator (DIEP) flap, and compared the findings with the border of flap perfusion detected by ICG imaging. Left breast reconstruction using a free DIEP flap was performed for a 60-year-old woman. The DIEP flap was vascularized by a perforator vessel coursing to the right abdominis muscle. First, Diagnogreen (5 mg; Daiichi Sankyo Co., Tokyo, Japan) was intravenously injected, and the ICG fluorescence perfusion border detected by PDE-neo (Hamamatsu Photonics, Hamamatsu City, Shizuoka, Japan) was determined. The ICG border was defined by two reconstructive surgeons after fluorescence had spread out for 2 minutes. Next, zones Ⅱ and Ⅳ of the DIEP flap, contralateral to the perforator, were evaluated using photographs obtained by SnapshotNIR. There were significant StO2 value differences between the ICG-negative area and ICG-positive area. This device can be widely applied in the noninvasive evaluation of flap viability.

Long term observation of de novo adipogenesis using novel bioabsorbable implants with larger size in a porcine model.

Introduction: The regeneration of adipose tissue in patients after breast cancer surgery would be desirable without the use of growth factors or cells to avoid potential recurrence and metastasis. We reported that prolate spheroidal-shaped poly-L-lactic acid (PLLA) mesh implants of approximately 18-mm polar diameter and 7.5-mm greatest equatorial diameter containing collagen sponge (CS) would be replaced by regenerated adipose tissue after implantation, thereby suggesting an innovative method for breast reconstruction. Our study aimed to evaluate the adipose tissue regeneration ability of implant aggregates in a porcine model. Methods: We prepared implant aggregates consisting of thirty PLLA mesh implants containing CS packed in a woven poly (glycolic acid) bag. The implant aggregates were inserted under the mammary glands in the porcine abdomen for a year. Single and double groups were classified by inserting either one or two implant aggregates on each side of the abdomen, respectively. Results: In both groups, the volume of the implant aggregates decreased over time, and the formation of adipose tissue peaked between 6 and 9 months. Histologically, the formation of adipose tissue was confirmed in the area that was in contact with native adipose tissue. Conclusions: Our implant aggregates could induce the autologous adipose tissue after long term implantation in vivo, without the use of any growth factor or cell treatment, presenting a potential novel method of breast reconstruction.

Real-world safety profile of eculizumab in patients with paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, or generalized myasthenia gravis: an integrated analysis of post-marketing surveillance in Japan.

Eculizumab is a C5 inhibitor approved for the treatment of paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and anti-acetylcholine receptor antibody-positive generalized myasthenia gravis (AChR + gMG) in Japan. We report integrated safety data from post-marketing surveillance in these three indications, focusing on commonly occurring adverse events (AEs) and infection-related AEs. Of 1219 patients registered, 1055 (PNH: 780; aHUS: 192; AChR + gMG: 83) had available safety data. Total eculizumab exposure was 3977.361 patient-years. AEs were reported in 74.03% of patients. AEs with an incidence of  ≥ 1.0 per 100 patient-years included hemolysis, headache, nasopharyngitis, renal impairment, anemia, pneumonia, upper respiratory tract inflammation, influenza, condition aggravated, and infection. The incidence of infection-related AEs was 21.30 per 100 patient-years, the most frequent types (≥ 1.0 per 100 patient-years) being nasopharyngitis, pneumonia, influenza, and infection. Meningococcal infections were reported in four patients (0.10 per 100 patient-years). Two patients died from meningococcal sepsis, with a mortality rate of 0.05 per 100 patient-years. This is the largest safety dataset on eculizumab in Japan derived from more than 10 years of clinical experience. No new safety signals were observed and the safety profile of eculizumab was consistent with that in previous clinical trials and international real-world safety analyses.

A high-hydrostatic pressure device for nevus tissue inactivation and dermal regeneration for reconstructing skin defects after giant congenital melanocytic nevus excision: a clinical trial.

Background: A novel treatment has been developed to reconstruct large skin defects caused by the excision of giant congenital melanocytic nevi. It involves the reimplantation of high-hydrostatic pressurized nevus tissue as a cell-inactivated autologous scaffold for dermal regeneration, followed by the implantation of cultured epithelial autografts on the regenerated dermis. Because this treatment has shown promise in a first-in-human clinical trial which used a prototype pressure machine, a novel pressure device was specifically designed for clinical use. Methods: In a prospective investigator-initiated clinical trial involving three patients, we evaluated the safety and efficacy of the skin regeneration treatment using a pressure device. All three patients underwent surgical excision of the nevus tissue, primary reimplantation of the inactivated nevus tissue, and secondary implantation of cultured epithelial autografts. Results: Engraftment of inactivated nevus tissue and cultured epithelial autografts was successful in all three cases, with over 90% epithelialization at 8 weeks post-surgery. No serious adverse events or device malfunction were observed during the trial. Conclusion: The novel pressure device safely and effectively enabled dermal regeneration using the nevus tissue as an autologous scaffold. This innovative approach offers several advantages, including reduced invasiveness due to minimal sacrifice of normal skin for skin grafting and high curative potential resulting from full-thickness removal of the nevus tissue.

Kaposi's Sarcoma-Associated Herpesvirus ORF67.5 Functions as a Component of the Terminase Complex.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a double-stranded DNA (dsDNA) gammaherpesvirus with a poorly characterized lytic replication cycle. However, the lytic replication cycle of the alpha- and betaherpesviruses are well characterized. During lytic infection of alpha- and betaherpesviruses, the viral genome is replicated as a precursor form, which contains tandem genomes linked via terminal repeats (TRs). One genomic unit of the precursor form is packaged into a capsid and is cleaved at the TR by the terminase complex. While the alpha- and betaherpesvirus terminases are well characterized, the KSHV terminase remains poorly understood. KSHV open reading frame 7 (ORF7), ORF29, and ORF67.5 are presumed to be components of the terminase complex based on their homology to other terminase proteins. We previously reported that ORF7-deficient KSHV formed numerous immature soccer ball-like capsids and failed to cleave the TRs. ORF7 interacted with ORF29 and ORF67.5; however, ORF29 and ORF67.5 did not interact with each other. While these results suggested that ORF7 is important for KSHV terminase function and capsid formation, the function of ORF67.5 was completely unknown. Therefore, to analyze the function of ORF67.5, we constructed ORF67.5-deficient BAC16. ORF67.5-deficient KSHV failed to produce infectious virus and cleave the TRs, and numerous soccer ball-like capsids were observed in ORF67.5-deficient KSHV-harboring cells. Furthermore, ORF67.5 promoted the interaction between ORF7 and ORF29, and ORF29 increased the interaction between ORF67.5 and ORF7. Thus, our data indicated that ORF67.5 functions as a component of the KSHV terminase complex by contributing to TR cleavage, terminase complex formation, capsid formation, and virus production. IMPORTANCE Although the formation and function of the alpha- and betaherpesvirus terminase complexes are well understood, the Kaposi's sarcoma-associated herpesvirus (KSHV) terminase complex is still largely uncharacterized. This complex presumably contains KSHV open reading frame 7 (ORF7), ORF29, and ORF67.5. We were the first to report the presence of soccer ball-like capsids in ORF7-deficient KSHV-harboring lytic-induced cells. Here, we demonstrated that ORF67.5-deficient KSHV also formed soccer ball-like capsids in lytic-induced cells. Moreover, ORF67.5 was required for terminal repeat (TR) cleavage, infectious virus production, and enhancement of the interaction between ORF7 and ORF29. ORF67.5 has several highly conserved regions among its human herpesviral homologs. These regions were necessary for virus production and for the interaction of ORF67.5 with ORF7, which was supported by the artificial intelligence (AI)-predicted structure model. Importantly, our results provide the first evidence showing that ORF67.5 is essential for terminase complex formation and TR cleavage.

Delaying the third dose of Japanese aluminum-free hepatitis A vaccine Aimmugen elicits effective immune responses against hepatitis A in adults.

Inactivated aluminum-adsorbed hepatitis A vaccines such as Havrix, Vaqta, and Avaxim are commonly used worldwide. These vaccines are typically administered in a two-dose series (at 0 and 6-12 months). However, a lyophilized inactivated aluminum-free hepatitis A vaccine, Aimmugen, which is approved in Japan, is typically administered in a three-dose series (at 0, 2-4, and 24 weeks). Hence, individuals visiting endemic hepatitis A areas receive the primary two doses of Aimmugen before traveling and the third booster dose much later. It is currently uncertain whether boosting with a delayed third dose of Aimmugen is effective, or whether a new vaccination schedule should instead be initiated. Therefore, we investigated the anti-hepatitis-A viral immune response of adult travelers who received the third dose of Aimmugen more than 24 weeks after the first dose. Participants were vaccinated with the third dose of Aimmugen more than 2 years after the first two doses. Antibody titers were measured at Day 0 (prevaccination) and at 28-42 days after the third dose of Aimmugen. Twenty-nine adult participants were enrolled in the study (14 men and 15 women; mean age ± standard deviation age, 36.2 ± 8.1 years). The interval between the first two doses and the third dose was 3-14 years. The seroprotection rate (i.e., the percentage of participants with anti-hepatitis A virus antibody titers ≥ 10 mIU/mL) was 96.6 % (28/29) at Day 0 and increased to 100 % (29/29) at Days 28-42. Geometric mean concentration increased from 105 to 4,013 mIU/mL. We demonstrated that delaying the third dose of Aimmugen still elicited effective immune responses after priming with two doses of the vaccine. Trial registration: UMIN Clinical Trials Registry (UMIN-CTR): MIN000013624. Registered 03 April 2014. https://center6.umin.ac.jp/cgi-bin/ctr/ctr_view_reg.cgi?recptno=R000015906.

Carbon-ion radiotherapy for inoperable upper tract ureteral cancer.

AIM: This study aimed to report initial results of hypofractionated carbon-ion radiotherapy (C-ion RT) for inoperable upper tract ureteral cancer. METHODS: Retrospective chart review was performed for five consecutive patients with medically inoperable ureter cancer that was treated with radical C-ion RT between December 2013 and December 2014. The median age of the patients was 80 years (range, 68-84 years). The reasons for inoperability were advanced age, post-contralateral nephrectomy, alcoholic cirrhosis, both advanced age and contralateral renal function degeneracy, and pneumonia. The median size of tumor was 2.8 cm (range, 2.2-4.0 cm). Diagnostic imaging did not identify lymph node metastases or distant metastases in any case. All patients underwent C-ion RT (52.8 Gy relative biological effectiveness; 12 fractions in 3 weeks). The clinical target volume encompassed the growth tumor volume with a 5-mm margin bilaterally; there was a 40-mm margin craniocaudally but the clinical target volume did not encompass the whole ureter. RESULTS: Within a median follow-up time of 32.9 months (range, 24-36 months), two patients died and three remained alive. Neither local recurrence nor regional lymph node metastases were observed. Secondary bladder tumor was observed in four patients, and one patient had a liver metastasis. Grade 1 hematuria was observed in two patients, and Grade 3 pyelonephritis was observed in one patient as acute toxicity. Ureteral obstruction was observed in two patients. CONCLUSION: C-ion RT might be a useful treatment option for inoperable ureter cancer.

CD8-positive Tumor-infiltrating Lymphocytes and Prognosis in Radiotherapy for Uterine Cervical Squamous Cell Carcinoma.

BACKGROUND/AIM: Prognostic factors, including CD8-positive tumor-infiltrating lymphocytes (CD8+TILs), in definitive radiotherapy (RT) for squamous cell carcinoma (SqCC) of the uterine cervix need to be studied. This study aimed to explore these factors in a retrospective cohort. PATIENTS AND METHODS: Patients with SqCC who underwent definitive RT comprising external beam RT and intracavitary brachytherapy at our facility between April 2006 and November 2013 were evaluated. CD8 immunohistochemistry was performed in pre-treatment biopsy samples to analyze the prognostic significance of CD8+TILs in the tumor nest. Positive staining was defined as at least one CD8+ lymphocyte infiltrating the tumor area in the specimen. RESULTS: In total, 150 consecutive patients were included. Among them, 66 (43.7%) patients had International Federation of Gynecology and Obstetrics (FIGO, 2008 edition) stage IIIA or higher progressive disease. The median follow-up period was 61 months. In the entire cohort, the 5-year cumulative rates of overall survival (OS), progression-free survival (PFS), and pelvic recurrence-free rate (PRFR) were 75.6%, 69.6%, and 84.8%, respectively. Of the 150 patients, 120 (80.0%) patients were CD8+TIL positive. The independent favorable prognostic factors were FIGO stage I or II disease, administration of concurrent chemotherapy, and CD8+TILs for OS (p=0.028, 0.005, and 0.038, respectively); FIGO stage I or II disease and CD8+TILs for PFS (p=0.015 and <0.001, respectively); and CD8+TILs for PRFR (p=0.017). CONCLUSION: The presence of CD8+TILs in the tumor nest may be a favorable prognostic factor of survival after definitive RT in patients with SqCC of the uterine cervix.

Development of a novel regenerative therapy for malignant bone tumors using an autograft containing tumor inactivated by high hydrostatic pressurization (HHP).

Surgical resection of malignant bone tumors leads to significant defects in the normal surrounding tissues that should be reconstructed to avoid amputation. Our research aimed to inactivate osteosarcoma (OS)-affected bone to obtain autologous bone grafts for bone defect reconstruction using a novel therapy called high hydrostatic pressurization (HHP) therapy. The key points are complete tumor death and preservation of the non-denatured native extracellular matrix (ECM) and bone tissue by HHP. Previously, we found that HHP at 200 MPa for 10 min can completely inactivate cells in normal skin and skin tumors, including malignant melanoma and squamous cell carcinoma while maintaining their original biochemical properties and biological components. Based on our previous research, this study used HHP at 200 MPa for 10 min to eradicate OS. We prepared an OS cell line (LM8), pressurized it at 200 MPa for 10 min, and confirmed its inactivation through morphological observation, WST-8 assay, and live/dead assay. We then injected OS cells with or without HHP into the bone marrow of the murine tibia, after which we implanted tumor tissues with or without HHP into the anterior surface of the tibia. After HHP, OS cells did not proliferate and were assessed using a live/dead assay. The pressurized cells and tumors did not grow after implantation. The pressurized bone was well prepared as tumor-free autologous bone tissues, resulting in the complete eradication of OS. This straightforward and short-pressing treatment was proven to process the tumor-affected bone to make a transplantable and tumor-free autologous bone substitute.

Characterization of Oral Microbiota Following Chemotherapy in Patients With Hematopoietic Malignancies.

Oral microbiota may be associated with serious local or systemic medical conditions resulting from chemotherapy. This study was conducted to evaluate the changes in the oral microbiota following the initiation of chemotherapy in patients with hematopoietic malignancies and to identify the characteristics of the oral microbiota associated with oral mucositis. Oral samples were collected from 57 patients with hematopoietic malignancies at 2 time points: before the start of chemotherapy and 8 to 20 days after the start of chemotherapy, when chemotherapy-induced oral mucositis often occurs, and 16S rRNA metagenomic analyses were performed. Comparative and linear discriminant analysis effect size (LEfSe) analyses were used to determine the characteristic bacterial groups before and after the initiation of chemotherapy and in those who developed oral mucositis. The alpha and beta diversities of oral microbiota before and after the initiation of chemotherapy differed significantly (operational taxonomic unit index, P < .001; Shannon's index, P < .001; unweighted UniFrac distances, P = .001; and weighted UniFrac distances, P = .001). The LEfSe analysis revealed a group of bacteria whose abundance differed significantly before and after the initiation of chemotherapy. In the group of patients who developed oral mucositis, a characteristic group of bacteria was identified before the start of chemotherapy. In conclusion, we characterized the oral microbiota associated with the initiation of chemotherapy in patients with hematopoietic malignancies. In addition, our findings suggest that oral microbiota composition before the start of chemotherapy may be associated with oral mucositis. The results of this study emphasize the importance of oral management focusing on the oral microbiota during chemotherapy in patients with hematologic malignancies.

Calreticulin Upregulation in Cervical Cancer Tissues From Patients After 10 Gy Radiation Therapy.

Purpose: Understanding the immune response during radiation therapy (RT) in a clinical setting is imperative for maximizing the efficacy of combined RT and immunotherapy. Calreticulin, a major damage-associated molecular pattern that is exposed on the cell surface after RT, is presumed to be associated with the tumor-specific immune response. Here, we examined changes in calreticulin expression in clinical specimens obtained before and during RT and analyzed its relationship with the density of CD8+ T cells in the same patient set. Methods and Materials: This retrospective analysis evaluated 67 patients with cervical squamous cell carcinoma who were treated with definitive RT. Tumor biopsy specimens were collected before RT and after 10 Gy irradiation. Calreticulin expression in tumor cells was evaluated via immunohistochemical staining. Subsequently, the patients were divided into 2 groups according to the level of calreticulin expression, and the clinical outcomes were compared. Finally, the correlation between calreticulin levels and density of stromal CD8+ T cells was evaluated. Results: The calreticulin expression significantly increased after 10 Gy (82% of patients showed an increase; P < .01). Patients with increased calreticulin levels tended to show better progression-free survival, but this was not statistically significant (P = .09). In patients with high expression of calreticulin, a positive trend was observed between calreticulin and CD8+ T cell density, but the association was not statistically significant (P = .06). Conclusions: Calreticulin expression increased after 10 Gy irradiation in tissue biopsies of patients with cervical cancer. Higher calreticulin expression levels are potentially associated with better progression-free survival and greater T cell positivity, but there was no statistically significant relationship between calreticulin upregulation and clinical outcomes or CD8+ T cell density. Further analysis will be required to clarify mechanisms underlying the immune response to RT and to optimize the RT and immunotherapy combination approach.

Establishment of a keratinocyte and fibroblast bank for clinical applications in Japan.

Regenerative medicine products using allogeneic cells, such as allogeneic cultured epidermis (allo-CE), have become a more critical therapeutic method for the treatment of burns. However, there are no clinically available allo-CE products in Japan. Therefore, establishing a quality-controlled cell bank is mandatory to create regenerative medical products using allogeneic cells. In this study, we selected ten patients from the Department of Plastic Surgery of Kyoto University Hospital to become cell donors. We performed medical interviews and blood sampling for the donor to ensure virus safety. We examined the tissues and isolated cells by performing a nucleic acid test (NAT). To establish a master cell bank, quality evaluation was performed according to the International Conference of Harmonization (ICH) Q5A. Serological tests of the blood samples from the ten donors showed that two of them were ineligible. The cells registered in the cell bank were found to be compatible after virus testing was performed, and a master cell bank was constructed. Hence, we established a keratinocyte and fibroblast bank of clinically usable human cultured cells in Japan for the first time.

Comparison of Wound Healing Effect of Skin Micrograft Impregnated into Two Kinds of Artificial Dermis in a Murine Wound Model.

A micrograft (MG) suspension produced by the Rigenera protocol has been used to stimulate tissue regeneration. Recently, a combination therapy of an artificial dermis and skin MG has been used to promote angiogenesis and granulation tissue formation in the artificial dermis. There are no reports comparing the differences in MG impregnation efficiency between different artificial dermis products. Therefore, we compared the impregnation of skin MG in Pelnac Gplus and Integra. Methods: Skin MG was prepared from the skin of C57BL/6JJcl mice using Rigeneracons and administered onto Pelnac Gplus and Integra sheets. The amount of MG suspension impregnated in Pelnac Gplus and Integra was evaluated. Pelnac Gplus and Integra sheets combined with MG were applied to murine defects, and wound area, neoepithelium length, granulation tissue formation, and newly formed capillaries were compared with the control groups on days 7 and 14. Results: The weight percentage of the MG absorbed by Pelnac Gplus and Integra was 88.8% ± 3.5% and 28.2% ± 7.0%, respectively (P < 0.05). In the in vivo experiment, the area of newly formed granulation tissue and both the number and area of newly formed capillaries in the PelnacG + MG group were significantly larger than those in the control group at 14 days after implantation (P < 0.05). Conclusions: Skin MG was successfully impregnated into Pelnac Gplus by simple administration but not into Integra. Administration of skin MG into the Pelnac Gplus promoted granulation formation and angiogenesis. Pelnac Gplus was more suitable than Integra in the combination therapy.

The Contribution of Kaposi's Sarcoma-Associated Herpesvirus ORF7 and Its Zinc-Finger Motif to Viral Genome Cleavage and Capsid Formation.

During Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection, lytic-related proteins are synthesized, viral genomes are replicated as a tandemly repeated form, and subsequently, capsids are assembled. The herpesvirus terminase complex is proposed to package an appropriate genome unit into an immature capsid, by cleavage of terminal repeats (TRs) flanking tandemly linked viral genomes. Although the mechanism of capsid formation in alpha- and betaherpesviruses are well-studied, in KSHV, it remains largely unknown. It has been proposed that KSHV ORF7 is a terminase subunit, and ORF7 harbors a zinc-finger motif, which is conserved among other herpesviral terminases. However, the biological significance of ORF7 is unknown. We previously reported that KSHV ORF17 is essential for the cleavage of inner scaffold proteins in capsid maturation, and ORF17 knockout (KO) induced capsid formation arrest between the procapsid and B-capsid stages. However, it remains unknown if ORF7-mediated viral DNA cleavage occurs before or after ORF17-mediated scaffold collapse. We analyzed the role of ORF7 during capsid formation using ORF7-KO-, ORF7&17-double-KO (DKO)-, and ORF7-zinc-finger motif mutant-KSHVs. We found that ORF7 acted after ORF17 in the capsid formation process, and ORF7-KO-KSHV produced incomplete capsids harboring nonspherical internal structures, which resembled soccer balls. This soccer ball-like capsid was formed after ORF17-mediated B-capsid formation. Moreover, ORF7-KO- and zinc-finger motif KO-KSHV failed to appropriately cleave the TR on replicated genome and had a defect in virion production. Interestingly, ORF17 function was also necessary for TR cleavage. Thus, our data revealed ORF7 contributes to terminase-mediated viral genome cleavage and capsid formation. IMPORTANCE In herpesviral capsid formation, the viral terminase complex cleaves the TR sites on newly synthesized tandemly repeating genomes and inserts an appropriate genomic unit into an immature capsid. Herpes simplex virus 1 (HSV-1) UL28 is a subunit of the terminase complex that cleaves the replicated viral genome. However, the physiological importance of the UL28 homolog, KSHV ORF7, remains poorly understood. Here, using several ORF7-deficient KSHVs, we found that ORF7 acted after ORF17-mediated scaffold collapse in the capsid maturation process. Moreover, ORF7 and its zinc-finger motif were essential for both cleavage of TR sites on the KSHV genome and virus production. ORF7-deficient KSHVs produced incomplete capsids that resembled a soccer ball. To our knowledge, this is the first report showing ORF7-KO-induced soccer ball-like capsids production and ORF7 function in the KSHV capsid assembly process. Our findings provide insights into the role of ORF7 in KSHV capsid formation.

Adjustable biodegradability of low-swelling hydrogels prepared from recombinant peptides based on human collagen type 1.

An ideal hydrogel for tissue engineering and regenerative therapy is cytocompatible, biocompatible, and has low-swelling characteristics. Recently, a novel low-swelling hydrogel with a homogenous structure was developed by crosslinking a recombinant peptide, modeled on human collagen type 1 (RCPhC1), with a four-arm polyethylene glycol (tetra-PEG). Here, we hypothesized that the biodegradability of the RCPhC1 hydrogel was adjustable by altering its initial polymer concentration. Three types of RCPhC1 hydrogels were prepared using the initial polymer at different concentrations, and their morphology, swelling ratio, collagenase degradability, cytocompatibility, biocompatibility, and biodegradability were compared. The results revealed a low swelling ratio. The higher the concentration of the initial polymer, the longer it took for it to be degraded by collagenase. The average cell viability ratio was over 92% when using the direct contact method, which suggests that the hydrogels have excellent cytocompatibility. No death, tumorigenesis, exposure of the implants, or skin necrosis associated with the subcutaneous implantation of the hydrogels was found in mice in vivo. Moreover, histological evaluation revealed the formation of a thin fibrous capsule, which suggests an acceptable biocompatibility. Furthermore, as hypothesized, it was confirmed that the biodegradability can be adjusted by changing the initial polymer concentration. Collectively, the ability to fine-tune the biodegradability of RCPhC1 hydrogels demonstrates their potential for use in various clinical applications.

Comparison of the Therapeutic Effects of [211At]NaAt and [131I]NaI in an NIS-Expressing Thyroid Cancer Mouse Model.

Astatine (211At) is an alpha-emitter with a better treatment efficacy against differentiated thyroid cancer compared with iodine (131I), a conventional beta-emitter. However, its therapeutic comparison has not been fully evaluated. In this study, we compared the therapeutic effect between [211At]NaAt and [131I]NaI. In vitro analysis of a double-stranded DNA break (DSB) and colony formation assay were performed using K1-NIS cells. The therapeutic effect was compared using K1-NIS xenograft mice administered with [211At]NaAt (0.4 MBq (n = 7), 0.8 MBq (n = 9), and 1.2 MBq (n = 4)), and [131I]NaI (1 MBq (n = 4), 3 MBq (n = 4), and 8 MBq (n = 4)). The [211At]NaAt induced higher numbers of DSBs and had a more reduced colony formation than [131I]NaI. In K1-NIS mice, dose-dependent therapeutic effects were observed in both [211At]NaAt and [131I]NaI. In [211At]NaAt, a stronger tumour-growth suppression was observed, while tumour regrowth was not observed until 18, 25, and 46 days after injection of 0.4, 0.8, and 1.2 MBq of [211At]NaAt, respectively. While in [131I]NaI, this was observed within 12 days after injection (1, 3, and 8 MBq). The superior therapeutic effect of [211At]NaAt suggests the promising clinical applicability of targeted alpha therapy using [211At]NaAt in patients with differentiated thyroid cancer refractory to standard [131I]NaI treatment.

Comparison of the gastric microbiome in Billroth I and Roux-en-Y reconstructions after distal gastrectomy.

The changes in gastric microbiota following reconstruction after gastrectomy have not been reported. This study aimed to compare the gastric microbiota following Billroth I and Roux-en-Y reconstructions after distal gastrectomy. We enrolled 71 gastrectomized patients with gastric cancer; 31 and 40 underwent Billroth I and Roux-en-Y reconstructions, respectively. During upper gastrointestinal endoscopy, gastric fluid was collected immediately before and 6 months after distal gastrectomy. Deoxyribonucleic acid isolated from each sample was evaluated using 16S ribosomal ribonucleic acid metagenomic analysis. Analysis revealed that the gastric microbiota's species richness (expressed as the alpha diversity) was significantly lower after than before distal gastrectomy (operational taxonomic units, p = 0.001; Shannon index, p = 0.03). The interindividual diversity (beta diversity) was significantly different before and after distal gastrectomy (unweighted UniFrac distances, p = 0.04; weighted UniFrac distances, p = 0.001; Bray-Curtis, p = 0.001). Alpha and beta diversity were not significantly different between Billroth I and Roux-en-Y reconstructions (observed operational taxonomic units, p = 0.58; Shannon index, p = 0.95; unweighted UniFrac distances, p = 0.65; weighted UniFrac distances, p = 0.67; Bray-Curtis, p = 0.63). Our study demonstrated significant differences in gastric microbiota diversity, composition, and community before and after distal gastrectomy but no difference between Billroth I and Roux-en-Y reconstruction after distal gastrectomy.

Phase I Study of Tremelimumab Monotherapy or in Combination With Durvalumab in Japanese Patients With Advanced Solid Tumors or Malignant Mesothelioma.

BACKGROUND: The primary objective of this phase I, open-label trial was to assess safety and tolerability of tremelimumab monotherapy and combination therapy with durvalumab in Japanese patients with advanced cancer. Tremelimumab is a fully human monoclonal antibody against CTLA-4 in clinical trials; durvalumab is a monoclonal antibody against PD-L1 for the treatment of bladder and lung cancer. METHODS: In part 1, tremelimumab 3 or 10 mg/kg was given every 4 weeks (Q4W) for 6 doses, and thereafter every 12 weeks until discontinuation (n = 8); subsequently tremelimumab 10 mg/kg Q4W for 6 doses/Q12W and thereafter until discontinuation was administered in 41 patients with malignant pleural or peritoneal mesothelioma (MPM). In part 2, tremelimumab 10 mg/kg (Q4W for 6 doses followed by Q12W for 3 doses) was given in combination with durvalumab 15 mg/kg (Q4W for 13 doses) in cohort 1 (n = 4). In cohort 2 (n = 6), tremelimumab 1 mg/kg (Q4W for 4 doses) was given in combination with durvalumab 20 mg/kg (Q4W for 4 doses followed by 10 mg/kg Q2W for 22 doses), while in cohort 3 (n = 6), fixed-dose tremelimumab 75 mg Q4W for 4 doses plus durvalumab 1500 mg Q4W for 13 doses was given. RESULTS: In part 1, no dose-limiting toxicities (DLTs) for tremelimumab 3 or 10 mg/kg (Q4W for 6 doses/Q12W thereafter until discontinuation) were observed. Six (75%) patients reported treatment-related adverse events (trAEs). In the MPM dose-expansion cohort, 38 (92.7%) patients reported trAEs. In part 2, one DLT (Grade 4 myasthenia gravis) was reported for tremelimumab 10 mg/kg (Q4W for 6 doses/Q12W for 3 doses) plus durvalumab 15 mg/kg (Q4W for 13 doses). One DLT (Grade 4 hyperglycemia) was reported for tremelimumab 75 mg (Q4W for 4 doses) plus durvalumab 1500 mg (Q4W for 13 doses). Fourteen (87.5%) patients reported trAEs. Tremelimumab demonstrated low immunogenicity; 1 (16.7%) patient developed antidrug antibodies. CONCLUSION: Tremelimumab 10 mg/kg (Q4W/Q12W), tremelimumab 1 mg/kg (Q4W) plus durvalumab 20 mg/kg (Q4W/10 mg/kg Q2W), and fixed-dose tremelimumab 75 mg (Q4W) plus durvalumab 1500 mg (Q4W) were safe and tolerable.ClinicalTrials.gov Identifier: NCT02141347 (https://clinicaltrials.gov/ct2/show/NCT02141347).

Double-layer omics analysis of castration- and X-ray-resistant prostate cancer cells.

Castration-resistant prostate cancer shows resistance to not only androgen deprivation therapy (ADT) but also X-ray therapy. On the other hand, carbon ion beams have a high biological effect and are used for various cancers showing resistance to X-ray therapy. The purposes of this study are to clarify the difference in the sensitivity of Castration-resistant prostate cancer to X-ray and carbon ion beams and to elucidate the mechanism. The androgen-insensitive prostate cancer cell line LNCaP-LA established by culturing the androgen-sensitive prostate cancer cell line LNCaP for 2 years in androgen-free medium was used for this study. First, colony formation assays were performed to investigate its sensitivity to X-ray and carbon ion beams. Next, DNA mutation analysis on 409 cancer-related genes and comprehensive transcriptome analysis (RNA-seq) were performed with a next-generation sequencer. Lethal dose 50 values of X-rays for LNCaP and LNCaP-LA were 1.4 Gy and 2.8 Gy, respectively (P < 0.01). The Lethal dose 50 values of carbon ion beams were 0.9 Gy and 0.7 Gy, respectively (P = 0.09). On DNA mutation analysis, AR mutation was observed specifically in LNCaP-LA. From RNA-seq, 181 genes were identified as differentially expressed genes (DEGs; FDR <0.10, P < 0.00076) between LNCaP and LNCaP-LA. Function analysis suggested that cell death was suppressed in LNCaP-LA, and pathway analysis suggested that the NRF2-pathway involved in intracellular oxidative stress prevention was activated in LNCaP-LA. LNCaP-LA showed X-ray resistance compared to LNCaP and sensitivity to carbon ion beams. The AR mutation and the NRF2-pathway were suggested as causes of resistance.