Oncological long-term outcomes of laparoscopic versus open gastrectomy for cT3-4 gastric cancer at surgical staging: a propensity-score matched cohort study.

BACKGROUND: The oncological efficacy of laparoscopic surgery for advanced gastric cancer (AGC) has been evaluated by several randomized trials. However, the inclusion of earlier-stage disease was a limitation in previous studies. METHODS: Patients with cT3-4 gastric cancer, determined by surgical staging to minimize migration of earlier stages, treated at a tertiary cancer center from 2009 to 2018 were included. Based on the surgical approach, the patients were divided into two groups: the laparoscopic gastrectomy (LG) and the open gastrectomy (OG) and matched for age, sex, macroscopic appearance (type 4 or non-type 4), body mass index, estimated tumor size, clinical stage T3'T4, clinical N stage, pathologic T stage (T3 or T4), and type of surgery (total or distal gastrectomy). RESULTS: 588 patients (221 LG, 367 OG) were included in the analysis. After 1:1 propensity-score matching, 386 patients (193 LG, 193 OG) were assigned for analysis. In the LG group, operation time was longer with lower blood loss. The incidence of postoperative complications (≥ grade III) did not differ significantly between the groups (OG: 8.3%, vs. LG: 9.3%). Overall survival (OS) was longer in the LG group (5-year OS: 79.3 vs. 73% HR 0.66, 95% CI 0.44-0.99, P = 0.0497). Relapse-free survival (RFS) did not show a statistical difference (5-year RFS: 69.5 vs. 68.7 HR 0.88, 95% CI 0.62-1.26, P = 0.487). Subgroup analysis for OS also demonstrated equivalent outcomes. CONCLUSION: LG demonstrates comparable safety and efficacy to OG for advanced gastric cancer at surgical staging, with similar rates of severe complications and long-term oncological outcomes. Further research is needed to validate these findings, particularly for total gastrectomy and for patients from Western populations.

Evaluation of Endoscopic Response Using Deep Neural Network in Esophageal Cancer Patients Who Received Neoadjuvant Chemotherapy.

BACKGROUND: We previously reported that endoscopic response evaluation can preoperatively predict the prognosis and distribution of residual tumors after neoadjuvant chemotherapy (NAC). In this study, we developed artificial intelligence (AI)-guided endoscopic response evaluation using a deep neural network to discriminate endoscopic responders (ERs) in patients with esophageal squamous cell carcinoma (ESCC) after NAC. METHOD: Surgically resectable ESCC patients who underwent esophagectomy following NAC were retrospectively analyzed in this study. Endoscopic images of the tumors were analyzed using a deep neural network. The model was validated with a test data set using 10 newly collected ERs and 10 newly collected non-ER images. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the endoscopic response evaluation by AI and endoscopists were calculated and compared. RESULTS: Of 193 patients, 40 (21%) were diagnosed as ERs. The median sensitivity, specificity, PPV, and NPV values for ER detection in 10 models were 60%, 100%, 100%, and 71%, respectively. Similarly, the median values by the endoscopist were 80%, 80%, 81%, and 81%, respectively. CONCLUSION: This proof-of-concept study using a deep learning algorithm demonstrated that the constructed AI-guided endoscopic response evaluation after NAC could identify ER with high specificity and PPV. It would appropriately guide an individualized treatment strategy that includes an organ preservation approach in ESCC patients.

Exposure to a Postoperative Hypercoagulable State Predicts Poor Prognosis After Transthoracic Esophagectomy in Patients with Esophageal Cancer.

PURPOSE: The contribution of postoperative coagulation-fibrinolysis status to prognosis is yet to be fully investigated. Thus, in this study, we aimed to elucidate the relationship between postoperative hypercoagulable state (PHS) after transthoracic esophagectomy and long-term outcome in patients with esophageal cancer. METHODS: Patients with esophageal cancer who underwent transthoracic esophagectomy were selected from a prospectively maintained database. Based on the trend of postoperative plasma fibrin-fibrinogen degradation product (FDP) levels, patients with PHS were identified. The prognostic significance of PHS was evaluated via multivariate analysis using the Cox regression model. RESULTS: Based on the plasma FDP levels of 172 patients that reached a plateau between POD5 and POD7, we calculated the mean FDP value of POD5, 6, and 7, setting a median value as a cutoff. Consequently, 87 patients were classified as PHS. The overall survival (OS) in the PHS group was determined to be significantly lower than in the non-PHS group (5-year OS; 68% and 80%, p = 0.012). Recurrence-free survival (RFS) in the PHS group was significantly lower than in the non-PHS group (5-year RFS; 60% and 79%, p = 0.017). Using the pathological stage as a covariate in the multivariate analysis, PHS was an independent prognostic factor of OS [hazard ratio (HR) 2.517, p = 0.009] and RFS (HR 1.905, p = 0.041). CONCLUSIONS: PHS was found to be an independent negative prognostic factor in patients with esophageal cancer. Possible improvement of the oncological outcome by early postoperative intervention with anticoagulants should be explored in clinical trials.

Invasion of Chicken Anemia Virus in Specific-Pathogen-Free Chicken Flocks and Its Successful Elimination from the Colony.

A specific-pathogen-free (SPF) chicken colony was maintained with successive groups a month apart in age. The absence of specific pathogens, including chicken anemia virus (CAV), was confirmed through periodic serological tests for each group. However, some groups became CAV seropositive. The procedures of removing seropositive and the adjacent seronegative chickens followed with chemically disinfecting the housing did not halt CAV outbreaks. The full genome sequence of the CAV strain that appeared was closely related to low-virulence isolates in China. The outbreaks of CAV decreased with an increase in the seropositive chicken population, indicating that the progeny is protected from CAV infection by maternal anti-CAV antibodies. The persistence of CAV in erythroid and lymphoid tissues or reproductive tissues from CAV seropositive chickens was examined in chickens of various ages using polymerase chain reaction (PCR). Since a low persistence of CAV was observed in the colony, we isolated eggs from CAV seropositive hens through artificial insemination using semen collected from roosters and confirmed as CAV-free by PCR. Fertilized eggs were transferred to a new SPF facility and used for generating CAV-free progeny. To date, chickens reared in the new facility have been CAV-free for longer than two years. Redirection of eggs from seropositive hens was an effective means of eliminating CAV from chickens.

Production of cloned NIBS (Nippon Institute for Biological Science) and α-1, 3-galactosyltransferase knockout MGH miniature pigs by somatic cell nuclear transfer using the NIBS breed as surrogates.

BACKGROUND: Nuclear transfer (NT) technologies offer a means for producing the genetically modified pigs necessary to develop swine models for mechanistic studies of disease processes as well as to serve as organ donors for xenotransplantation. Most previous studies have used commercial pigs as surrogates. METHOD AND RESULTS: In this study, we established a cloning technique for miniature pigs by somatic cell nuclear transfer (SCNT) using Nippon Institute for Biological Science (NIBS) miniature pigs as surrogates. Moreover, utilizing this technique, we have successfully produced an α-1, 3-galactosyltransferase knockout (GalT-KO) miniature swine. Fibroblasts procured from a NIBS miniature pig fetus were injected into 1312 enucleated oocytes. The cloned embryos were transferred to 11 surrogates of which five successfully delivered 13 cloned offspring; the production efficiency was 1.0% (13/1312). In a second experiment, lung fibroblasts obtained from neonatal GalT-KO MGH miniature swine were used as donor cells and 1953 cloned embryos were transferred to 12 surrogates. Six cloned offspring were born from five surrogates, a production efficiency of 0.3% (6/1953). CONCLUSIONS: These results demonstrate successful establishment of a miniature pig cloning technique by SCNT using NIBS miniature pigs as surrogates. To our knowledge, this is the first demonstration of successful production of GalT-KO miniature swine using miniature swine surrogates. This technique could help to ensure a stable supply of the cloned pigs through the use of miniature pig surrogates and could expand production in countries with limited space or in facilities with special regulations such as specific pathogen-free or good laboratory practice.

Effect of aqueous olanexidine versus alcohol-based chlorhexidine for surgical skin antisepsis on incidence of surgical site infections in gastrointestinal surgery: multicentre randomised controlled clinical trial (OEDO trial) protocol.

INTRODUCTION: Surgical site infections (SSIs) are among the most common nosocomial infections in surgery patients. Two types of preparations, povidone-iodine and chlorhexidine-alcohol, are commonly used in preoperative antiseptic procedures worldwide. However, there are inconsistencies among international guideline recommendations concerning skin antiseptics. This trial aimed to evaluate the superiority of olanexidine, which reduced SSI rates more than povidone-iodine in our previous randomised trial, over chlorhexidine-alcohol in clean-contaminated surgery. METHODS AND ANALYSIS: This multicentre randomised controlled clinical trial will compare two antiseptics (1.5% olanexidine and 1.0% chlorhexidine-alcohol) to prevent SSI in clean-contaminated gastrointestinal surgeries with surgical wounds. On providing consent, patients aged <18 years will be included. The primary outcome will be the postoperative 30-day overall SSI rate, while the secondary outcomes will be the postoperative 30-day superficial incisional SSI rate, deep incisional SSI rate, organ/space SSI rate, positive bacterial wound culture rate, cultured bacterial strains, rates of intervention-related toxicity and allergic events (eg, erythema, pruritus, dermatitis and other symptoms of allergy around the region disinfected by the antiseptic during surgery), rate of reoperations due to SSI, medical economic effect indicators (based on health insurance claims) and hospital duration. The Mantel-Haenszel method will be used to estimate the adjusted risk ratio and its 95% CI for the primary analysis, which will compare the treatment effects. ETHICS AND DISSEMINATION: The protocol was approved by the Institutional Review Board of Keio University School of Medicine and subsequently by the board of each participating site. Participant recruitment began in January 2023. The final results will be published in medical journals after international peer review. TRIAL REGISTRATION NUMBER: UMIN000049712.

In vitro effect of recombinant human tumor necrosis factor-alpha on canine neutrophil apoptosis.

Neutrophils (polymorphonuclear leukocytes: PMNs) are essential for the host defense against various infections and are often injurious to the host, causing inflammatory diseases where tumor necrosis factor-alpha (TNF-alpha) is suggested to play an important role. Since an effect of TNF-alpha on canine PMN apoptosis has not been studied, canine PMNs were stimulated with recombinant human (rh)TNF-alpha in the present study to investigate the effect of TNF-alpha on canine PMN apoptosis. PMN apoptosis and function to produce ROS were assessed by flow cytometry. Delayed apoptosis was observed in the PMNs treated with rhTNF-alpha at 100 ng/ml, accompanied by retention of capability to produce ROS. However, PMN apoptosis was accelerated by rhTNF-alpha combined with cycloheximide. Therefore, it is indicated that TNF-alpha is able to activate anti- and pro-apoptotic pathways in PMNs and that the inhibition of PMN apoptosis by TNF-alpha requires protein synthesis in the PMNs.

Apoptosis of canine mammary tumor cells induced by small interfering RNA (siRNA) against Bcl-xL gene.

The inhibition of Bcl-xL mRNA expression and the acceleration of apoptotic cell rates in canine mammary tumor cell line (CF33) by the small interfering RNA (siRNA) were analyzed. The level of Bcl-xL transcripts in CF33 was decreased when cultured with siRNA, suggesting that siRNA might inhibit the expression of Bcl-xL mRNA in the CF33. Apoptotic cell rates in CF33 cultured with siRNA in Oligofectamine medium, with double strand RNA in Oligofectamine medium, without siRNA in Oligofectamine medium and in DMEM alone were 60.9%, 30%, 28.7% and 11.6% at 48-hr incubation, respectively, when evaluated by TUNEL assay. From these results, it was suggested that canine Bcl-xL might be an anticancer target of canine tumors.

Cloning of canine toll-like receptor 9 and its expression in dog tissues.

Toll-like receptor 9 (TLR9) is activated by bacterial DNA and induces production of inflammatory cytokines. In this study, canine TLR9 cDNA was cloned and sequenced. Further, the expression of TLR9 mRNA was investigated in canine tissues. The full-length cDNA for the canine TLR9 gene was 3419bp encoding 1032 amino acids. The similarity of canine TLR9 with those of cat, cattle, human, mouse and pig was 90, 84.3, 83.6, 76.3 and 85.2% at the nucleotide level, and 90.5, 82.7, 81.4, 74.8 and 85.7% at the amino acid level. By reverse transcription-polymerase chain reaction (RT-PCR) analysis, canine TLR9 mRNA was detected in venous blood mononuclear cells and lymph node and weakly in spleen, whereas it was not detected in kidney, liver, pancreas, lung, small intestine, large intestine, heart, skin, muscle, stomach and bladder.

In vitro effect of recombinant human granulocyte colony-stimulating factor on canine neutrophil apoptosis.

Apoptosis is essential in eliminating neutrophils (polymorphonuclear leukocytes: PMNs) in animals. The suppression of PMN apoptosis is believed to be beneficial in eradicating pathogens and is implicated in the pathogenesis of human inflammatory diseases. In the present study, canine PMNs were stimulated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to investigate the in vitro effect on the apoptosis of canine PMNs. Apoptotic cell rates were assessed by flow cytometry in relation to the ability of PMNs to produce reactive oxygen species (ROS). Canine PMN apoptosis was markedly suppressed by rhG-CSF treatment, in association with the retention of the PMN ability to produce ROS. The addition of cycloheximide abolished this suppression by rhG-CSF. Moreover, canine PMNs, which were stimulated by rhG-CSF, expressed high levels of anti-apoptotic mcl-1 gene mRNA, as quantified by real-time polymerase chain reaction method. The results suggest that PMNs, stimulated by G-CSF, could work effectively over a longer period to eliminate pathogens, and that the prolongation of the PMN life-span might occasionally aggravate tissue injuries in dogs. In addition, the suppression of PMN apoptosis seems to be mediated by the induction of anti-apoptotic mcl-1 gene expression.

High expression of Bcl-xL in delayed apoptosis of canine neutrophils induced by lipopolysaccharide.

The expression of Bcl-2 family members, Bcl-xL, Bcl-2, Mcl-1 and Bax was investigated in delayed apoptosis of canine neutrophils induced by lipopolysaccharide (LPS). Apoptotic cell rates in neutrophils stimulated by LPS (100 ng/ml) were measured at 24 h incubation by TUNEL assay. The incidence of apoptotic neutrophils stimulated by LPS at 24 h incubation was 17.0+/-2% and that in non-stimulated neutrophils was 29.9+/-3%. By real-time quantitative PCR analysis, it was indicated that Bcl-xL and Bax levels in canine neutrophils were significantly affected by LPS stimulation. The levels of Bcl-xL, Bcl-2, Mcl-1 and Bax transcripts at 9 h incubation in neutrophils stimulated by LPS (100 ng/ml) were increased by about 80.4-, 1.9-, 1.4- and 5.3-folds, in comparison to those in non-stimulated neutrophils, respectively. These results indicated that Bcl-xL was proved have an important role in the inhibition of canine neutrophil apoptosis by LPS.

Effect of antineoplastic drugs on the expression of Bcl-2 and Bcl-xL genes in the feline T-cell leukemia cell line.

The Bcl-2 gene is the first member of a rapidly expanding family of genes that regulate apoptosis. Bcl-2 has been shown to repress cell death triggered by a diverse array of stimuli including chemotherapy and gamma-irradiation. Chemotherapy of feline lymphoma is generally carried out with antineoplastic drugs, which are reported to induce apoptosis in tumor cells. However, the precise apoptotic signals, induced by chemotherapeutic drugs against feline tumors have not been fully characterized. Therefore, we have evaluated the expression of Bcl-2 and Bcl-xL in FT-1 upon in vitro treatment with these drugs. In the present study, full length of feline Bcl-xL gene was sequenced, and the expressions of Bcl-2 and Bcl-xL mRNAs in feline lymphoma cell line (FT-1) cultured with doxorubicin, prednisolone or vincristine were investigated. Feline Bcl-xL clone was 1163 base pairs in length and encoded 233 amino acids. The predicted amino acid sequence was 99.1%, 98.7%, 96.1%, 97.4%, 97.0% and 97.9% homologous to predicted Bcl-xL of dog, human, mouse, pig, rat and sheep, respectively. The levels of Bcl-2 transcripts at 24h incubation in FT-1 stimulated with doxorubicin (0.3mug/ml), prednisolone (0.2mug/ml) and vincristine (5ng/ml) were increased to about 41.0-, 62.0- and 11.1-fold to those in non-stimulated FT-1, respectively. On the other hand, the level of Bcl-xL transcripts at 24h incubation in FT-1 stimulated by doxorubicin and prednisolone were significantly increased about 4.2- and 5.8-folds to the controls and inducible level of Bcl-xL by vincristine was decreased about 0.35-folds.

Molecular Design of Bisphosphonate-Modified Proteins for Efficient Bone Targeting In Vivo.

To establish a rational molecular design for bisphosphonate (BP)-modified proteins for efficient bone targeting, a pharmacokinetic study was performed using a series of alendronate (ALN), a nitrogen-containing BP, modified proteins with various molecular weights and varying degrees of modification. Four proteins with different molecular weight-yeast glutathione reductase (GR; MW: 112,000 Da), bovine serum albumin (BSA; MW: 67,000 Da), recombinant human superoxide dismutase (SOD; MW: 32,000 Da), and chicken egg white lysozyme (LZM; MW: 14,000 Da)-were modified with ALN to obtain ALN-modified proteins. Pharmacokinetic analysis of the tissue distribution of the ALN-modified and unmodified proteins was performed after radiolabeling them with indium-111 (111In) by using a bifunctional chelating agent. Calculation of tissue uptake clearances revealed that the bone uptake clearances of 111In-ALN-modified proteins were proportional to the degree of ALN modification. 111In-GR-ALN and BSA-ALN, the two high-molecular-weight proteins, efficiently accumulated in bones, regardless of the degree of ALN modification. Approximately 36 and 34% of the dose, respectively, was calculated to be delivered to the bones. In contrast, the maximum amounts taken up by bone were 18 and 13% of the dose for 111In-SOD-ALN(32) and LZM-ALN(9), respectively, because of their high renal clearance. 111In-SOD modified with both polyethylene glycol (PEG) and ALN (111In-PEG-SOD-ALN) was efficiently delivered to the bone. Approximately 36% of the dose was estimated to be delivered to the bones. In an experimental bone metastasis mouse model, treatment with PEG-SOD-ALN significantly reduced the number of tumor cells in the bone of the mice. These results indicate that the combination of PEG and ALN modification is a promising approach for efficient bone targeting of proteins with a high total-body clearance.

Treatment with an adenosine uptake inhibitor attenuates glomerulonephritis in mice.

This study evaluated the effects of KF24345 (3-[1-(6,7-diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride), a novel adenosine uptake inhibitor, on experimental glomerulonephritis induced in mice by two intravenous injections of rabbit anti-mouse glomerular basement membrane antiserum. Mice with glomerulonephritis showed continuous proteinuria and the histological evaluation revealed glomerular and tubular damage at 7 weeks after the first antiserum injection. KF24345 as well as prednisolone and cyclophosphamide significantly inhibited proteinuria and glomerular damage when it was orally administered once a day from 2 to 7 weeks. Prednisolone elevated plasma bilirubin and glutamic-pyruvic transaminase levels, and cyclophosphamide decreased erythrocytes. Moreover, both prednisolone and cyclophosphamide decreased spleen and thymus weights. KF24345 did not show this kind of side effects. These results demonstrate that KF24345 ameliorates glomerulonephritis with minimal side effects in mice, suggesting that the adenosine uptake inhibitor may be useful for the treatment of glomerulonephritis.

KF24345, an adenosine uptake inhibitor, ameliorates the severity and mortality of lethal acute pancreatitis via endogenous adenosine in mice.

Adenosine protects against cellular damage and dysfunction under several adverse conditions including inflammation and ischemia. In this study, we examined the effects of 3-[1-(6,7-diethoxy-2-morpholinoquinazolin-4-yl)piperidin-4-yl]-1,6-dimethyl-2,4(1H,3H)-quinazolinedione hydrochloride (KF24345), an adenosine uptake inhibitor, on experimental acute pancreatitis induced by choline-deficient and ethionine-supplemented diet in mice. KF24345, administered with the diet onset and every 24 h thereafter, prevented hyperamylasemia, acinar cell injury and serum tumor necrosis factor-alpha elevation and ultimately decreased mortality. Therapeutic treatment with KF24345, which started 32 h after the diet onset, also decreased mortality. The beneficial effect of KF24345 on mortality was abolished by the pretreatment with 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385), a selective adenosine A(2A) receptor antagonist. An intravenous injection of KF24345 at 48 h after the diet onset increased plasma adenosine concentrations in mice with acute pancreatitis. These results suggest that KF24345 shows anti-pancreatitis effects via endogenous adenosine and adenosine A(2A) receptors. The adenosine uptake inhibition could be a new therapeutic approach for acute pancreatitis.

Canine Bcl-xL gene and its expression in tumor cell lines.

The canine Bcl-xL gene was cloned and sequenced. Canine Bcl-xL cDNA clone was 1252 bp in length, and encoded 233 deduced amino acides. The predicted canine Bcl-xL amino acid sequence shared 99.6%, 97.0%, 97.9%, 98.7% and 98.3% homology with that of human, mouse, rat, sheep and pig Bcl-xL, respectively. RT-PCR analysis revealed that canine Bcl-xL mRNA was constitutively expressed in CL-1 (canine lymphoma) and GL-1 (canine B cell leukemia) cell lines.

Characterization of canine caspase-3.

The canine caspase-3 gene was cloned and sequenced. The canine caspase-3 cDNA clone was 1473 base pairs in length and encoded 277 amino acids. The predicted amino acid sequence showed 88.4%, 88.0%, 85.9%, 65.9% and 60.6% homology with that of human, pig, mouse, chicken and zebra fish caspase-3, respectively. The caspase-3 mRNA was confirmed to express in skin, lymph node, bone marrow, small intestine and lung from a healthy dog by RT-PCR analysis.