Tranilast inhibits the function of cancer-associated fibroblasts responsible for the induction of immune suppressor cell types.

Cancer-associated fibroblasts (CAFs) are the dominant stromal component in the tumour microenvironment (TME), playing critical roles in generation of pro-tumourigenic TME; however, their contribution to suppression of antitumour immune responses has not been fully understood. To elucidate the interaction between CAFs and immune suppressor cells, we examined whether inhibition of CAFs function would impair the induction of immune suppressor cell types in vitro. In this study, we applied an anti-allergic and antifibrotic agent tranilast, which is used clinically, and evaluated a potential of tranilast to serve as a CAFs inhibitor. CAFs that had been isolated from E.G7 or LLC1 tumour-bearing mice were cultured in the presence of tranilast, and thereafter, CAFs functions on the secretion of some soluble factors as well as the induction of immune suppressor cells were evaluated. As a result, tranilast inhibited the proliferation of CAFs and reduced the levels of stromal cell-derived factor-1, prostaglandin E2 and transforming growth factor-ß1 from CAFs in a dose-dependent manner. On the other hand, tranilast exerted no inhibitory effects on immune cells at doses under 100 µm. The induction of regulatory T cells and myeloid-derived suppressor cells from their progenitor cells was suppressed in the medium that CAFs had been cultured in the presence of tranilast; however, these findings were not observed when those progenitor cells were cultured in the medium containing tranilast alone. These data demonstrate that tranilast inhibits CAFs function, which is responsible for the induction of immune suppressor cells, and possesses a potential to serve as a specific CAFs inhibitor.

Imaging of sub-surface defect in CFRP laminate using A0-mode Lamb wave: Analytical, numerical and experimental studies.

Lamb wave propagation in the anisotropic material is characterized by the prominent directivity of wave energy transfer governed by the fiber direction. Due to this anisotropic behavior, it is difficult to define the location of defects by using the arriving time of reflected signals. In this article, A0-mode Lamb wave-based damage detection technique has been illustrated which can detect the overlapping region of incident and scattered wave in the vicinity of the finite defect region in CFRP composite plate-like structure. A 5-cycle Hanning windowed tone burst of 30 kHz has been allowed to propagate through a 2 mm thickness [0/90]4S CFRP plate with subsurface cylindrical defect. In the near field region of the defect, the incoming and reflected wave overlaps and the dynamic shear strains of the out-of-plane displacement evaluated consequently. A covariance matrix is developed consisting of the shear strains. The proposed technique can detect the overlapping regions by measuring the determinant of covariance matrix, thus the image of the defect can be reconstructed. In this article, the analytical model of the proposed wavelet-based technique for the subsurface cylindrical defect is discussed and their physical meanings are investigated through numerical and experimental studies in a cross-ply laminate.

Human cord blood cells transplanted into chronically damaged liver exhibit similar characteristics to functional hepatocytes.

Human umbilical cord blood (CB) cells have many advantages as a source for stem cell transplantation because of immaturity and availability. It has been reported that CB cells transplanted into an injured liver displayed hepatocyte-like phenotypes. However, there have been few studies to characterize CB-derived hepatocyte-like cells (HLCs). In this study, CB cells were transplanted into mice with 2 types of liver damage: transient and chronic damage. We analyzed the expression of hepatic differentiation markers in CB-derived HLCs. In the liver of NOD/SCID mice with transient damage, CB-derived HLCs were detected infrequently at 3 weeks after transplantation. In contrast, in the liver of SCID mice damaged chronically by a urokinase-type plasminogen activator transgene under the control of albumin promotor/enhancer (ALB-uPA/SCID mice), more human HLCs colonized the host liver compared with hosts with transiently damaged livers. The CB-derived HLCs in both the transiently and the chronically damaged liver expressed a few markers of human hepatocytes, whereas the transcripts related to mature hepatic functions, including cytochrome P450s, were detected only in the ALB-uPA/SCID mice. These data indicated that CB cells were able to display a similar phenotype to functional hepatocytes in the recipient liver with chronic damage. CB cells may represent a transplantable source for chronic decompensated liver disease.

Isolation of hepatocyte-like cells from mouse embryoid body cells.

We previously reported that embryoid body (EB) cells derived from embryonic stem (ES) cells are capable of differentiating into functional hepatocyte-like cells both in vitro and in vivo. Because transplantation of EB-derived cells into the liver via the spleen resulted in a low incidence of teratoma formation, purification of hepatocyte-like cells is required to prevent teratoma formation. The aim of this study was to purify hepatocyte-like cells from cultured EBs. For the isolation of hepatocyte-like cells, EBs cultured for 15 days were treated with trypsin-EDTA. The disaggregated cells were plated on a gelatin-coated dish as a monolayer. These cells were separated by Percoll gradient centrifugation, enriched by magnetic cell sorting, and purified by FACS. The purified hepatocyte-like cells in monolayer cultures were positive for immunostaining for albumin and expressed albumin mRNA, but not Oct3/4 mRNA. Transplantation of the purified hepatocyte-like cells derived from mouse ES cells might be an effective treatment for liver failure.

Beneficial effect of tetrahydrobiopterin on the survival of rats exposed to hepatic ischemia-reperfusion injury.

INTRODUCTION: The protective role of nitric oxide (NO) against hepatic ischemia-reperfusion (I/R) injury remains controversial. In this study we investigated the effect of tetrahydrobiopterin (BH4) on the survival of rats exposed to an hepatic I/R injury. METHODS: The rats were subjected to 100 minutes of 70% hepatic ischemia 30 minutes after administration of BH4 or saline. A specific inducible NO synthase (iNOS) blocker, 1400W, was used to evaluate endogenous iNOS. NOS protein measured the histological appearance of the liver by Western blotting, and survival was evaluated after reperfusion. RESULTS: The 1-week survival rate was 60% among the BH4 group and 10% for the saline group. The serum ALT and bilirubin values in the BH4 group were significantly lower than the saline group. Histological examination of the liver revealed only a small necrotic area in the BH4 group as opposed to massive necrosis and cell infiltration in the saline group. Injection of 1400W significantly decreased the prolongation of survival produced by BH4. CONCLUSIONS: BH4 significantly improved the survival rate, the histological findings, and the liver function, thereby reducing liver failure. Western blotting showed a higher level of iNOS protein in the BH4 group than the saline group, 1400W suppressed this effect of BH4. Taken together, these observations suggest that NO derived from reactions driven by BH4-induced iNOS exerts a protective effect against reperfusion injury.

Immunosuppressive effect of long-term drainage of thoracic duct on immunological memory in adult thymectomized rats.

Profound reduction of the recirculating lymphocyte pool using thoracic duct drainage (TDD), a method developed by Gowans et al, has been shown to be of limited immunosuppressive value when applied in experimental as well as in clinical settings across major histocompatibility antigen complex (MHC) differences. This limitation is due to the observation that animals, in particular mice, are normally not able to have the drainage last longer than 8 to 10 days. However, using a simple modification of TDD, we have established a long-term TDD method, ie, more than 20 days. Combining this long-term TDD with adult thymectomy, we have examined the life span of naive and memory T cells specific for the minor histocompatibility antigen H-Y in female lewis rats. Furthermore, we demonstrated that memory T cells specific for the H-Y antigen do not appear to be recirculating lymphocytes.

Comparison of the vector systems for gene transduction into rat dendritic cells and peritoneal exudate cells.

Specialized antigen-presenting cells (APC), known as dendritic cells (DC), play a pivotal role in initiating primary immune responses. It has been reported that several vector systems, including adenoviral vectors, retroviral vectors, Hemagglutinating Virus of Japan (HVJ)-related vectors, and electroporation, are able to transduce genes into mouse and human DC. This has not been achieved for rat DC. To our knowledge, there is no direct evidence to support the view that the currently used vector systems are able to transduce genes into mature DC. Because most, if not all, gene transfer studies investigating DC or DC-related cell populations are carried out using heterogeneous groups of cells, it is therefore very important to determine to what extent gene transduction occurs in rat DC, and also selected mature DC (CD161a+ fully mature DC). In this study, we provide evidence that none of 4 vector systems are able to transfer genes into fully mature rat DC, which are derived from bone marrow cells (BMC), driven by Flt3/Flk2 ligand and interleukin (IL)-6, and purified by CD161a. Nevertheless, the most efficient gene transduction was observed in the developing DC progenitor cells during the long-term culture of rat BMC, and its gene expression was successfully achieved after 2 weeks of culture only with a human immunodeficiency virus (HIV)-based lentiviral vector system. The most critical time point for lentiviral gene transduction was around the 7th day from the beginning of culture with lentiviral vectors. Rat peritoneal exudate cells (PEC) and another cell line (K562) were easily transducted by adenoviral vectors and lentiviral vectors.

Phenotype and functional identity of GM-CSF-independent dendritic cells generated by long-term propagation of DC progenitor cells in bone marrow cells and skin Langerhans cells.

Evidence is provided that dendritic cells (DC) generated by either long-term bone marrow cell (BMC) culture with Flt3L and interleukin-6 (IL-6), or after short-term BMC culture with granulocyte macrophage-colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), contain heterogeneous cell populations of admixed DC and Mphi, regardless of the cytokine source. By employing GM-CSF-independent culture systems with the aid of Flt3/Flk-2 ligand and IL-6 and phenotypic characterization of BMC-derived DC and skin Langerhans cells (LC), revealed similar phenotypes. Furthermore, CD103 (OX62), which is widely used for rat DC separation, was found to be insufficient to enrich DC, due to downregulation of the marker. In this regard, the most efficient selection of rat DC, was obtained by CD161a (NKR-P1A), a member of the C-type lectin family. Despite the phenotypic similarity with BMC-derived DC, the nucleus of LC showed a distinct morphology. A large population of DC generated by Flt3L/IL-6 from GM-CSF receptor-deficient mice by do not express NK1.1 (NKR-P1B and NKR-P1C). The profiles for BMC-derived DC were the same as for skin Langerhans cells.

Studies on the most efficient vector systems for gene transduction into dendritic cells.

Specialized antigen-presenting cells (APC), known as dendritic cells (DC), play a pivotal role in initiating primary immune responses. Several vector systems, including adenoviral vectors, retroviral vectors, hemagglutinating virus of Japan-related vectors, and the electroporation, have been shown to transduce genes into mouse and human but not rat DC. However, there is no direct evidence to support the view that the currently used vector systems are able to transduce genes into mature DC. Inasmuch as most, if not all, gene transfer studies investigating DC or DC-related cell populations are performed employing heterogeneous-groups of cells, it is therefore important to determine the extent to which gene transduction occurs in bona fide DC. In this study, we provide evidence that none of these vector systems are able to transfer genes into mature rat DC, which are derived from bone marrow cells (BMC), driven by Flt3/Flk2 ligand and IL-6, and purified with CD161a. Nevertheless, the most efficient gene transduction was observed with developing DC progenitor cells during long-term culture of rat BMC. Successful gene transfer was achieved after 2-week culture with an HIV-based lentiviral vector system.

Teratoma formation and hepatocyte differentiation in mouse liver transplanted with mouse embryonic stem cell-derived embryoid bodies.

We previously reported that mouse embryonic stem (ES) cells are capable of differentiating into hepatocytes in cultured embryoid bodies (EBs) and that hepatocytes generate in the recipient liver injected with cultured day-9 EB cells via spleen without the formation of a teratoma. Because ES cells frequently form teratomas in recipient mice, we investigated incidence of teratoma formation when day-9 EBs derived from ES cells were transplanted directly into the subcapsule of mouse liver. In contrast to injection of day-9 EB cells through the portal vein via the spleen, direct subcapsular injection of cultured day-9 EB cells into liver, and even of cultured day-15 EBs, resulted in an high incidence of teratoma in the liver. In teratomas of livers injected directly with day-15 EBs, hepatocytes were detected singly and in clusters. These results imply that undifferentiated cells capable of developing into teratomas exist in cultured EBs, and even in cultured day-15 EBs containing differentiated hepatocytes.

Trigger digits-associated carpal tunnel syndrome: relationship between carpal tunnel release and trigger digits.

Of 875 idiopathic carpal tunnel syndrome (CTS) cases, 101 (11.5%) required trigger digit release operations within three years before and/or after carpal tunnel release (CTR); these 101 cases were investigated, retrospectively. Trigger digit release (TDR) was performed most often after the CTR, especially within three months. Next most common was at the same time as the CTR. The TDR performance rate after CTR was 5.9%. The nerve conduction study (NCS) comparison between trigger digits-associated CTS and isolated CTS showed that pre-operative distal motor latency was significantly more delayed in trigger digits-associated CTS, while there was no evidence of any difference due to age or gender. The difference of operative method (open or endoscopic procedure) did not influence the incidence rate of trigger digits after the CTR. This study suggested that trigger digits-associated CTS has a previously developed wide-ranging narrowing of the flexor tendon sheath.

DNA synthesis in hepatocytes during liver allograft rejection in rats.

We have investigated DNA synthesis in hepatocytes after orthotopic liver transplantation in four rat combinations--DA into DA (isogeneic), DA into PVG (allogeneic, nonrejector), DA into BN (allogeneic, rejector), and DA into (BN X PVG)F1 (allogeneic, intermediate nonrejector). The methods used were assay of thymidine kinase activity in graft homogenate and staining of hepatocytes in liver sections by an antibromodeoxyuridine monoclonal antibody. Both demonstrated increased DNA synthesis in grafts in all combinations except isogeneic, and showed that its intensity and timing paralleled the occurrence of the rejection reaction. The significance of hepatocyte regeneration after liver grafting is discussed.

31P NMR assessment of orthotopic rat liver transplant viability. The effect of warm ischemia.

The relationship between NMR visible high energy phosphates and transplant outcome for the case of liver damage by warm ischemia was investigated. In vivo 31P nuclear magnetic resonance (NMR) spectroscopy of rat liver was performed before the induction of warm ischemia in the donor and 20 min after reestablishment of portal blood flow in the recipient. Pretransplant damage was varied by subjecting the livers to 0, 15, 30, or 60 min of warm ischemia prior to harvesting. In the controls (0 min warm ischemia), 4 of 4 rats survived transplantation (one week survival end-point) and the mean NTP recovery was 94 +/- 8%; 3 of 6 rats survived in the 15 min warm ischemia group. Mean NTP recovery was 77 +/- 20% in the 15 min survival subgroup and 32 +/- 20% in the nonsurvival subgroup. Of 6 rats, 1 survived in the 30 min group. NTP recovery was 44% for the 30 min survivor and 37 +/- 5% in the nonsurvival subgroup. Of 4 rats, 1 survived in the 60 min warm ischemia group. NTP recovery was 56% for the 60 min survivor and 28 +/- 7% in the nonsurvival subgroup. Overall, there was a significant difference between the mean NTP recovery of the survival and nonsurvival subgroups (78 +/- 21% versus 31 +/- 18%, P < 0.001). The dividing line between the survival and nonsurvival groups was approximately 50% NTP recovery. Of 9 rats with liver NTP recovery greater than 50%, 8 survived while 10 of 11 rats with less than 50% recovery died. NMR visible NTP recovery 20 min after the reestablishment of portal blood flow was a good indicator of transplant outcome in the case of rat liver damage by warm ischemia.

Hepatic microcirculatory changes after reperfusion in fatty and normal liver transplantation in the rat.

The hepatic microcirculation in fatty and normal liver grafts in ACI rats was investigated using in vivo microscopy. Six groups were studied. They were: normal and fatty control livers (sham operated), 6-hr cold University of Wisconsin solution (UW)-preserved fatty and normal liver grafts (survival conditions, fatty and normal liver grafts), 18-hr cold UW-preserved fatty livers (nonsurvival conditions, fatty liver graft), and 24-hr cold UW-preserved normal livers (nonsurvival conditions, normal liver grafts). Fatty livers in all groups were found to have narrow and irregular sinusoids with blood cell adhesions to endothelial cells. The number of adhesions increased as the preservation time increased. Sinusoidal blood flow area decreased as the preservation time increased and was correlated with survival in both normal and fatty liver grafts. The phagocytic activity of Kupffer cells (corrected for flow) increased as the preservation time increased. The phagocytic Kupffer cell activity of the 18-hr preserved fatty liver group was greater than the activity of any other group. These features may cause liver cell death and contribute to primary graft nonfunction after transplantation of a fatty liver.

A rat fatty liver transplant model.

A rat model of fatty liver transplantation has been developed to study primary nonfunction in fatty liver grafts. ACI rats were fed with a diet deficient in choline and methionine for 7, 14, 28, and 42 days. Fat content in the pretransplant livers was examined by gas chromatography and histology. The main constituent of the fatty droplets was determined to be triglyceride. The triglyceride concentration reached a maximum by day 14 and remained constant for an additional 28 days. Histology revealed an absence of necrosis in 14- and 28-day fatty livers but scattered hepatocytic necrosis and inflammation in 42-day fatty livers. After being given cold (UW stored, 4 degrees C) or warm (37 degrees C) ischemia, the fatty liver was orthotopically transplanted into normal ACI rats. The one-week survival of fatty liver grafts after 6, 12, 18, and 24 hr cold preservation was 5/5, 5/6, 3/8, 0/6 for 14-day fatty liver and 5/5, 4/6, 0/8, 0/6 for 42-day fatty livers. The survival of normal liver grafts was 5/5, 6/6, 5/9, 2/8, respectively. Increased survival rate was correlated with the absence of hepatocytic necrosis. The survival after 15 and 30 min warm ischemia prior to transplant was 5/5, 2/6 for normal liver grafts and 4/7, 0/6 for 28-day fatty liver graft, respectively. Fatty livers were less resistant to damage induced by cold or warm ischemia.

Influences of chronic tobacco smoke inhalation on aging and oxidant-antioxidant balance in the senescence-accelerated mouse (SAM)-P/2.

We studied the influences of chronic tobacco exposure on aging and oxidant-antioxidant balance in two different strains of mice, hitherto called SAM (senescence-accelerated mice). One is a senescence-prone strain, "SAM-P/2," and another is a senescence-resistant strain, "SAM-R/1." We used 100 male mice--20 young (12 weeks of age) mice and 30 mature (24 weeks of age) mice from each strain. Half of each series were housed in a Hamburg II machine and exposed to tobacco smoke inhalation for five weeks. The result was that fewer of the mature SAM-P/2 survived compared with the mature SAM-R/1 after chronic tobacco inhalation. The grading of senility in the mature SAM-P/2 was also significantly higher than that in the mature SAM-R/1. The reduction of glutathione contents of blood and liver after tobacco exposure in the mature SAM-P/2 was greater than that in the young SAM-P/2 and the mature SAM-R/1. Moreover, oxygen radical generation of total blood cells stimulated by phorbol-myristate-acetate or opsonized zymosan showed a greater increase in the mature SAM-P/2 compared to the young SAM-P/2 and the mature SAM-R/1. These results indicate that the senescence-prone strain (SAM-P/2) was more susceptible to tobacco smoke exposure than the resistant strain (SAM-R/1). The impaired oxidant-antioxidant balance in the SAM-P/2 may therefore contribute to the process of senescence acceleration.

Analysis of connective tissue macromolecular components in Ishibashi rat skin: role of collagen and elastin in cutaneous aging.

The Ishibashi (IS) rat, established from cross-breeding between Wistar and wild rats, has a unique skin appearance, with wrinklings and furrows indicative of skin aging appearing at the age of 12 weeks. To understand the underlying mechanism of the formation of wrinkles, macromolecular components of connective tissue, collagen and elastin, in the young (5-6-week-old) and the aged (23-30-week-old) IS rat skins were biochemically analyzed. Hydroxyproline and isodesmosine contents in the aged IS rats were reduced 22% (P < 0.05) and 37% (P < 0.05) compared to the young rats, whereas no significant differences in the contents of both macromolecules in control Sprague-Dawley (SD) rats were seen. The relative content of type III collagen was unaltered between the young and aged skins of both IS and SD rats. A relative decrease in the intact elastin molecule (65 kDa) and a relative increase in the elastin fragments with lower molecular weights were observed in the aged IS rat skin by immunoblotting method. These results indicate that the reduction in collagen and elastin contents and increased degradation of elastin molecules in the aged IS rat skin could be related to the formation of wrinkles. Thus, the IS rat may provide a useful model for the study of skin aging.

Hepatic resection under the intermittent selective portal branch occlusion by balloon catheter.

BACKGROUND: In hepatic resection, it is important to control intrahepatic blood flow to minimize blood loss. Intermittent and selective vascular occlusion, if possible, are advisable. STUDY DESIGN: For this purpose, we created the double balloon catheter, which when introduced into a lobar or a smaller branch of the intrahepatic portal vein through a branch of the ileocolic vein, made it possible to occlude these branches temporarily during hepatic resection. The small balloon located at the tip of the catheter made it easy to introduce the catheter to the portal branch selectively, under the guidance of ultrasonography. Another balloon was inflated intermittently to occlude selective portal blood flow. Using this technique, hepatic resection was achieved in 18 consecutive patients: 13 with hepatocellular carcinomas (11 with cirrhosis, two with chronic hepatitis), one with cholangiocellular carcinoma, three with metastatic carcinomas, and one with intrahepatic calculi. RESULTS: In these cases, 19 hepatic resections were performed; two left hepatectomies, one extended right hepatectomy, one right hepatectomy, six segmentectomies, eight subsegmentectomies, and one partial hepatectomy. In each case, well demarcated hepatic tissue delineated by ischemic change was removed with minimal bleeding and little impairment to the residual hepatic tissue, resulting in a good postoperative course. CONCLUSIONS: This double balloon catheter can replace the dissection of the hepatoduodenal ligament for hepatic resection, which causes bleeding, especially in patients with cirrhosis, and results in less cell injury of the residual hepatic parenchyma.

Laparoscopic enucleation of an insulinoma of the pancreas tail.

A 29 year-old woman with a tumor of the pancreatic tail was referred to our institute. The tumor was confirmed to be a solitary benign insulinoma that protruded from the pancreas and was distant from the main pancreatic duct. A laparoscopic enucleation was performed with Laparoscopic Coagulating Shears (LCS). The postoperative course was uneventful. The laparoscopic enucleation for benign pancreatic tumor was thought to be a feasible procedure when the appropriate instruments were used.

Hand-assisted laparoscopic hepatic resection.

Thanks to recent advances, performance of liver resection is now possible using laparoscopic procedures. However, still there are some difficulties to overcome. The hand-assisted method lends safety and reliability to the laparoscopic procedure. A 54-year-old man diagnosed with hepatocellular carcinoma (HCC) was referred for hepatectomy. Angiography with computed tomography (CT) scans revealed a 2-cm hepatocellular carcinoma (HCC) at segment V, close to the gallbladder. A hand-assisted laparoscopic hepatic resection was performed. Four 10-mm trocars, one for wall lifting and three for working, were placed in the upper abdomen. A small incision was added at the right side of umbilicus, and the operator's left hand was inserted through it. A microwave tissue coagulator and laparoscopic ultrasonic dissector were used for liver resection. Total operation time was 162 min; blood loss was 20 g. The postoperative course was uneventful, and the postoperative hospital stay was 7 days. We thus demonstrated that laparoscopic liver resection is safer and easier when the hand of the operator can be inserted into the abdomen. The small incision does not greatly diminish the benefits that accrue from minimally invasive laparoscopic surgery. The hand-assisted procedure allows better access to the tumor. In addition, hand assistance restores the sense of touch to the operator and is an effective means of controlling sudden and unexpected bleeding.