A Novel Three-Step Approach for Secure Splenectomy During Laparoscopic Total Gastrectomy for Gastric Cancer.

BACKGROUND: Splenectomy during total gastrectomy increases operative morbidity (Nakata et al. in Surgical endoscopy 7:1817-1822, 2015). Establishing a safe approach to laparoscopic splenectomy is one of the most urgent issues in the treatment of proximal advanced gastric cancer, which invades to the greater curvature (Kawamura et al. in Gastric Cancer 3:662-668, 2015). We developed a novel three-step procedure for splenectomy during laparoscopic total gastrectomy (LTG). METHODS: Splenectomy consisted of three steps. Step 1 (dorsal approach): The pancreatic tail and spleen were mobilized. This step delineates the dissection plane and the anatomy around the pancreatic tail. Step 2 (suprapancreatic approach): The suprapancreatic peritoneum was incised to fenestrate to the mobilized space. The no. 11d station was dissected. The inferior branch of the splenic artery was exposed. Step 3 (splenic hilum approach): The spleen was lifted up to straighten the splenic hilum. The aim was to prolong the splenic vasculature and enable the surgeon to transect splenic vasculatures easily despite their anatomical diversity. Division of the splenic branches promotes mobility of the pancreatic tail, enabling precise dissection and preservation of its blood supply. RESULTS: Of 45 patients with gastric cancer who underwent LTG, seven underwent concurrent splenectomy. In all cases, splenectomy was successfully accomplished. The median operation time, duration of splenectomy, blood loss, number of total retrieved lymph nodes, lymph node counts from stations 10 and 11d, and drain amylase levels on the third postoperative day were 382 min, 94 min, 30 ml, 51, 5, 5, and 158 IU/L, respectively. Postoperative morbidity more severe than Clavien-Dindo grade 2 occurred in one case, with no pancreas-related morbidity. No mortality or conversion occurred. CONCLUSIONS: This laparoscopic procedure allows adequate nodal dissection and safe splenectomy.

Laparoscopic transhiatal lymphadenectomy in the lower mediastinum for adenocarcinoma of the esophagogastric junction.

Laparoscopic transhiatal esophagogastrectomy is difficult because the lower mediastinum is so deeply located that the operative field is narrow and restricted by surrounding organs. Therefore, we performed lymphadenectomy with opening of the bilateral mediastinal pleura to maintain safety and obtain better exposure of lymph nodes and important organs. We will present our technique for laparoscopic lower mediastinal lymphadenectomy and reconstruction for cancer of the esophagogastric junction. Five abdominal ports were used. Retraction of the left lobe of the liver exposed the esophageal hiatus. A long, narrow gastric tube (3 cm wide) was formed, and regional abdominal lymph nodes (No. 1, 2, 3a, 7, 8a, 9, 19, and 20) were resected. The diaphragmatic hiatus was widely split and the opened bilateral mediastinal pleura enabled better exposure for lymph node dissection and reconstruction. The level where the inferior vena cava passed through the diaphragm into the chest was used as a landmark to identify supradiaphragmatic (No. 111) and lower thoracic paraesophageal nodes (No. 110), which were completely retrieved with this procedure. The posterior mediastinal nodes (No. 112pulR, 112pulL, and 112aoA) were also retrieved with bilateral opening of the mediastinal pleura and dissection of the inferior pulmonary ligaments. An esophagogastric tube anastomosis with pseudo-fornix was made with a no-knife linear stapler to prevent postoperative reflux esophagitis. This approach enabled safe and accurate laparoscopic lower mediastinal nodal dissection. With the advantage of a narrow gastric tube, the good working space made tension-free anastomosis possible.

Prospective registry for laparoscopic liver resection.

INTRODUCTION: Laparoscopic liver resection (LLR) has been widely performed throughout the world. Although prospective registry studies to clarify the safety of LLR have been feasible, no prior multicenter prospective study has addressed this issue. We have conducted a multicenter prospective cohort study to reveal the current status of LLR in Japan. METHODS: From April 2015 to March 2016, candidates for LLR were preoperatively enrolled at 12 institutions. The primary end-point was surgical safety, which was evaluated based on surgical factors and on short-term and midterm outcomes. RESULTS: A total of 102 patients were enrolled. Planned laparoscopic procedures included 96 pure laparoscopies, 1 hand-assisted laparoscopy, and 5 hybrid techniques. Non-anatomical partial resection or left lateral sectionectomy were performed in almost all cases. The median duration of surgery was 221 min. The median blood loss was 80.5 mL. Conversion was required for four patients (3.9%). The 90-day postoperative morbidities with grades more severe than II in the Clavien-Dindo classification were observed in six patients (5.9%). The median postoperative hospital stay was 9.5 days. No cases involved reoperation or mortality. CONCLUSION: Minor resection of LLR has been performed safely. To ensure the safe dissemination of LLR, including for major resection, a larger multicenter prospective study is required.

Asialo GM1 positive CD8+ T cells induce skin allograft rejection in the absence of the secondary lymphoid organs.

BACKGROUND: Secondary lymphoid organs are considered to be the only organs in which APCs and naïve T cells interact to initiate adaptive immune responses. Aly/aly mice are autosomal recessive mutants of C57BL/6 mice, and lack lymph nodes and Peyer's patches. In this study, we investigated immune responses to skin allografts in splenectomized aly/aly mice, which lack secondary lymphoid organs completely, and examined the effect of anti-asialo GM1 (AsGM1) antibodies on these responses. METHODS: Skin allografts were transplanted to 1) heterozygous aly/+ mice, which had normal secondary lymphoid organs, 2) splenectomized aly/+ mice, 3) aly/aly mice, and 4) splenectomized aly/aly mice, with and without anti-AsGM1 antibody treatment. Graft survival time and alloreactive antibody production were investigated. RESULTS: Heterozygous aly/+ mice and splenectomized aly/+ mice rejected skin allografts acutely. Aly/aly mice also rejected skin allografts, but at a later time than aly/+ mice. Sixty percent of splenectomized aly/aly mice rejected skin allografts within 120 days. Serial administration of anti-AsGM1 antibodies prevented skin allograft rejection in splenectomized aly/aly mice during the same 120-day period of observation. After ceasation of anti-AsGM1 antibody treatment, skin allografts were rejected; we observed a simultaneous increase in AsGM1 expression on CD8+ T cells. Alloreactive antibodies were detected in both splenectomized aly/aly mice that rejected skin allografts and in splenectomized aly/aly mice that accepted skin allografts after treatment with anti-AsGM1 antibodies. CONCLUSIONS: Cytotoxic and humoral immune responses to skin allografts could be initiated despite the absence of secondary lymphoid organs. AsGM1+ cells were important effector cells in secondary lymphoid organ-independent skin allograft rejection.

Absence of PERV infection in baboons after transgenic porcine liver perfusion.

BACKGROUND: Xenotransplantation offers great promise to supplement the shortage of human organs available for transplant, but cross-species infection is a substantial concern. Porcine endogenous retrovirus (PERV), in particular, is thought to pose a risk as a potential pathogen to humans. We evaluated whether PERV is capable of infecting nonhuman primates in vivo after extracorporeal porcine liver perfusion (ECLP). METHODS: Livers were harvested from six human decay-accelerating factor (h-DAF) transgenic piglets and perfused with fresh baboon blood via the portal vein and the hepatic artery. Six healthy baboons underwent direct cross-circulation with the ECLP for 13 to 24 h without immunosuppression. Peripheral blood and bone marrow of baboons were sampled periodically until the baboons were euthanized for the examination of various organ tissue samples. Genomic DNA was extracted from those samples and tested for PERV and pig-specific centromeric DNA sequences by quantitative PCR. Validation showed that the assay could detect one copy of PERV in a background of 150,000 baboon cells, and it was quantitative over a range from 10 to 10(6) copies of PERV. RESULTS: PERV sequences were detected in a high number (4.4 x 10(3)-1.6 x 10(4)/1 microg) in peripheral leukocyte DNA during the initial phases of ECLP, but they disappeared within 1 week. Bone marrow DNA contained PERV sequences longer than peripheral blood, but PERV signals became negative within 1 month. No PERV DNA relapse was seen over the course of this study. Pig-specific centromeric sequences were also detected in the same manner. At 6 months or 1 year after ECLP, no PERV or pig-specific centromeric sequences were detected in the genomic DNA obtained from the following organs: skin, lymph nodes, spleen, liver, pancreas, kidney, heart, and lung. CONCLUSIONS: ECLP did not result in PERV infection or pig-cell microchimerism in baboons.

Assessment of donor liver steatosis: pathologist or automated software?

Steatosis in donor liver biopsy specimens has been shown to correlate with graft dysfunction after orthotopic liver transplantation. This 2-part (laboratory pilot, clinical retrospective) study compared the traditional interpretation of steatosis by a pathologist with an automated measurement determined by an image analysis system. In our pilot study, Sprague-Dawley rats were studied prospectively by feeding them a choline-deficient diet for up to 7 days. In our clinical group, data from 49 consecutive recipients of cadaveric liver transplantation were reviewed retrospectively. In both studies, the percentages of microvesicular fat, macrovesicular fat, and total fat content within liver biopsy specimens were determined by an automated image analysis software program and a pathologist using the same set of slides. The association between fat content of the donor liver and patient survival and graft survival, along with levels of aspartate aminotransferase, alanine aminotransferase, prothrombin time, and total bilirubin after transplantation, were also examined in the clinical study. A direct correlation was observed between levels of macrovesicular fat determined by a pathologist and the automated software using livers from rats fed a choline-deficient diet and livers from deceased donors. A significant association was observed between macrovesicular fat content in the donor liver biopsy and graft survival by both techniques. We conclude that an image analysis system can be used to automate the determination of fat content in liver biopsy specimens, and that its findings correlate with both the visual interpretation by a pathologist and graft survival. Further study is needed to determine the role of an automated technique in the evaluation of donor livers for transplantation.

Membrane pore size impacts performance of a xenogeneic bioartificial liver.

BACKGROUND: We have developed a novel bioartificial liver (BAL) composed of porcine hepatocyte spheroids in a reservoir design. A semipermeable membrane is used to protect the spheroids from immune-mediated damage. This study was designed to assess the influence of membrane pore size on performance of the spheroid reservoir BAL. METHODS: Eight healthy dogs were studied during primary and secondary exposures to the spheroid reservoir BAL using membranes with small (10 nm) or large (200 nm) pores. BAL performance was assessed by multiple functional assays. Spheroids were examined microscopically before and after all BAL treatments. Titers of xenoreactive antibody were monitored until elective death of animals on day 42. RESULTS: Viability and functional performance of spheroids were significantly greater after all BAL treatments that used membranes with 10-nm versus 200-nm pores. Reduced performance in the 200 nm group was associated with 7.7-fold and 78.0-fold rise in xenoreactive antibody titers after first and second treatments, respectively. Dogs in the 10 nm group remained hemodynamically stable during all BAL treatments, whereas those in the 200 nm group experienced acute hypotension (P<0.001) during second BAL exposures. Microscopic examination of spheroids after BAL treatments indicated that deposition of canine proteins, including complement, was associated with reductions in both viability and functional performance of the BAL. CONCLUSIONS: The elicited immune response of healthy dogs to a xenogeneic BAL was blocked and BAL performance significantly improved by reducing the permeability of the BAL membrane.

Suppressed complement activation in human decay accelerating factor transgenic porcine liver cross-circulated with nonhuman primates.

BACKGROUND: We developed an extracorporeal liver perfusion (ECLP) system as a liver-assist device. In this study, we evaluated the safety of the ECLP using human decay accelerating factor (hDAF) transgenic porcine livers in healthy baboons. METHODS: Livers were isolated from five hDAF transgenic pigs and five nontransgenic pigs for the ECLP. Ten cross-circulations between the ECLP and healthy baboons were performed without immunosuppressive agents. Cross-circulation was discontinued in any of the following circumstances: elevated hepatic arterial (>200 mm Hg) or portal (>60 mm Hg) perfusion pressure, massive exudate from the graft liver, mild macroscopic hemolysis, thrombocytopenia, or 24-hr well-conditioned cross-circulation. RESULTS: The cross-circulations with nontransgenic porcine livers were discontinued at 4.4+/-1.2 hr (mean+/-standard deviation) because of high perfusion pressure (n=2) or hemolysis (n=3). Three cross-circulations with hDAF transgenic porcine livers were performed for 24 hr; the other two cross-circulations were discontinued at 13 and 17 hr because of massive exudate and thrombocytopenia, respectively. The duration was 20.4+/-5.1 hr. Deposition of membrane attack complex in the hDAF transgenic porcine liver was less than that in the nontransgenic liver, although immunoglobulin-M deposition was comparable. The porcine livers showed no apparent interlobular bleeding or lobular necrosis. All porcine livers maintained bile production during the cross-circulation. No baboons showed any serious complications after the cross-circulation. CONCLUSION: The hDAF transgenic porcine liver reduced complement activation in xenoperfusion with healthy nonhuman primate blood and led to extended duration of cross-circulation.

Apoptotic cell death and function of cryopreserved porcine hepatocytes in a bioartificial liver.

We have previously shown that cryopreservation leads to increased apoptotic death of porcine hepatocytes intended for use in a bioartificial liver (BAL). This study was designed to determine if a broad-spectrum caspase inhibitor, IDN-1965, reduced apoptosis and increased function of cryopreserved porcine hepatocytes in static culture or in a BAL. Porcine hepatocytes were studied immediately after isolation and after 2 weeks of cryopreservation in liquid nitrogen using medium supplemented with 25 micromol/L IDN-1965 or vehicle. Both apoptotic and necrotic cells were observed in cultures of fresh and cryopreserved hepatocytes, but the percentage of apoptotic cells increased after cryopreservation. Cryopreservation in IDN-1965 improved hepatocyte viability and reduced apoptotic cell death determined by TUNEL assay. Cryopreservation of hepatocytes in IDN-1965 was also associated with reduced caspase 3-like activity, decreased release of cytochrome c from mitochondria, and a slower decline in mitochondrial membrane potential after thawing. These markers of apoptosis were lowest after cryopreservation when IDN-1965 was added to both the culture and cryopreservation medium. Functional markers of hepatocyte activity (albumin production, diazepam metabolism, urea production) were also increased after cryopreservation and culture of hepatocytes in medium supplemented with 25 micromol/L IDN-1965. Cryopreservation of porcine hepatocytes in the presence of caspase inhibitor IDN-1965 was associated with reduced apoptosis and improved function of porcine hepatocytes in both static culture and a perfused BAL. These data demonstrate that inhibition of apoptosis also preserves cell function.

Membrane barrier of a porcine hepatocyte bioartificial liver.

Pores in the membrane of a bioartificial liver (BAL) allow it to function as a semipermeable barrier between its contents (i.e., liver cells) and components of the recipient's immune system. This study is designed to assess the influence of pore size on immune response to a BAL containing porcine hepatocytes. Sixteen healthy dogs were divided into four groups (four dogs per group) based on pore size of the BAL membrane and level of exposure to porcine hepatocytes. Group 1 dogs were administered porcine hepatocytes by intraperitoneal injection and served as positive controls. Group 2 dogs were exposed to porcine hepatocytes in a large-pore (200-nm) BAL, and group 3 dogs were exposed to porcine hepatocytes in a small-pore (10-nm) BAL. Group 4 dogs were exposed to a no-cell (unloaded) BAL and served as negative controls. Intraperitoneal injection of hepatocytes or 3 hours of BAL hemoperfusion was performed day 0 and 3 weeks later on day 21. Biochemical, humoral, and cellular measures of immune response were collected until day 44. The initiation of BAL hemoperfusion was associated with a rapid decline in CH(50) levels of complement and transient neutropenia and thrombocytopenia during all BAL exposures. Xenoreactive antibody response to BAL was increased by use of membranes with large pores and secondary exposures. Skin testing on day 42 showed a delayed-type hypersensitivity response to porcine hepatocytes that also correlated with level of previous antigen exposure. BAL treatment was associated with both immediate and elicited immunologic responses. The immediate response was transient and not influenced by membrane pore size, whereas elicited responses were influenced by pore size of the BAL during previous exposures.