Effects of preconditioning on ischemia/reperfusion injury of hepatocytes determined by immediate early gene transcription.

Ischemia reperfusion (I-R) of the liver induces various events leading to cell death (apoptosis) and subsequent cells proliferation. Recent experimental studies have described the protective effect of ischemic preconditioning (IPC) on I-R injury of the liver. However, the mechanisms involved in this protection remain unknown. The protein products of immediate early genes (IEGs) behave as crucial transcriptional regulators not only in apoptosis but also in cell proliferation. Here, we evaluated the effects of IPC on IEG transcription after I-R injury, using a rat liver I-R injury model. Injury to hepatocytes was evaluated by measuring serum levels of aspartate transaminase (AST), alanine transaminase (ALT), and lactate dehydrogenase (LDH) and that to endothelial cells by plasma concentration of hyaluronic acid (HA). The extent of necrosis was evaluated by H&E staining, while cell proliferation and apoptosis were evaluated by proliferating cell nuclear antigen (PCNA) and terminal deoxy(d)-UTP nick end labelling (TUNEL) staining, respectively. Alterations in the transcription of IEGs (c-fos and c-jun) were examined by Northern blotting. Rats subjected to 40-min liver ischemia, preceded by 10-min preconditioning, showed significantly lower AST, ALT, LDH, and HA levels at 6 h after I-R than untreated animals (P < 0.05; n at least 5 rats per group). The percentage of necrotic areas at 24 h after I-R was significantly lower in IPC-treated animals than in the controls. The numbers of apoptotic cells at 24 h after I-R and the numbers of PCNA-positive cells at 24 and 48 h after I-R were significantly lower in IPC-treated rats than in controls. Transcription levels of IEGs were low in IPC-treated rats, particularly c-jun at 1 and 1.5 h after I-R (P < 0.05). Our results indicate that IPC provides a significant protective effect on for liver cells against I-R injury and that its effect is evidenced by a significant decrease in the transcription levels of IEGs following the insult.

Induction of immediate early genes after partial hepatectomy in cholestatic liver.

The protein products of immediate early genes (IEGs) behave as transcriptional regulators and play an important role in the regulation of gene expression associated with liver cell proliferation and apoptosis. The aim of this study was to examine whether the transcription of IEGs was induced by obstructive jaundice during liver regeneration after 70% partial hepatectomy, and to examine their association with animal survival, cell proliferation, and apoptosis. Obstructive jaundice (OJ) was induced in rats by common bile duct ligation (CBDL), and 70% partial hepatectomy was performed 5 days after CBDL (OJ group). Changes in the induction of the IEGs, c-fos and c-jun, were compared between control and OJ groups in relation to survival before and after partial hepatectomy. Cell proliferation and apoptosis were evaluated by proliferating cell nuclear antigen (PCNA) immunohistological staining and by an in situ TdT-mediated d-UTP-digoxigerin nick-end labeling (TUNEL) method. The 2-week survival in the OJ group (2/7) was significantly less that of the sham operation (control) group (7/7). Enhanced induction of IEGs was evident in the OJ group after partial hepatectomy compared with findings in the control group. The PCNA-labeling index (LI) in the OJ group was increased after partial hepatectomy, but only minimally, compared with that in control animals. Apoptotic cells appeared in bile ducts and surrounding hepatocytes after partial hepatectomy in the OJ group, although apoptotic cells were rare in the control group. IEG transcription does occur after partial hepatectomy in jaundiced liver but it is enhanced and sustained, and leads to apoptosis rather than leading to the efficient proliferation of hepatocytes.

Lymph node metastasis-related carbohydrate epitopes of gastric cancer with submucosal invasion.

This study was conducted to examine the lymph node metastasis-related carbohydrate epitopes of cancer cells in primary lesions of gastric cancer with submucosal invasion (sm gastric cancer). A total of 118 formalin-fixed and paraffin-embedded surgical specimens were studied. Carbohydrate epitopes were detected histochemically using 17 kinds of biotin-labeled lectins and three kinds of monoclonal antibodies (mAbs) against Thomsen-Friedenreich antigen (HB-T1), Tn (HB-Tn1), and sialyl Tn antigens (HB-STn1). The node-positive group showed significantly lower reactivities with Ricinus communis I (RCA-I), Lotus tetragonolobus (Lotus), Triticum vulgaris (WGA), and Concanavalia ensiformis (ConA), and higher reactivities with HB-STn1. When sm gastric cancer was classified into differentiated and undifferentiated types, lower reactivities with RCA-I and ConA were observed in the differentiated type cancers, whereas lower reactivities with Lotus, WGA, and higher reactivity with HB-STn1 were observed in the undifferentiated type cancers. Nodal status was able to be predicted with high precision by a combination of these carbohydrates and histological subtypes. Since neuraminidase treatment rarely changed the reactivity of cancer cells with these lectins, we conclude that the loss of certain kinds of carbohydrates that are restricted by histological subtypes is related to lymph node metastasis in sm gastric cancer.

Clinicopathologic features of local and metastatic recurrences in primary lung adenocarcinoma.

We attempted to construct the contour of recurrence in primary lung adenocarcinoma with clinicopathologic variables based on data of 131 patients with completely resected primary lung adenocarcinoma. In univariate analysis, tumor size (more or less 3 cm in diameter), p-T, p-N, pathological stage, differentiation, ly factor and v factor were chosen for prognostic predictors. In multivariate analysis, v factor and p-N were independent variables of local recurrence and metastatic recurrence, respectively. The examination of significant correlation among clinicopathologic variables in terms of 5-year survival rates of patients showed that tumor size, p-T, ly factor and v factor were profoundly related to local recurrence, whereas ly factor, differentiation and p-N were linked to distant metastasis. We therefore examined an additive effect of tumor size, differentiation and vascular invasion on recurrence. The results demonstrated that neither local nor metastatic recurrences were found in patients with well differentiated adenocarcinoma less than 3 cm in diameter if vascular invasion was negative. We conclude that vascular (ly factor and v factor) is central to lung adenocarcinoma recurrence. The vascular invasion is a powerful predictor of recurrence in less than 3 cm diameter, well differentiated adenocarcinoma of the lung.

Sialyl-Tn antigen as a useful predictor of poor prognosis in patients with advanced stomach cancer.

Sialyl-Tn antigen (STn) expression was studied immunohistochemically in 211 primary advanced gastric carcinomas. The overall rate of positive STn staining was 17% (35/211), and positive STn staining was found not to be correlated with tumor size, depth of invasion, lymph node metastasis, liver metastasis, or peritoneal metastasis. However, patients with tumors that were immunoreactive for STn demonstrated significantly lower survival (P < 0.05). Multivariate analysis revealed that STn staining was an independent prognostic factor. From these findings we conclude that careful followup and intense postoperative therapy are required for patients with advanced gastric cancer who have positive immunoreactivity for STn.

The differential induction of two immediate early genes, c-fos and c-jun, after systemic hypovolemic shock/resuscitation in the rat liver and kidney.

The aim of this study was to investigate the expression of the immediate early genes (IEGs), c-fos and c-jun, in the rat kidney and liver in two types of hemorrhage shock/resuscitation models. In the first group, hemorrhagic shock was induced by the withdrawal of blood through the carotid artery. A mean arterial blood pressure (MAP) of 40mmHg was maintained for 1h before blood was reperfused. In the second group, the MAP was maintained at the same level for 2h. Animals were resuscitated with Ringer's lactate solution. In the first group, a rapid and transient induction of c-fos and c-jun mRNAs in both the liver and kidney was observed, peaking 0 to 2 h after reperfusion. In the second group, a more protracted pattern of induction was evident in both organs. In both models, the induction of c-fos mRNA was distinctly different in the liver and kidney. These results indicated, first, that with respect to IEG expression, organs respond differently to a systemic shock/resuscitation stimuli, and second, that alterations in the pattern of IEG expression might represent an indication of the degree of organ damage or the repair processes subsequent to hypotension/reperfusion.

Coronary vascular bed perfusion with a polyethylene glycol-modified hemoglobin-encapsulated liposome, neo red cell, in rats.

Whether hemoglobin (Hb) encapsulated liposomes have vasoconstrictive activity remains controversial. We therefore examined the vascular activity of a liposome Hb, Neo red cell (NRC), in a simple in vitro model of Langendorff perfusion of the rat heart using Krebs-Henseleit (KH) solution as the perfusate. In the KH solution, NRC (Hb at 1 mg/ml), however, induced an immediate and abnormal increase in perfusion pressure. Histological examinations revealed that embolisms were the likely cause of this disturbance. Inorganic crystals formed by the mixing of NRC with the perfusate were a possible source of the embolisms. We found that the addition of bovine serum albumin to the perfusate was effective in avoiding embolic events. This protocol was used to compare the vasoconstrictive properties of unmodified bovine Hb and NRC. Unmodified bovine Hb (1 mg/ml) caused an increase in perfusion pressure and a decrease in the duration of bradykinin-induced relaxation. In contrast, NRC (Hb at 1 mg/ml) had no such vasoconstrictive effects. These results provide the first information regarding perfusion of the circulatory vascular bed by NRC and further evidence that the encapsulation of Hb into liposomes is an effective approach to modulate Hb-related vasoconstrictive activity.

Effect of liposome encapsulated hemoglobin "neo red cells" on severe hemorrhagic shock.

We induced shock by exsanguination and administered Neo Red Cells (NRC) after 30 minutes to experimentally examine the efficacy of NRC on severe shock with respect to hemodynamics and oxygen transport capacity. Seven beagles were used for this experiment. After intravenous anesthesia, intratracheal intubation was performed, and inhalation of 50% oxygen was administered. Animals were exsanguinated through a vein at a rate of 30 mL/min. Animals showing systolic blood pressure of 60 to 69 mmHg were regarded as being in shock. After animals were left untreated for 30 minutes, NRC was administered. This was then repeated. Administration of NRC at a 1.5-fold dose compared to the exsanguinated blood volume was required for animals to recover from shock. Animals tolerated shock 3 times, but did not recover from the 4th shock. Although NRC with approximately one third the viscosity of whole blood was administered, vascular resistance was increased and cardiac output was decreased, resulting in progression of heart failure. In addition, oxygen consumption increased with shock. NRC satisfied oxygen requirements by compensating for the decrease in cardiac output with an increase in AV difference, but erythrocytes were insufficient to increase difference in arterial and venous oxygen content (AV difference), and did not supply sufficient volume of oxygen.

[Recurrent thymic carcinoid tumor with multiple endocrine neoplasia syndrome--a case report].

A 55-year-old male with multiple endocrine neoplasia syndrome accompanied by hyperparathyroidism and hypergastrinemia was admitted because of local recurrence of thymic carcinoid tumor and a parathyroid adenoma. The recurrent thymic carcinoid tumor replaced anterior mediastinum, invaded brachiocephalic vein and superior vena cava, a disseminated nodule was found at pericardium. After induction chemotherapy using carboplatin and etoposide the operation was performed. The parathyroid tumor and recurrent thymic carcinoid tumor were removed completely together with brachiocephalic vein, superior vena cava, pericardium and anterior chest wall. Superior vena cava was replaced with synthetic graft and chest wall was reconstructed. The patient is alive and well 22 months after surgery without recurrence. Immunohistochemistry of removed specimens revealed parathormone and gastrin secreted from the parathyroid adenoma but not from the carcinoid tumor. Careful survey of systemic endocrine organs is necessary in case of thymic carcinoid tumor. Aggressive surgery in locally recurrent thymic carcinoid without distant metastasis must be considered.

Prenatal determination of human platelet antigen type 4 by DNA amplification of amniotic fluid cells.

To predict a fetus at risk for neonatal alloimmune thrombocytopenia (NATP) caused by human platelet antigen (HPA)-4 incompatibility, we applied a sequence-specific polymerase chain reaction (PCR-SSP). We were able to determine the HPA-4 genotype of three infants at risk using amniotic fluid cells without the need for fetal blood sampling. The HPA-4 genotypes of amniotic fluid cells determined in this way were completely concordant with the genotype and phenotype of infants' venous blood samples obtained after delivery. Therefore, this technique is also convenient to a fetus at risk in the antenatal management of NATP induced by HPA-4 incompatibility.

Soybean agglutinin binding as a useful prognostic indicator in stomach cancer.

Soybean agglutinin (SBA)-binding carbohydrate expression was studied immunohistochemically in 353 primary gastric carcinomas. The overall rate of positive SBA staining was 23% (81/353), and positive SBA staining was significantly correlated with tumor size, macroscopic tumor type, depth of invasion, lymph node metastasis, and venous invasion (P < 0.05). Moreover, patients with tumors that were immunoreactive for SBA demonstrated significantly higher survival (P < 0.01). From these findings we conclude that careful follow-up and intense postoperative therapy are required for patients with gastric cancers that lack immunoreactivity for SBA.

An EDTA-associated anti-B agglutinin: the role of ionized calcium.

BACKGROUND: It is believed that EDTA-dependent panagglutination is associated with free carboxylic acids that support reactions of rare autoagglutinins. CASE REPORT: An ABO typing discrepancy occurred in an 88-year-old patient. The specificity of his autoagglutinin was demonstrated by panel cell study and absorption tests using normal donors' red cells or immunoadsorbents coated with A, B, or O substances. Inhibition assays were performed to determine whether the autoagglutinin was inhibited by ionized calcium or carboxylic acids. The autoagglutinin had anti-B specificity when tested in the presence of EDTA. It was neutralized by group B secretor saliva and adsorbed by crystalline silica coated with simple B substances with or without EDTA, although it was absorbed by group B red cells only in the presence of EDTA. The agglutinating activity was stronger at 25 degrees C (titer 64) than at 37 degrees C (titer 16) and was destroyed by treatment of the serum with dithiothreitol, which suggests that the autoagglutinin is IgM. This activity also appeared in the patient's serum after dialysis and in an eluate obtained after adsorption with simple B substances, and it was inhibited by the addition of CaCl2 at 0.5 mM or higher concentrations. This suggests that the agglutination is not dependent on EDTA but, rather, on the concentration of ionized calcium. The autoagglutinin failed to react with group B red cells treated with glutaraldehyde for 10 minutes. CONCLUSION: An anti-B autoagglutinin was shown to have caused an ABO typing discrepancy in the presence of EDTA. These results suggest that autoagglutination requires an environment with low levels of ionized calcium, but not the presence of carboxyl groups.

An improved technique for low anterior resection using a PDS endoloop.

We describe herein the results of performing a new technique of low anterior resection of the rectum using a PDS endoloop, on ten patients with rectal cancer. This technique involves first preparing the rectosigmoid colon with an anvil as in the conventional low anterior resection; then, after the stapler is inserted transanally, two endoloops are solid over the colon and rectum. The rectum is ligated by pushing the knot of the endoloop and a second knot is applied 2 cm proximal to the first. Finally, the rectum is cut and the stapler is closed and fired to make a circular end-to-end anastomosis. The level of the anastomosis ranged from 2.5 to 6 cm with a mean of 4.7 cm in the ten patients, only one of whom developed a minor anastomotic leakage postoperatively. Moreover, no patient has developed local recurrence or distant metastasis to date. In summary, this technique offers certain advantages that allow the operation to be done with more skill and safety in a narrow pelvis.

[Respiratory failure after surgery for esophageal cancer and hypercoagulability].

Operation of esophageal cancer accompanies a big surgical stress and postoperative pulmonary complications such as respiratory failure are observed at high frequencies. On the other hand, when a big stress is added to a body, a state of hypercoagulation in which blood coagulation mechanism is abnormally enhanced appears and this state is closely related to organ failures but has many unknown points. So, we examined 39 patients given excision of esophageal cancer with respect to occurrence of postoperative respiratory failures, participation of coagulopathy in aggravation and their degrees before and after the operation to find out the relationship between postoperative respiratory failure and the state of hypercoagulation. We gave a diagnosis of respiratory failure to the patients whose respiratory index exceeded 1.5 on the day after operation but did not show atelectasis or hydrothorax. As a result, respiratory failures were observed in 7 out of 39 cases (17.9%). When a risk score (RS) of postoperative respiratory failure was determined using multivalent analysis (quantification type II) with preoperative factors such as age and function of heart, lung, liver, and kidney as well as preoperative blood coagulation factors, RS = 2.87 (antithrombin-III (AT-III) less than 75%) +1.89 (age over 70 years) +1.78 (respiratory index over 0.15) +1.44 (serum albumin less than 3.0 mg/dl) +1.28 (cardiac index less than 3.0 l/min/m2) was obtained and a drop in preoperative AT-III was considered a risk for occurrence of post operative respiratory failure. In contrast, referring changes in postoperative value of blood coagulation factors, a distinct rise in fibrinogen (FBG) appeared in early stage after operation in the respiratory failure group compared to the control group (p < 0.01). Moreover, recovery in AT-III of blood coagulation factors and in plasminogen and antiplasmin of fibrinolytic factors was delayed (p < 0.05) indicating promotion of postoperative hypercoagulation state and delay in recovery of coagulation-fibrinolysis factors. However, participation of platelet factors was absent.

[Experimental studies on the influence of duodenal contents on the mucosa of gastric remnant].

Using the PCNA staining method, we examined the histopathological changes and cytokinetics on the gastric mucosa of the gastric remnant at the anastomosis 10, 20 and 50 weeks after gastrectomy. We conducted the distal gastrectomy in rats and divided them into three groups: Group 1, in which "a sham" operation was performed; Group 2, in which reflux of duodenum fluids was prevented by Roux-en Y anastomosis; and Group 3, in which reflux of duodenum fluids was allowed by gastro-jejunal anastomosis. Adenocarcinoma developed only at the anastomosis of the afferent loops in animals of Group 3 directly exposed to duodenum fluid. The PCNA labeling index at the anastomosis of the afferent loop was high from the early stages. In addition, labeling index tended to increase gradually, and PCNA positive cells were diffusely scattered, which indicated acceleration of cytosis. From these results, we concluded that the reflux of duodenum fluid was essential for the development of cancer in the remnant stomach. Moreover, cytosis was closely related to the development of epithelial cancer of the gastric mucosa at the anastomosis of the remnant stomach.

Pharmacological approaches to reduce perioperative transfusion requirements in the aged.

Although iron deficiency is undoubtedly the commonest cause of anaemia even in elderly people, the aetiology is not always clear owing to various underlying diseases. Correction of anaemia is sometimes needed before surgery. The use of drugs that may influence blood coagulation, such as aspirin (acetylsalicylic acid), should be checked. Perioperative allogenic blood transfusion can often be avoided by the use of autologous blood and improved surgical techniques. Autologous blood donations are preferable in cases of planned surgery. Epoetin (recombinant human erythropoietin) in combination with iron supplementation facilitates the donation of autologous blood, even in elderly patients. Another method of avoiding allogenic blood transfusion is the collection and reuse of the blood a patient sheds in operations. During and/or after surgery, many haemostatic agents are available. Moreover, recent developments in gene engineering have enabled the utilisation of recombinant cytokines and coagulation factors. Further work remains to be done to define the proper use of these agents.

Effect and safety of liposome-encapsulated hemoglobin neo red cells (NRCs) as a perfusate for total cardiopulmonary bypass.

We developed a liposome encapsulated hemoglobin named Neo Red Cells (NRC), NRC (1) readily circulates, (2) shows a high oxygen transport efficiency, and (3) has a strong capsule membrane. In this study, we evaluated the effect of NRC as a priming solution for total cardiopulmonary bypass (TCPB) using a dog. The TCPB was started after removal of 57.1-73.3% of autologous blood and continued for 7 hours. During TCPB using NRC, the vascular resistance (VR) decreased to 1/4 of VR when red blood cells (RBC) were used. This change suggests that NRC, the viscosity of which is lower than that of RBC, reduced the load on the circulation system. The oxygen volume delivered by NRC was higher than that delivered by RBC, resulting in a greater oxygen consumption with NRC. During TCPB using NRC, the serum LDH level was lower than that using RBC. So we concluded that NRC compensated for the reduction in the oxygen transport ability, which is a disadvantage of dilution TCPB, and further increased the circulation improving effect and anti-hemolytic effect, which are advantages of the procedure. It, thus, enhanced both the safety and effectiveness of dilution TCPB.