Functional and phenotypic analysis of tumor-infiltrating lymphocytes isolated from human primary and metastatic liver tumors and cultured in recombinant interleukin-2

Tumor-infiltrating lymphocytes (TIL) were isolated from 40 of 51 consecutive human liver tumor samples (primary hepatocellular carcinoma, 16 of 18; metastatic, 23 of 29; benign, one of four). Functional and phenotypic characteristics of fresh and recombinant interleukin-2 (rIL-2)-expanded TIL were evaluated. The expansion of TIL from hepatic tumors in the presence of 1000 units/ml of rIL-2 was possible in 60% of cases. In comparison to TIL from metastatic liver tumors, TIL obtained from primary liver tumors expanded faster and better in rIL-2 cultures. Expanded TIL from primary tumors had significantly higher cytotoxicity against K562 targets, but not Raji targets, than those from metastatic tumors. Cytotoxicity against fresh autologous tumor targets was detected in seven of eight cultures tested. TIL from primary tumors retained antitumor reactivity significantly longer in culture. The optimal in vitro cytotoxicity was achieved between days 20 and 60 of culture in the presence of rIL-2. Antitumor activity was associated with the increase in these TIL cultures of a cell population expressing the Leu19 antigen with or without the CD3 antigen. The frequency of the CD3+Leu19+ population showed a bimodal distribution during culture: the first peak of CD3+Leu19+ cells occurred between days 30 and 60 and was associated with the increased antitumor activity; the second peak occurred after day 60 and was not associated with activity. These findings demonstrate that TIL from most human hepatic tumors can be successfully isolated, cultured in rIL-2, and enriched in Leu19+ effectors. In addition, these TIL upon IL-2 activation in vitro are capable of lysing fresh autologous and/or allogeneic tumor targets.     

Case of ruptured gastroduodenal arterial pseudoaneurysm with penetration to the duodenum, successfully treated with transcatheter arterial embolization]

A 77-year-old man, who underwent segmental pancreatectomy for intraductal papillary mucinous adenoma in 2001, was referred to our hospital with complaints of hematemesis and melena on January, 2004. Emergency upper gastrointestinal endoscopy showed a pulsating submucosal protrusion in the duodenal bulb, which was identified as a gastroduodenal arterial aneurysm measuring 1.5cm on abdominal CT imaging. Transcatheter arterial embolization of the aneurysm with metallic coils was successfully performed. Periodically repeated endoscopic examination has revealed the coils protruding into the duodenal lumen without any serious complication.     

Clinico-pathological study of collagen-related pulmonary lesions in cases of open lung biopsy]

We studied the clinico-pathological correlation of collagen disease-related pulmonary lesions to examine the pathological and radiological features of collagen lung, and the effect of steroid therapy. Ten open lung biopsy cases were examined; 4 male, and 6 female. The mean age was 55 years old. Seven cases developed pulmonary shadows after the diagnosis of collagen disease, and 3 cases showed pulmonary shadow prior to diagnosis. Pathologically, 6 cases proved to be bronchiolitis obliterans organizing pneumonia (BOOP), 3 cases were chronic interstitial pneumonia (UIP), and 1 case was acute interstitial pneumonia. All cases had inflammatory thickening of the interstitium involving the pleura, bronchial wall, and perivascular connective tissue. Half of the cases had bronchiolar inflammatory lesions. Radiologically BOOP cases showed either localized ground glass shadows, or diffuse reticulonodular shadows predominantly in the lower lung fields with shrinkage of affected areas. UIP cases showed reticulonodular shadows, and active UIP cases showed overlapping ground glass shadows. Steroids were administered in cases of BOOP and active UIP, and all cases showed improvement. We consider that open lung biopsy is of use in the diagnosis of some cases and in assessing whether steroid therapy is indicated.     

Ross procedure in an infant weighing 4.5 kg

A 6 month-old male infant (weight: 4.5 kg) with congenital aortic stenosis underwent aortic valve replacement with a pulmonary autograft (Ross procedure). The right ventricular outflow tract (RVOT) was reconstructed with a polytetrafluoroethylene (PTFE)-valved equine pericardial conduit. At the age of 5, re-RVOT reconstruction with an equine pericardial patch bearing a PTFE monocusp was required because of severe pulmonary stenosis resistant to 2 attempts of percutaneous transluminal pulmonary valvotomy. Currently, at the age of 8, the degree of aortic regurgitation is trivial and the pulmonary autograft is free of functional deterioration despite somatic growth.     

Systemic hemodynamics and oxygen transport and consumption during the anhepatic phase of orthotopic liver transplantation in dogs

During the anhepatic phase of orthotopic liver transplantation, the veno-venous bypass has been used to keep systemic hemodynamics (SHD) in a stable condition. In this study, changes of SHD and oxygen transport and consumption (OTC) during the anhepatic phase used the passive bypass (PB) with Anthron bypass tubes were compared with those used the pump-driven bypass (PDB) with a centrifugal pump in mongrel dogs. Moreover the effects of the increased instillation rate and administration of dobutamine on SHD and OTC were evaluated. The portal venous and inferior vena caval pressure were increased in PB group, but not in PDB group. Whenever PB or PDB was used, cardiac index and oxygen consumption were markedly decreased caused by hypovolemia. In PDB group trebled the instillation rate, SHD and oxygen consumption were not improved. These results suggested that the primarily cardiovascular depression during the anhepatic phase was related to the disturbance of SHD. When dobutamine was administered and the instillation rate was trebled in PDB group, SHD and OTC were maintained in a favorable state. It is concluded that PDB, administration of dobutamine and sufficient instillation are advantageous to maintain systemic hemodynamic stability during the anhepatic phase of orthotopic liver transplantation.     

Investigation of the anti-complement agents, FUT-175 and K76COOH, in discordant xenotransplantation

Abstract: We examined whether hyperacute rejection (HAR) of a discordant xenograft in a nonhuman primate model could be inhibited by the anticomplement agents, FUT-175 (FUT) and K76COOH (K76). The inhibitory effect of FUT and K76 on baboon sera was studied in vitro by i) complement-mediated hemolysis of sheep erythrocytes (by measuring serum CH50) and ii) cytotoxicity to cultured pig kidney (PK15) cells. The in vivo administration of FUT (at 0.2–25 mg/kg/h i.v. continuously) and K76 (50 mg/kg i.v. bolus) allowed evaluation of the serum levels of these drugs. Both FUT and K76 inhibited serum CH50 in a concentration-dependent manner. An enhanced effect was obtained by combining K76 with FUT therapy. High concentrations of FUT (>10^-4 M) and K76 (>10³ μxg/ml) were necessary to suppress serum CH50 to <5% of the normal level. However, PK15 cytotoxicity remained at >50% in the presence of i) 10^-4 M of FUT, ii) 10³ μg/ml of K76, and iii) 10^-6 M of FUT + 10³ μg/ml of K76. Pig heart transplantation (HTX) was performed in two baboons receiving FUT (1 mg/kg/h i.v. continuously) and K76 (at 200 mg/kg ×1 or 400 mg/kg + 200 mg/kg × 2 i.v, respectively). Cytotoxicity of the serum to PK15 cells at the time of HTX showed 39% and 1% cell death, respectively, in these two baboons, and the CH50 level was 1% (of control level) and 0%, respectively. Graft survival was 4.5 hours and 10 hours (with death of the baboon), respectively (compared with a mean of 29 minutes in control experiments). Both excised grafts showed typical features of hyperacute rejection. Immunopathological studies revealed deposition of C1q, C3d, C6, properdin, and Factor B, demonstrating that complement activation was not fully inhibited by FUT and K76. We conclude that i) FUT and K76 are indeed potent complement inhibitors, ii) the dosages of FUT and K76 necessary to suppress complement-mediated injury cannot be extrapolated from previously reported data obtained from serum CH50 levels, and iii) higher (possibly toxic) dosages will be required to inhibit complement activation completely. It seems unlikely that HAR will be prevented by these drugs alone, although they may be beneficial when combined with other forms of therapy.     

"Spontaneous" pneumocephalus associated with aerobic bacteremia

Three cases of "spontaneous" pneumocephalus suspected to have resulted from aerobic bacteremia caused by Enterobacter cloacae, Escherichia coli, and Klebsiella aerogenes are reported. In two cases, the E. cloacae and K. aerogenes were isolated from the cerebrospinal fluid. These cases were characterized by a rapid accumulation of air, without niveau, in the subarachnoid space and ventricles.     

Fine structure of soft and hard tissues involved in eye migration in metamorphosing Japanese flounder (Paralichthys olivaceus)

The body of a Japanese flounder (Paralichthys olivaceus) changes from a symmetrical to an asymmetrical form during metamorphosis. To obtain detailed information on the mechanisms of the migration of the right eye to the left side, soft and hard tissues in the head of larval flounders were examined using transmission electron microscopy (TEM). Retrorbital vesicles (Rvs) are pairs of sac-like structures under the eyes. It has been suggested that the asymmetrical development of Rvs, with the right (blind) one being bigger than the left, is the driving force behind eye migration. The present study revealed that the ultrastructure of the Rv sheath is quite similar to that of a lymphatic capillary. Thus, it is possible that the Rv is a part of the lymph system, and is probably related to the secondary vascular system in teleosts. If we assume that the Rv sheath has a high permeability to liquid, similar to lymphatic capillaries, it is not plausible that the active expansion of the Rv pushes the eyeball. On the other hand, the pseudomesial bar (Pb) is a bone that is unique to flounders and is present only on the right (blind) side. At the beginning of eye migration, an aggregation of fibroblast-like cells is observed in the dermis under the right eye, where the Pb will subsequently be formed. These cells have a well-developed rough endoplasmic reticulum (rER) and mitochondria, and are probably responsible for formation of the thick layers of collagen fibrils around them. Since it is unlikely that the active expansion of the Rv causes eye migration, the role played by the Pb and its rudiment becomes more significant in right eye migration in the Japanese flounder becomes more significant.     

Relationship between glucose transporter and changes in the absorptive system in small intestinal absorptive cells during the weaning process

In the present study, we investigated the changes in the localization of the glucose transporter GLUT2 and the fructose transporter GLUT5 in small intestinal absorptive cells during postnatal development, especially during the weaning period, using immunohistochemistry and confocal laser scanning microscopy. In the jejunum, GLUT2 was observed within the apical and basolateral membrane domain of absorptive cells, especially in the middle part of the villi. In the suckling rat ileum, GLUT2 was found within the apical and basolateral membrane domain of absorptive cells, but after 18 or 19 days after birth, GLUT2 was found mainly within the apical membrane domain. GLUT5 was observed within the apical membrane domain of absorptive cells in the suckling rat jejunum. In the 18- or 19-day-old rat jejunum, GLUT5 was localized within the apical and basolateral membrane domain of absorptive cells in the lower part of the villi, but after weaning, GLUT5 was found within the apical and basolateral membrane domain of absorptive cells throughout the entire villi. In the suckling rat ileum, there was little GLUT5 in the absorptive cells. In the 18- or 19-day-old rat ileum, GLUT5 was localized within the apical membrane domain of absorptive cells in the lower part of the villi, but after weaning, GLUT5 was observed mainly within the apical membrane domain of absorptive cells throughout the entire villi. These results suggest that the localization of glucose transporters corresponds with a shift from neonatal-suckling to weaned absorptive cells during postnatal development.     

Development of the articular cavity in the rat temporomandibular joint with special reference to the behavior of endothelial cells and macrophages

Previous developmental studies on the temporomandibular joint (TMJ) have proposed several hypotheses on the formation of its articular cavity. However, detailed information is meager. The present study examined the formation process of the articular cavity in the rat TMJ by immunocytochemistry for CD31, RECA-1, and ED1, which are useful cellular markers for endothelial cells and monocyte/macrophage lineages, respectively. The upper articular cavity formation had begun by embryonic day 21 (E21) and was completed at postnatal day 1 (P1) in advance of the lower cavitation; the latter took place from P1 to P3. The occurrence and distribution pattern of the CD31-, RECA-1-, and ED1-positive cells differed between the upper and lower articular cavity-forming areas: the ED1-positive cells exclusively occurred in the area of the prospective upper articular cavity prior to its formation, while no ED1-positive cell appeared in the lower cavity-forming area. In contrast, the CD31- and RECA-1-positive endothelial cells were restricted to the lower cavity-forming area (never the prospective upper cavity) at E19 and diminished thereafter. Throughout the cavity formation, we failed to find any apoptotic cells in the cavity formation area, indicating no involvement of apoptosis in the cavity formation in TMJ. The present findings on the behaviors of endothelial cells and ED1-positive cells show a possibility of different mechanism in the cavity formation between the upper and lower articular cavities in the rat TMJ. The appearance of ED1-reactive cells and temporal vascularization may play crucial roles in the upper and lower articular cavity formation, respectively.