Beneficial Effects of Combined Use of Extracorporeal Membrane Oxygenation and Hypothermic Machine Perfusion in Porcine Donors after Cardiac Death for Liver Transplantation.

Grafts from donors after cardiac death (DCD) have greatly contributed to expanding the donor organ pool. This study aimed to determine the benefits of subnormothermic extracorporeal membrane oxygenation (ECMO) and hypothermic machine perfusion (HMP) in a porcine model of DCD liver. Female domestic crossbred Large Yorkshire and Landrace pigs weighing approximately 20 kg were used. The abdominal aorta and inferior vena cava were cannulated and connected to an ECMO circuit for in situ perfusion of the abdominal organs at 22 °C for 60 min, 45 min after cardiac death. The pigs were divided into the cold storage (CS) group (n = 3), where liver grafts were preserved at 4 °C, and the HMP group (n = 3), where liver grafts were preserved by HMP at 8-10 °C. After 4 h of preservation, liver function was evaluated using an isolated liver reperfusion model for 2 h. Although the difference was insignificant, the liver effluent enzyme levels in the HMP group were lower than those in the CS group. Furthermore, morphological findings showed fewer injured hepatocytes in the HMP group than in the CS group. The combined use of in situ subnormothermic ECMO and HMP was beneficial for the functional improvement of DCD liver grafts.

Combined Use of Subnormothermic Extracorporeal Support and Hypothermic Oxygenated Machine Perfusion for Liver Graft After Cardiac Death in Pigs.

BACKGROUND: The use of grafts from donors after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool. This study aims to determine the benefits of extracorporeal membrane oxygenation (ECMO) and hypothermic oxygenated machine perfusion (HOPE) in a large animal model of DCD liver. METHODS: After cardiac arrest, the abdominal aorta and the inferior vena cava were cannulated and connected to an ECMO circuit. Porcine livers were perfused in situ with ECMO at 22°C for 60 minutes after 45 minutes of cardiac death. Then, the livers were perfused for 4 hours by cold storage (CS) or HOPE. In group 1, non-in situ ECMO and grafts were preserved by HOPE. In group 2, in situ ECMO and grafts were preserved by HOPE. In group 3, in situ ECMO and grafts were preserved by CS. After preservation, all grafts were evaluated using an isolated reperfusion model (IRM) with autologous blood for 2 hours. RESULTS: During HOPE, aspartate aminotransferase (AST) levels and hepatic arterial pressure in group 2 tended to be lower than in group 1. Hematoxylin-eosin staining findings after HOPE showed more massive sinusoidal congestion and hepatocyte cytoplasmic vacuolization in group 1 than in group 2. The AST and LDH levels in group 2 at the start-up of IRM tended to be lower than in group 1. CONCLUSIONS: The combined use of in situ subnormothermic ECMO and HOPE is essential for the functional recovery of DCD liver grafts.

Applicability of the histidine-tryptophan-ketoglutarate solution as a machine perfusion solution for marginal liver grafts.

BACKGROUND AND AIM: There are very few reports comparing the use of the University of Wisconsin solution and histidine-tryptophan-ketoglutarate solution as machine perfusion solutions for marginal liver grafts. We aimed to clarify whether the use of the histidine-tryptophan-ketoglutarate solution in hypothermic machine perfusion improves the split-liver graft function in a large animal model. METHODS: Porcine split-liver grafts were created by 75% liver resection. Hypothermic machine perfusion experimental groups were divided as follows: Group 1, perfusate, University of Wisconsin gluconate solution (UW group; n = 5), and Group 2, perfusate, histidine-tryptophan-ketoglutarate solution (HTK group; n = 4). After 4 h of preservation, the liver function was evaluated using an isolated liver reperfusion model for 2 h. RESULTS: In the HTK group, the portal vein and hepatic artery resistance during hypothermic machine perfusion and the portal vein resistance during isolated liver reperfusion were lower than those in the UW group. In addition, the total Suzuki score for hepatic ischemia-reperfusion injury in the HTK group was significantly better than that in the UW group. The number of anti-ETS-related genes staining-positive sinusoid epithelial cell nuclei in the HTK group was higher than that in the UW group (not significant). CONCLUSIONS: The histidine-tryptophan-ketoglutarate solution can be perfused with lower vascular resistance than the University of Wisconsin solution, reducing shear stress and preventing sinusoid epithelial cell injury in marginal grafts used as split-liver grafts.

The experience of 100 cases of 8K/two-dimensional laparoscopic colorectal surgery - The evaluation of 8K/two-dimensional laparoscopy and how to use it.

Background: The resolution of 8K ultra-high-definition imaging technology (7680 × 4320 pixels) is 16-fold higher than the current high-definition technology (1920 × 1080 pixels). 8K/two-dimensional (2D) laparoscopy was clinically available in 2014, but few reports concerning its application have been published. The aim of this study was to evaluate the appropriate methods of usage and problems learned from clinical use of 8K/2D laparoscopy. Subjects and Methods: The patients were 100 colorectal surgery patients who underwent 8K/2D laparoscopy at Asahikawa Medical University Hospital between November 2018 and March 2021. We evaluated the effectiveness, operating conditions, methods and issues of 8K/2D laparoscopy. Results: The median age was 68.5 years. The primary disease was malignancy of the left side of the colon and rectum in 92 patients. The right-sided colectomy was performed in five cases, total proctocolectomy of ulcerative colitis was performed in 3 cases. The proper application of 8K/2D laparoscopy can be achieved by adhering to certain tips, such as darkening the operation room and keeping an appropriate distance from the monitor. Regarding intraoperative complications caused by the 8K/2D laparoscope, skin burns due to heat from the tip of the laparoscope were observed in one patient. There were no cases of complications due to the 8K/2D laparoscopy. Conclusion: 8K/2D laparoscopy can be used safely in colorectal surgery. There are still some tips for proper use, such as keeping an appropriate distance to the monitor and darkening the room. However, 8K/2D laparoscopy can provide delicate images and can be used without any operational problems.

A case of percutaneous transhepatic stomal varices embolization and partial splenic artery embolization for rectal cancer after CAPOX/BEV chemotherapy: the summary of the stomal varices related to oxaliplatin administration.

Capecitabine and oxaliplatin (CAPOX) plus bevacizumab (BEV) therapy (CAPOX/BEV) is a standard treatment recommended as the first-line treatment for colorectal cancer recurrence. Recently, sinusoidal obstruction syndrome (SOS) and resulting portal hypertension have been reported as important side effects of oxaliplatin. We herein report a rectal cancer patient who underwent percutaneous transhepatic stoma variceal embolization (PTO) and partial splenic artery embolization (PSE) for stomal variceal bleeding and splenomegaly due to portal hypertension caused by SOS after CAPOX therapy. A 43-year-old man who underwent robot-assisted laparoscopic abdominoperineal resection for advanced lower rectal cancer was started on CAPOX/BEV therapy for early recurrence 1 month after surgery. In the sixth course, splenomegaly rapidly worsened, stomal varices appeared, and the stoma began bleeding. At 5 months after the appearance of stomal varices, the splenomegaly worsened, the frequency of stomal bleeding increased, and PTO was performed. Five months later, PSE was performed for splenomegaly and thrombocytopenia. At 5 months since the PSE, the stoma bleeding has not recurred, and the thrombocytopenia has been corrected. The patient has been able to continue chemotherapy. We suggest that staged treatment by PTO and PSE be considered an important treatment option for stomal varices and splenomegaly associated with SOS.

The evaluation of the correlation between origami crane training and Fundamentals of Laparoscopic Surgery (FLS).

Objective: How does making origami cranes under a dry box affect Fundamentals of Laparoscopic Surgery (FLS) scores in medical students? Design: Four medical students from Asahikawa Medical University (tertiary hospital) participated. They made origami cranes under a dry box (origami crane training) five days per week for four weeks. The time required to make each origami crane (origami crane time) and degree of completion were evaluated. FLS scores were measured before training and on days 5, 10, 15, and 20. We examined the relationship between "origami crane training" and FLS scores. Results: At the beginning of the experiment, none of the participants could complete the origami crane, but they were able to complete it in 31 ± 7 min on day 20. The Total FLS score was 164 ± 48 before the start of training, and 1107 ± 112 on day 20. The average scores of the students closely approached the Proficiency Level for the FLS tasks of peg transfer, loop ligation and extracorporeal ligation (103→228, 61→137, 0→259). The change over time in the average of the increase in Total FLS Score (difference from the first time and each week's score) improved significantly in four weeks (P < 0.01). Conclusions: Origami crane training improved the medical students' FLS scores. We thought that origami crane training mainly enhanced hand-eye coordination and bi-hand coordination.

A stealth antigen SPESP1, which is epigenetically silenced in tumors, is a suitable target for cancer immunotherapy.

Recent studies have revealed that tumor cells decrease their immunogenicity by epigenetically repressing the expression of highly immunogenic antigens to survive in immunocompetent hosts. We hypothesized that these epigenetically hidden "stealth" antigens should be favorable targets for cancer immunotherapy due to their high immunogenicity. To identify these stealth antigens, we treated human lung cell line A549 with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5Aza) and its prodrug guadecitabine for 3 d in vitro and screened it using cDNA microarray analysis. We found that the gene encoding sperm equatorial segment protein 1 (SPESP1) was re-expressed in cell lines including solid tumors and leukemias treated with 5Aza, although SPESP1 was not detected in untreated tumor cell lines. Using normal human tissue cDNA panels, we demonstrated that SPESP1 was not detected in normal human tissue except for testis and placenta. Moreover, we found using immunohistochemistry SPESP1 re-expression in xenografts in BALB/c-nu/nu mice that received 5Aza treatment. To assess the antigenicity of SPESP1, we stimulated human CD4+ T-cells with a SPESP1-derived peptide designed using a computer algorithm. After repetitive stimulation, SPESP1-specific helper T-cells were obtained; these cells produced interferon-γ against HLA-matched tumor cell lines treated with 5Aza. We also detected SPESP1 expression in freshly collected tumor cells derived from patients with acute myeloid leukemia or lung cancer. In conclusion, SPESP1 can be classified as a stealth antigen, a molecule encoded by a gene that is epigenetically silenced in tumor cells but serves as a highly immunogenic antigen suitable for cancer immunotherapy.

A critical role of STING-triggered tumor-migrating neutrophils for anti-tumor effect of intratumoral cGAMP treatment.

Stimulator of interferon genes (STING) contributes to anti-tumor immunity by activating antigen-presenting cells and inducing mobilization of tumor-specific T cells. A role for tumor-migrating neutrophils in the anti-tumor effect of STING-activating therapy has not been defined. We used mouse tumor transplantation models for assessing neutrophil migration into the tumor triggered by intratumoral treatment with STING agonist, 2'3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Intratumoral STING activation with cGAMP enhanced neutrophil migration into the tumor in an NF-κB/CXCL1/2-dependent manner. Blocking the neutrophil migration by anti-CXCR2 monoclonal antibody impaired T cell activation in tumor-draining lymph nodes (dLNs) and efficacy of intratumoral cGAMP treatment. Moreover, the intratumoral cGAMP treatment did not show any anti-tumor effect in type I interferon (IFN) signal-impaired mice in spite of enhanced neutrophil accumulation in the tumor. These results suggest that both neutrophil migration and type I interferon (IFN) induction by intratumoral cGAMP treatment were critical for T-cell activation of dLNs and the anti-tumor effect. In addition, we also performed in vitro analysis showing enhanced cytotoxicity of neutrophils by IFN-ß1. Extrinsic STING activation triggers anti-tumor immune responses by recruiting and activating neutrophils in the tumor via two signaling pathways, CXCL1/2 and type I IFNs.

Phosphorylated vimentin as an immunotherapeutic target against metastatic colorectal cancer.

Colorectal cancer (CRC) patients with metastatic lesions have low 5-year survival rates. During metastasis, cancer cells often obtain unique characteristics such as epithelial-mesenchymal transition (EMT). Vimentin a biomarker contributes to EMT by changing cell shape and motility. Since abnormal phosphorylation is a hallmark of malignancy, targeting phosphorylated vimentin is a feasible approach for the treatment of metastatic tumors while sparing non-tumor cells. Recent evidence has revealed that both CD8 cytotoxic T lymphocytes (CTLs) and also CD4 helper T lymphocytes (HTLs) can distinguish post-translationally modified antigens from normal antigens. Here, we showed that the expression of phosphorylated vimentin was upregulated in metastatic sites of CRC. We also showed that a chemotherapeutic reagent augmented the expression of phosphorylated vimentin. The novel phosphorylated helper peptide epitopes from vimentin could elicit a sufficient T cell response. Notably, precursor lymphocytes that specifically reacted to these phosphorylated vimentin-derived peptides were detected in CRC patients. These results suggest that immunotherapy targeting phosphorylated vimentin could be promising for metastatic CRC patients.

Expression of placenta-specific 1 and its potential for eliciting anti-tumor helper T-cell responses in head and neck squamous cell carcinoma.

Placenta-specific 1 (PLAC1) is expressed primarily in placental trophoblasts but not in normal tissues and is a targetable candidate for cancer immunotherapy because it is a cancer testis antigen known to be up-regulated in various tumors. Although peptide epitopes capable of stimulating CD8 T cells have been previously described, there have been no reports of PLAC1 CD4 helper T lymphocyte (HTL) epitopes and the expression of this antigen in head and neck squamous cell carcinoma (HNSCC). Here, we show that PLAC1 is highly expressed in 74.5% of oropharyngeal and 51.9% of oral cavity tumors from HNSCC patients and in several HNSCC established cell lines. We also identified an HTL peptide epitope (PLAC131-50) capable of eliciting effective antigen-specific and tumor-reactive T cell responses. Notably, this peptide behaves as a promiscuous epitope capable of stimulating T cells in the context of more than one human leukocyte antigen (HLA)-DR allele and induces PLAC1-specific CD4 T cells that kill PLAC1-positive HNSCC cell lines in an HLA-DR-restricted manner. Furthermore, T-cells reactive to PLAC131-50 peptide were detected in the peripheral blood of HNSCC patients. These findings suggest that PLAC1 represents a potential target antigen for HTL based immunotherapy in HNSCC.

Intratumoral STING activations overcome negative impact of cisplatin on antitumor immunity by inflaming tumor microenvironment in squamous cell carcinoma.

Although cisplatin (CDDP) has been used as a major chemotherapeutic drug for head and neck squamous cell carcinoma (HNSCC), its impact on T-cell functions is controversial. Therefore, we investigated the immunologic effects of CDDP and antitumor effects by combination therapy of CDDP with a ligand for stimulator of interferon genes, cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Direct impacts of CDDP on T-cell functions were addressed by comparing T-cell functions between human subjects treated and untreated with CDDP. The immune responses and the efficacy of combination therapy using CDDP and cGAMP were assessed using BALB/c mice inoculated with mouse squamous cell carcinoma (SCC) cell lines. CDDP inhibited T-cell proliferation in a dose-dependent manner. T-cell functions of CDDP-treated HNSCC patients were comparable to those of healthy donors and CDDP-untreated HNSCC patients. In the mice bearing SCC cell lines, combination therapy using CDDP and cGAMP enhanced the gene expressions of CXCL9 and CXCL10 in the tumor tissues and inhibited tumor growth. The antitumor effect was cancelled by anti-CXCR3 monoclonal antibody. These findings suggest that the combination therapy using CDDP and an immunomodulating drug like cGAMP would be a rational cancer immunotherapy for patients with HNSCC.

PD-L1-specific helper T-cells exhibit effective antitumor responses: new strategy of cancer immunotherapy targeting PD-L1 in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) originates from squamous epithelium of the upper aerodigestive tract and is the most common malignancy in the head and neck region. Among HNSCCs, oropharynx squamous cell carcinoma (OSCC) has a unique profile and is associated with human papillomavirus infection. Recently, anti-programmed cell death-1 monoclonal antibody has yielded good clinical responses in recurrent and/or metastatic HNSCC patients. Therefore, programmed death-ligand 1 (PD-L1) may be a favorable target molecule for cancer immunotherapy. Although PD-L1-expressing malignant cells could be targeted by PD-L1-specific CD8+ cytotoxic T lymphocytes, it remains unclear whether CD4+ helper T lymphocytes (HTLs) recognize and kill tumor cells in a PD-L1-specific manner. METHODS: The expression levels of PD-L1 and HLA-DR were evaluated using immunohistochemical analyses. MHC class II-binding peptides for PD-L1 were designed based on computer algorithm analyses and added into in vitro culture of HTLs with antigen-presenting cells to evaluate their stimulatory activity. RESULTS: We found that seven of 24 cases of OSCC showed positive for both PD-L1 and HLA-DR and that PD-L1241-265 peptide efficiently activates HTLs, which showed not only cytokine production but also cytotoxicity against tumor cells in a PD-L1-dependent manner. Also, an adoptive transfer of the PD-L1-specific HTLs significantly inhibited growth of PD-L1-expressing human tumor cell lines in an immunodeficient mouse model. Importantly, T cell responses specific for the PD-L1241-265 peptide were detected in the HNSCC patients. CONCLUSIONS: The cancer immunotherapy targeting PD-L1 as a helper T-cell antigen would be a rational strategy for HNSCC patients.

Targeting phosphorylated p53 to elicit tumor-reactive T helper responses against head and neck squamous cell carcinoma.

The human T cell receptor is capable of distinguishing between normal and post-translationally modified peptides. Because aberrant phosphorylation of cellular proteins is a hallmark of malignant transformation, the expression of the phosphorylated epitope could be an ideal antigen to combat cancer without damaging normal tissues. p53 activates transcription factors to suppress tumors by upregulating growth arrest and apoptosis-related genes. In response to DNA damage, p53 is phosphorylated at multiple sites including Ser33 and Ser37. Here, we identified phosphorylated peptide epitopes from p53 that could elicit effective T helper responses. These epitope peptides, p5322-41/Phospho-S33 and p5322-41/Phospho-S37, induced T helper responses against tumor cells expressing the phosphorylated p53 protein. Moreover, chemotherapeutic agents augmented the responses of such CD4 T cells via upregulation of phosphorylated p53. The upregulation of phosphorylated p53 expression by chemotherapy was confirmed in in vitro and xenograft models. We evaluated phosphorylated p53 expression in the clinical samples of oropharyngeal squamous cell carcinoma and revealed that 13/24 cases (54%) were positive for phosphorylated p53. Importantly, the lymphocytes specific for the phosphorylated p53 peptide epitopes were observed in the head and neck squamous cell cancer (HNSCC) patients. These results reveal that a combination of phosphorylated p53 peptides and chemotherapy could be a novel immunologic approach to treat HNSCC patients.

[Small renal cell carcinoma presented with pleural metastasis and multiple bone metastasis ; a case report].

The patient is a 69-year-old male. His chief complaint was chest pain. Because imaging studies suggested pleural mesothelioma associated with multiple bone metastases, right pleural tumor resection was performed. Pathological diagnosis was metastatic pleural tumor, and renal cell carcinoma (RCC) was suspected as its origin. Dynamic computed tomography showed a small tumor in the right kidney. The tumor was 15 mm in diameter and consistent with RCC. Laparoscopic radical nephrectomy was performed for the right kidney. Pathological diagnosis was RCC, clear cell carcinoma with sarcomatoid component, T1aN0M1, stage IV. Sorafenib therapy was started 46 days after the operation as a systemic therapy, and stable disease has been maintained. Generally, small RCC is assumed to have a good prognosis. However, a small percentage of patients with small RCC have distant metastasis at the time of diagnosis, and the prognosis is reported to be poor. We report this case, and a review of the literature.