Characteristics of Immune Response to Tumor-Associated Antigens and Immune Cell Profile in Patients With Hepatocellular Carcinoma.

Host antitumor immune responses may be different between hepatocellular carcinoma (HCC) caused by metabolic disorders and HCC associated with hepatitis virus infection. In this study, we examined the immune response of tumor-associated antigen (TAA)-specific T cells and immune cell profile in patients with HCC separated by cause. Thirty-two patients with hepatitis B virus (HBV)-related HCC, 42 patients with hepatitis C virus-related HCC, and 18 patients with nonalcoholic steatohepatitis (NASH)-related HCC were analyzed. The frequencies of TAA-specific T cells, the expression levels of surface markers on each immune cell, and the expression of each TAA in HCC tissue were measured. The immune response to TAA and immune cell profile were markedly different among the three groups. The immune response to TAA in the NASH-related HCC group was weaker than the responses in the other two groups. In patients with NASH-related HCC, the frequencies of effector regulatory T cells (eTregs) and cluster of differentiation 8-positive (CD8+ ) T cells strongly expressing cytotoxic T-lymphocyte antigen (CTLA)-4 were high. The frequency of CD8+ T cells strongly expressing programmed cell death 1 was the highest in patients with HBV-related HCC. Among these immune cell profiles, the frequencies of C-X-C motif chemokine receptor 3+ eTregs and CTLA-4+ CD8+ T cells were inversely correlated with the strength of the TAA-specific T-cell immune response, and the restoration of TAA-specific T-cell responses by anti-CTLA-4 antibody was observed. Conclusion: The immune response to TAA were markedly different among the three groups, and a correlation with the immune cell profile was observed, suggesting that development of immunotherapy based on the etiology of HCC may lead to more effective treatment outcomes.

Serum C16:1n7/C16:0 ratio as a diagnostic marker for non-alcoholic steatohepatitis.

BACKGROUND AND AIMS: Accurate diagnosis of non-alcoholic steatohepatitis (NASH) from non-alcoholic fatty liver disease (NAFLD) is clinically important. Therefore, there is a need for easier ways of diagnosing NASH. In this study, we investigated the serum fatty acid composition and evaluated the possibility of using the serum fatty acid composition as a diagnostic marker of NASH. METHODS: The subjects were 78 NAFLD patients (non-alcoholic fatty liver [NAFL]: 30, NASH: 48) and 24 healthy individuals. Fatty acids extracted from the liver tissue and serum were identified and quantified by gas chromatography. In addition, we evaluated the relationship between serum and liver tissue fatty acid composition, patient background, and liver histology. The diagnostic performance of NASH was evaluated by calculating the area under the receiver operating characteristic (AUROC). RESULTS: The results of the fatty acid analysis showed the C16:1n7/C16:0 ratio to have the strongest correlation between serum and liver tissue (r = 0.865, P < 0.0001). The serum C16:1n7/C16:0 ratio in the NASH group was higher compared with that in the NAFL group (P = 0.0007). Evaluation of the association of the serum C16:1n7/C16:0 ratio with liver histology revealed significant correlation with lobular inflammation score, ballooning score, and fibrosis score. The AUROC for predicting NASH in all NAFLD patients was 0.7097. The AUROC was nearly equivalent even when the study subjects were restricted to patients with a fibrosis score ≤ 2 only (AUROC 0.6917). CONCLUSION: Measuring the serum C16:1n7/C16:0 ratio may be an effective non-invasive method for diagnosing NASH, particularly in its early stages.

[A case of gastric follicular lymphoma resected and diagnosed with laparoscopy and endoscopy cooperative surgery].

A woman in her 70s was diagnosed with a protruding mucosa-associated lymphoid tissue (MALT) lymphoma during a secondary health examination. After eradication of Helicobacter pylori, a biopsy revealed gastric follicular lymphoma (FL) and the lesion was still protruding one year later. 18F-fluorodeoxyglucose positron emission tomography showed focal nodular hypermetabolic activity, suggesting that FL may have transformed into a diffuse large B-cell lymphoma. Upper gastrointestinal endoscopy, colonoscopy, and capsule endoscopy showed no other lesions in the gastrointestinal tract, and bone marrow biopsy showed no permeation into the marrow. Therefore, this lesion, which appeared as a submucosal tumor, was limited to the stomach. Laparoscopy and endoscopy cooperative surgery was performed, because it allows for correct pathological diagnosis while removing only a minimal portion of the stomach wall. Histological findings showed follicular structures consisting of abnormal lymphoid cells. Immunohistochemical analysis revealed that neoplastic cells were positive for CD20, CD79a, Bcl-2, CD10, and c-MYC, but negative for CD3, CD5, and cyclin D1. Finally, we diagnosed this lesion as a primary gastric FL.

A rapidly growing, large, mature retroperitoneal teratoma in an adult male patient.

A 40-year-old man complaining of abdominal distention was referred to our hospital. Computed tomography of the abdomen demonstrated a very large abdominal mass with fat and calcification. The size of the mass rapidly increased from 30cm to 40cm over two weeks. The tumor was removed and diagnosed by pathological examination to be a retroperitoneal mature cystic teratoma that contained a 40-cm long, mature intestinal tract-like cyst, together with bone marrow and fat. The rapid growth of the tumor may have been caused by an increased secretion in the cyst.

A case of cytomegalovirus infection with splenic infarction and an esophageal ulcer in an immunocompetent adult.

BACKGROUND: Recently, morbidities due to primary cytomegalovirus (CMV) infection have increased in young Japanese adults because of decreased anti-CMV antibodies in them. CMV infections are typically resolved naturally in immunocompetent individuals, and complications rarely occur. Here we present the case of an immunocompetent adult with CMV infection complicated by splenic infarctions and an esophageal ulcer. CASE REPORT: A 37-year-old male complaining of a prolonged fever and liver injury was admitted to hospital for a closed examination. The patient had general malaise and mild appetite loss but no abdominal pain. Symptoms of infectious mononucleosis, including liver injury, appearance of atypical lymphocytes in the blood, and hepatosplenomegaly, were observed. A primary CMV infection was confirmed by CMV-IgM positive and CMV-IgG negative serological tests. Enhanced abdominal computed tomography confirmed hepatitis and splenic infarction, and an upper gastrointestinal endoscopy revealed an esophageal ulcer. The patient exhibited no predisposing risk factors for thrombosis, and he was diagnosed with splenic infarctions associated with CMV infection. Because the patient was immunocompetent, he underwent symptomatic therapy without antiviral or anticoagulant therapies. The treatment improved his overall condition. Including the present case, only 11 cases of CMV infections with splenic infarction in immunocompetent individuals have been reported. Contrary to what is observed in immunocompromised hosts, upper gastrointestinal lesions with CMV infection are rare in immunocompetent individuals. The esophageal lesion observed in our patient was a typical punched-out ulcer. The immunohistochemical staining of the tissue biopsies revealed that the ulcer was associated with CMV. CONCLUSION: Although splenic infarctions and esophageal ulcers are rare, they should be considered as potential complications accompanying CMV infection in immunocompetent individuals. The administration of symptomatic therapy should be considered even when the patient is immunocompetent.

Cleavage of Hsp70.1 causes lysosomal cell death under stress conditions.

Autophagy mediates the degradation of intracellular macromolecules and organelles within lysosomes. There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Heat shock protein 70.1 (Hsp70.1) exhibits dual functions as a chaperone protein and a lysosomal membrane stabilizer. Since chaperone-mediated autophagy participates in the recycling of ∼30% cytosolic proteins, its disorder causes cell susceptibility to stress conditions. Cargo proteins destined for degradation such as amyloid precursor protein and tau protein are trafficked by Hsp70.1 from the cytosol into lysosomes. Hsp70.1 is composed of an N-terminal nucleotide-binding domain (NBD) and a C-terminal domain that binds to cargo proteins, termed the substrate-binding domain (SBD). The NBD and SBD are connected by the interdomain linker LL1, which modulates the allosteric structure of Hsp70.1 in response to ADP/ATP binding. After the passage of the Hsp70.1-cargo complex through the lysosomal limiting membrane, high-affinity binding of the positive-charged SBD with negative-charged bis(monoacylglycero)phosphate (BMP) at the internal vesicular membranes activates acid sphingomyelinase to generate ceramide for stabilizing lysosomal membranes. As the integrity of the lysosomal limiting membrane is critical to ensure cargo protein degradation within the acidic lumen, the disintegration of the lysosomal limiting membrane is lethal to cells. After the intake of high-fat diets, however, ß-oxidation of fatty acids in the mitochondria generates reactive oxygen species, which enhance the oxidation of membrane linoleic acids to produce 4-hydroxy-2-nonenal (4-HNE). In addition, 4-HNE is produced during the heating of linoleic acid-rich vegetable oils and incorporated into the body via deep-fried foods. This endogenous and exogenous 4-HNE synergically causes an increase in its serum and organ levels to induce carbonylation of Hsp70.1 at Arg469, which facilitates its conformational change and access of activated µ-calpain to LL1. Therefore, the cleavage of Hsp70.1 occurs prior to its influx into the lysosomal lumen, which leads to lysosomal membrane permeabilization/rupture. The resultant leakage of cathepsins is responsible for lysosomal cell death, which would be one of the causative factors of lifestyle-related diseases.

Impact of common ALDH2 inactivating mutation and alcohol consumption on Alzheimer's disease.

Aldehyde dehydrogenase 2 (ALDH2) is an enzyme found in the mitochondrial matrix that plays a central role in alcohol and aldehyde metabolism. A common ALDH2 polymorphism in East Asians descent (called ALDH2*2 or E504K missense variant, SNP ID: rs671), present in approximately 8% of the world's population, has been associated with a variety of diseases. Recent meta-analyses support the relationship between this ALDH2 polymorphism and Alzheimer's disease (AD). And AD-like pathology observed in ALDH2-/- null mice and ALDH2*2 overexpressing transgenic mice indicate that ALDH2 deficiency plays an important role in the pathogenesis of AD. Recently, the worldwide increase in alcohol consumption has drawn attention to the relationship between heavy alcohol consumption and AD. Of potential clinical significance, chronic administration of alcohol in ALDH2*2/*2 knock-in mice exacerbates the pathogenesis of AD-like symptoms. Therefore, ALDH2 polymorphism and alcohol consumption likely play an important role in the onset and progression of AD. Here, we review the data on the relationship between ALDH2 polymorphism, alcohol, and AD, and summarize what is currently known about the role of the common ALDH2 inactivating mutation, ALDH2*2, and alcohol in the onset and progression of AD.

Implication of the cooking oil-peroxidation product "hydroxynonenal" for Alzheimer's disease.

Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that reduces cell injuries via detoxification of lipid-peroxidation product, 4-hydroxy-2-nonenal (hydroxynonenal). It is generated exogenously via deep-frying of linoleic acid-rich cooking oils and/or endogenously via oxidation of fatty acids involved in biomembranes. Although its toxicity for human health is widely accepted, the underlying mechanism long remained unknown. In 1998, Yamashima et al. have formulated the "calpain-cathepsin hypothesis" as a molecular mechanism of ischemic neuronal death. Subsequently, they found that calpain cleaves Hsp70.1 which became vulnerable after the hydroxynonenal-induced carbonylation at the key site Arg469. Since it is the pivotal aberration that induces lysosomal membrane rupture, they suggested that neuronal death in Alzheimer's disease similarly occurs by chronic ischemia via the calpain-cathepsin cascade triggered by hydroxynonenal. For nearly three decades, amyloid ß (Aß) peptide was thought to be a root substance of Alzheimer's disease. However, because of both the insignificant correlations between Aß depositions and occurrence of neuronal death or dementia, and the negative results of anti-Aß medicines tested so far in the patients with Alzheimer's disease, the strength of the "amyloid cascade hypothesis" has been weakened. Recent works have suggested that hydroxynonenal is a mediator of programmed cell death not only in the brain, but also in the liver, pancreas, heart, etc. Increment of hydroxynonenal was considered an early event in the development of Alzheimer's disease. This review aims at suggesting ways out of the tunnel, focusing on the implication of hydroxynonenal in this disease. Herein, the mechanism of Alzheimer neuronal death is discussed by focusing on Hsp70.1 with a dual function as chaperone protein and lysosomal stabilizer. We suggest that Aß is not a culprit of Alzheimer's disease, but merely a byproduct of autophagy/lysosomal failure resulting from hydroxynonenal-induced Hsp70.1 disorder. Enhancing ALDH2 activity to detoxify hydroxynonenal emerges as a promising strategy for preventing and treating Alzheimer's disease.

Vegetable Oil-Peroxidation Product 'Hydroxynonenal' Causes Hepatocyte Injury and Steatosis via Hsp70.1 and BHMT Disorders in the Monkey Liver.

Hsp70.1 has a dual function as a chaperone protein and lysosomal stabilizer. In 2009, we reported that calpain-mediated cleavage of carbonylated Hsp70.1 causes neuronal death by inducing lysosomal rupture in the hippocampal CA1 neurons of monkeys after transient brain ischemia. Recently, we also reported that consecutive injections of the vegetable oil-peroxidation product 'hydroxynonenal' induce hepatocyte death via a similar cascade in monkeys. As Hsp70.1 is also related to fatty acid ß-oxidation in the liver, its deficiency causes fat accumulation. The genetic deletion of betaine-homocysteine S-methyltransferase (BHMT) was reported to perturb choline metabolism, inducing a decrease in phosphatidylcholine and resulting in hepatic steatosis. Here, focusing on Hsp70.1 and BHMT disorders, we studied the mechanisms of hepatocyte degeneration and steatosis. Monkey liver tissues with and without hydroxynonenal injections were compared using proteomics, immunoblotting, immunohistochemical, and electron microscopy-based analyses. Western blotting showed that neither Hsp70.1 nor BHMT were upregulated, but an increased cleavage was observed in both. Proteomics showed a marked downregulation of Hsp70.1, albeit a two-fold increase in the carbonylated BHMT. Hsp70.1 carbonylation was negligible, in contrast to the ischemic hippocampus, which was associated with ~10-fold increments. Although histologically, the control liver showed very little lipid deposition, numerous tiny lipid droplets were seen within and around the degenerating/dying hepatocytes in monkeys after the hydroxynonenal injections. Electron microscopy showed permeabilization/rupture of lysosomal membranes, dissolution of the mitochondria and rough ER membranes, and proliferation of abnormal peroxisomes. It is probable that the disruption of the rough ER caused impaired synthesis of the Hsp70.1 and BHMT proteins, while impairment of the mitochondria and peroxisomes contributed to the sustained generation of reactive oxygen species. In addition, hydroxynonenal-induced disorders facilitated degeneration and steatosis in the hepatocytes.

Hsp70.1 carbonylation induces lysosomal cell death for lifestyle-related diseases.

Alzheimer's disease, type 2 diabetes, and non-alcoholic steatohepatitis (NASH) constitute increasingly prevalent disorders. Individuals with type 2 diabetes are well-known to be susceptible to Alzheimer's disease. Although the pathogenesis of each disorder is multifactorial and the causal relation remains poorly understood, reactive oxygen species (ROS)-induced lipid and protein oxidation conceivably plays a common role. Lipid peroxidation product was recently reported to be a key factor also for non-alcoholic steatohepatitis, because of inducing hepatocyte degeneration/death. Here, we focus on implication of the representative lipid-peroxidation product 'hydroxynonenal' for the cell degeneration/death of brain, pancreas, and liver. Since Hsp70.1 has dual roles as a chaperone and lysosomal membrane stabilizer, hydroxynonenal-mediated oxidative injury (carbonylation) of Hsp70.1 was highlighted. After intake of high-fat diets, oxidation of free fatty acids in mitochondria generates ROS which enhance oxidation of ω-6 polyunsaturated fatty acids (PUFA) involved within biomembranes and generate hydroxynonenal. In addition, hydroxynonenal is generated during cooking deep-fried foods with vegetable oils especially containing linoleic acids. These intrinsic and exogenous hydroxynonenal synergically causes an increase in its serum and organ levels to induce Hsp70.1 oxidation. As it is amphiphilic; being water-soluble but displays strong lipophilic characteristics, hydroxynonenal can diffuse within the cells and react with targets like senile and/or atheromatous plaques outside the cells. Hydroxynonenal can deepen and expand lysosomal injuries by facilitating 'calpain-mediated cleavage of the carbonylated Hsp70.1'. Despite the unique anatomical, physiological, and biochemical characteristics of each organ for its specific disease, there should be a common cascade of the cell degeneration/death which is caused by hydroxynonenal. This review aims to implicate hydroxynonenal-mediated Hsp70.1 carbonylation for lysosomal membrane permeabilization/rupture and the resultant cathepsin leakage for inducing cell degeneration/death. Given the tremendous number of worldwide people suffering various lifestyle-related diseases, it is valuable to consider how ω-6 PUFA-rich vegetable oils is implicated for the organ disorder.

Hydroxynonenal Causes Hepatocyte Death by Disrupting Lysosomal Integrity in Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: The lipid oxidation is a key factor for damaging hepatocytes and causing cell death. However, the mechanisms underlying hepatocyte death and the role of the most popular lipid peroxidation product 4-hydroxy-2-nonenal (HNE) in nonalcoholic steatohepatitis (NASH) remains unclear. METHODS: We demonstrated using hepatoma cell lines, a NASH mouse model, HNE-treated monkeys, and biopsy specimens from patients with NASH that HNE induced hepatocyte death by disintegrating the lysosomal limiting membrane. RESULTS: The degree of HNE deposition in human NASH hepatocytes was more severe in cases with high lobular inflammation, ballooning, and fibrosis scores, and was associated with enlargement of the staining of lysosomes in hepatocytes. In in vitro experiments, HNE activated µ-calpain via G-protein coupled receptor (GPR) 120. The resultant rupture/permeabilization of the lysosomal limiting membrane induced the leakage of cathepsins from lysosomes and hepatocyte death. The blockade of G-protein coupled receptor 120 (GPR120) or µ-calpain expression suppressed lysosomal membrane damage and hepatocyte death by HNE. Alda-1, which activates aldehyde dehydrogenase 2 to degrade HNE, prevented HNE-induced hepatocyte death. Intravenous administration of HNE to monkeys for 6 months resulted in hepatocyte death by a mechanism similar to that of cultured cells. In addition, intraperitoneal administration of Alda-1 to choline-deficient, amino-acid defined treated mice for 8 weeks inhibited HNE deposition, decreased liver inflammation, and disrupted lysosomal membranes in hepatocytes, resulting in improvement of liver fibrosis. CONCLUSIONS: These results provide novel insights into the mechanism of hepatocyte death in NASH and will contribute to the development of new therapeutic strategies for NASH.

Conservative treatment of hepatic portal venous gas resulting from non-occlusive mesenteric ischemia: a case report.

A 73-year-old man with severe intellectual disability, malnutrition, and hypoalbuminemia presented to our hospital after experiencing vomiting following dinner. Electrocardiography revealed a sinus rhythm. Plain abdominal radiography showed branching radiolucency in the liver. Abdominal computed tomography (CT) revealed branching gaseous foci of low density in the portal vein and its tributaries, suggesting the presence of hepatic portal venous gas (HPVG). Abdominal contrast-enhanced CT showed a segmental lack of contrast enhancement in the intestinal wall despite the absence of vascular occlusion in the main trunk and branches of the mesenteric artery. The patient was diagnosed with non-occlusive mesenteric ischemia (NOMI) accompanied by HPVG. Peripheral parenteral nutrition, antibiotic treatment, and human serum albumin were administered. The HPVG disappeared approximately 20 h after hospitalization. Intravascular dehydration associated with hypoalbuminemia was considered to be the cause of NOMI; the latter improved through the early correction of dehydration and hypoalbuminemia. The presence of HPVG is usually considered a diagnostic clue in patients with abdominal catastrophe and is associated with high mortality. However, the current case demonstrates the pitfalls of assessing the severity of the underlying condition based solely on the presence of HPVG.

Small bowel obstruction caused by intrauterine device infection.

A 39-year-old previously healthy woman was referred to our emergency department by a primary care doctor on suspected to be acute enteritis, complaining of fever, anorexia, lower abdominal pain, and frequent diarrhea. The day after admission, although frequent diarrhea stopped, the abdominal distension worsened. An abdominal radiograph revealed several dilated loops of the small bowel, suggested that small bowel obstruction (SBO) had developed. White blood cell count and c-reactive protein were markedly increased, and abdominal contrast-enhanced computed tomography scan showed localized severely edematous bowel mucosa, increased adipose tissue concentration in the pelvis, and a beaded low absorption area in the uterus. Gynecological examination revealed the presence of a pus-filled plastic intrauterine device (IUD) in the uterus. The patient confided that she had sex with her husband 2 days before the onset of symptoms. A diagnosis of SBO due to pelvic peritonitis caused by IUD infection during sexual activity was made. The SBO was cleared in 12 days with fasting, peripheral parenteral nutrition, antibiotic treatment, and insertion of an ileus tube. This case reminds us that it needs to consider disorders associated with the uterine appendages, in women of reproductive age with lower abdominal pain.

A case of small bowel adenocarcinoma that caused intestinal obstruction after administration of patency capsule.

An 80-year-old man was admitted to our hospital with iron deficiency anemia and exertional chest pain. Coronary artery angiography showed 90% stenosis in the middle left anterior descending branch; abdominal computed tomography (CT) showed enlarged mesenteric lymph nodes. Although his past medical history and results of imaging studies did not suggest intestinal stenosis, assessment of intestinal patency with the PillCam® patency capsule (tag-less PC) was performed. Thirty-three hours after administration, excretion of tag-less PC was not confirmed; an abdominal contrast-enhanced CT showed arrest of tag-less PC in the small bowel and thickening of the bowel wall, suggesting a small bowel tumor. Four days after administration of tag-less PC, he developed abdominal pain and vomiting. Intestinal obstruction was diagnosed by abdominal radiograph. A diagnosis of small bowel tumor with intestinal obstruction was made, and surgical resection was performed. The tumor was histologically an adenocarcinoma. It is necessary to carefully evaluate gastrointestinal patency before small intestine endoscopy especially in elderly people with reduced cardiopulmonary function and many underlying diseases.