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In order to clarify the effect of the Prandtl number on the heat transfer characteristics of dimples, large eddy simulations of fluid flow and heat transfer in a dimpled channel are conducted at Reynolds numbers (Reb) ranging from 1000 to 10,600 and Prandtl numbers (Pr) of 0.2, 0.7, and 3.0. Dimples having a print diameter of 2δ and a depth of 0.4δ, where δ is the channel half height, are arranged in a staggered pattern on one side-wall of the channel. As the Reynolds number decreases within a turbulent flow regime, the turbulent kinematic energy and the Reynolds shear stress decrease, whereas the absolute value of the temperature gradient in the wall-normal direction maintains a significantly large value at the downstream region above the dimple cavity. Moreover, the absolute value of the temperature gradient in the area becomes larger as the Prandtl number increases due to the low thermal diffusivity of the fluid. As a result, the production terms of the streamwise and vertical components of turbulent heat flux are broadly increased over the dimple cavity for R e b = 2000 and P r = 3.0 , and under this condition, the ratio of the local Nusselt number on the dimpled surface to that for a flat-channel flow is most increased at both the downstream region inside a dimple and the flat area between dimples, as compared to the other conditions considered herein. The thermal performance parameter, which is the ratio of the Nusselt number to the pressure drop as compared to the ratio for a flat-channel flow, takes a maximum value of 1.22 for this condition.

Electrical properties in dc-sputtered amorphous InGaZnO4films have been investigated using the thermopower and resistivity measurements. The amorphous InGaZnO4films shown-type conduction at room temperature, and electrical activation energy of ~0.47 eV. The resistivity and Seebeck coefficient at room temperature were ~2×104Ωm and 1.5 mV/K for as-deposited sample. The resistivity and Seebeck coefficient decreased to ~2×100Ω·m and 2.7 mV/K when the sample was annealed at 670770 K. Slow response of photocurrent was also observed. Partial crystallization and structural change cause the improvement of electron transport with annealing treatments.

Optical absorption in amorphous InGaZnO4films was examined by optical transmittance and photothermal spectroscopy using individual cantilever. The amorphous InGaZnO4films have optical bandgap of ~3.5 eV, and Urbach tail absorption and mid-gap absorption at 2.0-3.0 eV. Mid-gap absorption was reduced by thermal annealing at 300 °C in an atmosphere. This observation indicates that heat-induced structural change causes reduction of gap states in amorphous InGaZnO4films.

We aimed to reveal the regulatory function of macrophage scavenger receptor-A (MSR-A) in proinflammatory cytokine production by macrophages stimulated with mycobacterial cord factor (CF). By the culture with CF, MSR-A (+/+) alveolar macrophages and Kupffer cells produced TNF-alpha/MIP-1alpha in a time- and dose-dependent manner. However, the amounts of cytokines produced by them were much less compared to those produced by MSR-A (-/-) macrophages. Consistent with this, treatment of MSR-A (+/+) macrophages with anti-MSR-A antibody increased TNF-alpha production. Binding of CF to MSR-A was demonstrated by measuring the binding affinity. These results indicate that CF binds MSR-A, and MSR-A down-regulates TNF-alpha/MIP-1alpha production by activated macrophages, suggesting the role of this receptor in suppression of excessive inflammatory responses during mycobacterial infection.

In the portal tract of the regenerating liver after partial hepatectomy, vascular and bile ductular remodeling takes place in response to the portal hyperdynamic state and parenchymal hyperplasia. In order to reveal phenotypical changes in the portal fibroblasts, we immunohistochemically investigated neural cell adhesion molecules (NCAM) and alpha smooth muscle actin (alphaSMA) expression and the ultrastructural changes in them during liver regeneration. In the control rat liver, portal fibroblasts were negative for both NCAM and alphaSMA. They became positive for both markers two days after partial hepatectomy, increased in staining intensity, reached a maximum at three to four days, then decreased, being still clearly positive at 14 days. Under an electron microscope, portal fibroblasts from the regenerating liver had larger amounts of cytoplasm and rough endoplasmic reticulum than those from the control liver; thus they might be activated. Additionally, periportal hepatic stellate cells in the regenerating liver were activated with alphaSMA, but without NCAM. The present study has demonstrated that portal fibroblasts express NCAM and alphaSMA in the regenerating liver after partial hepatectomy via transformation into myofibroblasts following reconstruction of the portal tracts.

Aim: To clarify whether hepatocellular carcinoma (HCC) originates from hepatic progenitor cells and whether there is any correlation with the clinicopathologic factors of HCC, we reviewed 217 resected HCC specimens. Methods: Immunohistochemical examination of cytokeratin (CK) 7, CK19, CD34, and CD117 (c-KIT) was performed. Overexpression of CK7 and CK19 indicates differentiation from cholangiocellular and hepatic progenitor cells, while overexpression of CD34 and CD117 indicates hepatic stem cells. Fresh specimens were obtained from 20 HCC patients for mutation of the c-KIT gene. Results: CK7, CK19, and CD117 were positive in 41, 9.7, and 0.9% of the HCC specimens, respectively, and CD34 was never positive. None of the fresh HCC specimens demonstrated a c-KIT mutation. CK19 positivity was significantly correlated with a positive hepatitis B core antibody, and with poor survival outcome, and tended to correlate with poor histologic differentiation. Conclusion: These results suggest that: (i) about 10% of HCCs with typical histologic features originate from an intermediate hepatic progenitor cell, such as the canal of Hering and oval cells in the rat, or acquire the characteristics of cholangiocellular epithelium by metaplasia; (ii) HCC with typical histologic features rarely originates from hepatic stem cells, and (iii) patients with CK19-positive HCC have a poor prognosis.

Background The prognosis of hepatocellular carcinoma originating in or mainly involving the caudate lobe (caudate HCC) is generally poor. We reviewed the clinicopathologic findings of patients who underwent liver resection of caudate HCC and correlated the outcome with the surgical strategy. Methods Records of 402 patients who underwent liver resection for HCC were reviewed. The patients were divided into 2 groups. One group consisted of 15 patients who underwent liver resection for caudate HCC. The other group included 387 patients with HCC in a site other than the caudate lobe. Results Anatomic resection of Couinaud segment I or IX (a partial caudate lobectomy), conforming to portal anatomy, was performed in 13 patients with caudate HCC, and segmentectomies of segments I and IX (a total caudate lobectomy) were performed in 2 patients with caudate HCC. The incidence of postoperative complications was similar in the caudate HCC group and HCC in other sites group, with no operative deaths in the caudate HCC group. Tumor-free survival and cumulative survival were similar in the 2 groups. However, among patients with caudate HCC, tumor-free and cumulative survival were lower in patients with than without microscopic portal venous involvement ( P Conclusions Partial caudate lobectomy (anatomic resection of segment I or IX) along the portal system is an appropriate procedure for caudate HCC, especially in patients with impaired liver function or a small HCC. Patients with caudate HCC who have microscopic portal venous involvement may require adjuvant therapy as early recurrence is likely.

Activated hepatic stellate cells (HSCs) produce cyclooxygenase-2 (COX-2) protein to induce vascular endothelial growth factor (VEGF) production that participates in angiogenesis in injured liver. To reveal the unknown regulatory mechanism, we used hypoxic atmosphere mimicking injured-tissue microenvironment to induce VEGF expression in a rat hepatic stellate cell line (T6-HSCs). The present study showed that hypoxia up-regulated the protein levels of COX-2 and hypoxia-inducible factor-1-alpha (HIF-1alpha), but rapidly effected degradation of von Hippel-Lindau (vHL) protein. As a result, expression of VEGF in HSCs was markedly elevated; and pretreatment with COX-2 inhibitors (nimesulide or indomethacin) could significantly ameliorate the angiogenic event. Collectively, hypoxic HSCs increased accumulation of HIF-1alpha protein and induced VEGF expression in a time-dependent manner. Inhibition of COX-2 activities would prevent vHL protein from degradation and suppress HIF-1alpha up-regulation. Thus, vHL/HIF-1alpha has a regulatory role in COX-2-mediated VEGF production in hypoxic stellate cells in injured liver.

To better understand how benign hepatocellular tumors acquire nutrient vessels, we investigated the angioarchitecture of the smallest vessels in hepatocellular adenoma (HA) and focal nodular hyperplasia (FNH). In biopsy or resection specimens, microscopic angioarchitecture was characterized in terms of intratumoral fibrous septa and portal tract. Immunohistochemistry for CD34, factor-VIII-related antigen (F-VIII), type IV collagen (collagen IV), α-smooth muscle actin (α-SMA), and CD68 was performed. HA had no portal tract in fibrous septa although FNH showed portal tract in fibrous septa. The smallest vessels in HA stained diffusely for CD34, and arterial segments of the smallest vessels were stained for F-VIII, and collagen IV. Pericyte surrounding the smallest vessels in HA were stained for α-SMA. The smallest vessels in all but one FNH specimen stained for CD34, F-VIII, collagen IV, and α-SMA along portal tract or the artery. Both HA and FNH stained for CD68, as did normal liver. The results indicate that the smallest vessels in HA and FNH are incomplete capillaries, that have both characters of sinusoid and capillaries morphologically and phenotypically, i.e., capillarized sinusoid as in chronic hepatitis or early well-differentiated hepatocellular carcinoma. Benign hepatocellular tumors such as HA and FNH would acquire new vessels gradually by sinusoidal capillarization, not by rapid arterial angiogenesis.

Summary Background As seen in atopic dermatitis, allergic diseases often produce lesions both in the gastrointestinal tract and the skin, suggesting the involvement of an immunological relationship between the two organs in the pathogenesis. Objectives To study the role of gastric and epidermal Langerhans cells (LCs) in the sensitization and elicitation phases, respectively, of cutaneous delayed-type hypersensitivity (DTH) reactions to intragastrically administered hapten. Methods BALB/c mice, which were subjected to intragastric administration of trinitrochlorobenzene 5 days previously, received an elicitative challenge of the same hapten to the ear skin. Sections of the ear were immunostained for CD4 and CD8. Epidermal sheets of the ear and epithelial sheets of the forestomach were immunostained for I-A and observed under a confocal laser scanning microscope. Results Cutaneous DTH reactions were induced in mice, as demonstrated by an increase in ear thickness and a prominent infiltration of CD4+ and CD8+ lymphocytes at 24–36 h after the elicitative challenge. In the elicitation phase, epidermal LCs showed a significant increase in size, indicating in vivo activation, at 24 h. In the sensitization phase, gastric LCs increased in size at 2 h, became round at 6 h, and decreased in number at 24 h, possibly representing the sequential events of LC activation and migration from the epithelium. Conclusions The present study demonstrated that gastric LCs and epidermal LCs were activated in vivo in the sensitization and elicitation phases, respectively, of cutaneous DTH reactions in orally sensitized mice.

Apoptosis of T cells contributes to the immune homeostasis in inflamed organs. A prominent T-cell infiltration is usually seen in human chronic active hepatitis, being associated with liver fibrosis. In order to demonstrate T-cell apoptosis in the hepatic fibrotic tissue, we induced T-cell infiltration in the fibrotic liver of the rat by injecting concanavalin A (Con A), a T-cell mitogen. Lymphocytes increased in number with a peak at 1 day, preferentially distributing in the fibrotic tissue rather than the parenchyma. They consisted of CD4-positive and CD8-positive cells, and gave the feature of lymphoblasts. Double staining for CD3 and TUNEL demonstrated that T cells underwent apoptosis. Apoptotic cells were more frequent in the fibrotic livers than the normal livers, and were spatially associated with alpha-smooth muscle actin-positive myofibroblast-like cells that possibly derived from hepatic stellate cells (HSCs) and portal fibroblasts through activation. In vitro experiments demonstrated that lymphocyte apoptosis was more frequently induced in the co-culture of Con A-activated splenic T cells/activated HSCs compared to that induced in activated T cells/quiescent HSCs or resting T cells/activated HSCs. The present results indicate that T cells which have extravasated and infiltrated the hepatic fibrotic tissue undergo apoptosis probably through an interaction with myofibroblast-like cells, suggesting the regulatory role of the latter cells in T-cell accumulation in the fibrotic liver.

Granulomatous colitis is a major entity of human intestinal diseases. We previously reported that intravenous injection of mycobacterial cord factor (CF), a potent macrophage activator, induced pulmonary granulomas in mice with enhanced production of Th1 cytokines and chemokines. In this study we made a murine model of granulomatous colitis by intramural injection of CF. A single dose of 300 microg CF was injected into the wall of the rat and mouse colon in the form of liposomes. After 1 week granulomas developed at the injection site, extending from the subserosa to the lamina propria, and persisted for longer than 6 weeks. They were composed mainly of ED1-positive macrophages, which often underwent apoptosis, and CD4(+) and CD8(+) lymphocytes, which preferentially infiltrated around the macrophage accumulation. Myofibroblast proliferation was not prominent, and no appreciable fibrosis resulted after the decline of granulomas. Although the intestinal epithelium was involved in inflammation, tissue injuries such as mucosal erosion or ulceration were not induced. When granulomas were formed near the Peyer's patches, they invaded deeply into the lymphoid tissue, producing many small islands. The mesenteric lymph nodes also had many granulomatous islands in the cortex and medulla, but the liver and spleen displayed no granulomatous changes, suggesting that liposomal CF spreads via the lymphatic vessels from the injection site. The CF-induced colonic granulomas associated with mesenteric lymphadenitis will be useful for investigating human granulomatous colitis.

Sub-gap absorption spectra of amorphous InGaZnO4 films have been investigated using extended photothermal deflection spectroscopy from 0.31 to 3.4 eV. DC sputtered amorphous InGaZnO4 films show optical bandgap of ~ 3.6 eV, Urbach energy of ~ 0.25 eV, weak absorption tail around 2.37 eV and strong absorption peaks around 0.38 eV. The intensity of absorption tail around 2.37 eV increased and the intensity of the absorption peaks around 0.38 eV decreased with annealing temperature at 400 °C. We speculate that reduction of oxygen and hydrogen is caused by thermal treatment at 400 °C, and then oxygen vacancies form deep levels in the bandgap.

Unlike normal liver with the sinusoids, hepatocellular carcinomas (HCCs) possess capillaries. Whether these capillaries derive from the sinusoids remains unclear in human HCCs. This study aimed to examine sinusoidal capillarization in human HCCs and its relationship to the tumor size, arterialization and dedifferentiation. Thirty-eight HCCs with a diameter of 10 - 140 mm were pathologically and angiographically examined. By electron microscopy, the microvasculature of tumors was classified into sinusoidal, intermediate and capillary types, which were all negative, partially positive and all positive, respectively, for four parameters, i.e., endothelial defenestration, continuous basement membrane, lack of Kupffer cells, and lack of lipid-containing hepatic stellate cells. Well-, moderately and poorly differentiated HCCs displayed sinusoidal / intermediate / capillary types, intermediate / capillary types and only capillary type, respectively, suggesting the transition from the sinusoids to capillaries in well-differentiated (and probably moderately differentiated) HCCs. Furthermore, well-differentiated HCCs with a diameter of less than 30 mm often received preferential portal venous blood, while moderately and poorly differentiated ones were all supplied with arterial blood, indicating a relationship between dedifferentiation and arterialization. In contrast, the microvascular type displayed no significant relationship with tumor size or arterialization in well-differentiated HCCs. The present study has demonstrated that sinusoidal capillarization occurs in human well-differentiated HCCs and seems to be related to dedifferentiation of parenchymal tumor cells, but not to tumor size or arterialization.

Granulomatous inflammation is characterized morphologically by a compact organized collection of macrophages and their derivatives. It is classified as either a hypersensitivity type or a foreign-body type. Lipid components of theMycobacterium tuberculosiscell wall participate in the pathogenesis of infection. Strains ofM. tuberculosishave cord factor (trehalose 6,6′-dimycolate [TDM]) on their surface. To clarify host responses to TDM, including immunogenicity and pathogenicity, we have analyzed the footpad reaction, histopathology, and cytokine profiles of experimental granulomatous lesions in immunized and unimmunized mice challenged with TDM. In the present study, we have demonstrated for the first time that TDM can induce both foreign-body-type (nonimmune) and hypersensitivity-type (immune) granulomas by acting as a nonspecific irritant and T-cell-dependent antigen. Immunized mice challenged with TDM developed more severe lesions than unimmunized mice. At the active lesion, we found monocyte chemotactic, proinflammatory, and immunoregulatory cytokines. The level was enhanced in immunized mice challenged with TDM. This result implies that both nonimmune and immune mechanisms participate in granulomatous inflammation induced by mycobacterial infection. Taken together with a previous report, this study shows that TDM is a pleiotropic molecule against the host and plays an important role in the pathogenesis of tuberculosis.

Cell-mediated immunity is defined as a beneficial host response characterized by an expanded population of specific T cells, which, in the presence of antigens, produce cytokines locally. The activation and recruitment of cells into an area of inflammation is a crucial step in the development of DTH responses. DTH is immunologically a process similar to cell-mediated immunity, involving T cells and cytokines. CD4 T helper (Th) 1 cells, differentiated from naive Th cells by IL-12 and IL-18 produced from macrophages, play a regulatory role in the expression of DTH and activation of macrophages via interferon gamma generated by Th1 and natural killer cells. Macrophages accumulate at the site of DTH and become activated through the CD4 Th1 cell-cytokine-macrophage axis. However, DTH leads to pathologic responses, such as granulomatous inflammation, calcification, caseation necrosis, and cavity formation. Granulomas usually form as a result of the persistence of a nondegradable product or as the result of DTH responses. DTH is also required for host defense against etiologic agents, such as Mycobacterium tuberculosis. The expression of cell-mediated immunity/DTH is a double-edged sword that may contribute to both clearance of the etiologic agent and tissue damage.

Background/Aim Helicobacter pylori is the principal cause of gastritis and peptic ulcer disease. However, H. pylori-positive patients do not always have peptic ulcer. This study was carried out in order to determine the difference in host immune reaction to H. pylori between patients with peptic ulcer and those without. Methods Ten H. pylori-positive patients with peptic ulcer, 10 H. pylori-positive non-ulcer patients, and 10 healthy volunteers were examined for expression of surface molecules in peripheral blood mononuclear cells. The surface molecules were stained with immunofluorescence-labelled specific antibodies and analysed by flow cytometry. Results More mononuclear cells expressed molecules ICAM-1, VLA-4, Leu-M3 in H. pylori-positive ulcer patients than in non-ulcer patients and healthy volunteers. There were also more cells expressing CD28, SLe x , CD4, HLA-DR, and NU-B2 in H. pylori-positive ulcer patients than in non-ulcer patients and healthy volunteers. There were fewer cells expressing CD8 in H. pylori-positive ulcer patients than in non-ulcer patients and healthy volunteers. Conclusion H. pylori infection may cause immunological reactions which are reflected in peripheral mononuclear cells. However, the activity and characteristics of peripheral mononuclear cells, in terms of expression of adhesion molecules, may differ between ulcer and non-ulcer patients who are infected with H. pylori.

To demonstrate ultrastructural events in the early phase of hepatic metastasis of human colon carcinoma, we intrasplenically injected a highly metastasizable, human colon carcinoma cell line LM-H3 (1 x 10(6) cells) into nude mice, and electron microscopically investigated the hepatic metastasis. At 24 h, tumor cells adhered to the endothelial wall of terminal portal venules and periportal sinusoids. At 48-72 h, after extravasation, they deeply invaded the hepatic cell plate and the interstitial tissue of the portal tract, in which they underwent proliferation and made the metastatic foci. Tumor cells were linked with each other or with surrounding hepatocytes by desmosomes. Desmosomes were maintained during the mitosis. When invading tumor cells were exposed to the bile canaliculi, they generated microvilli on the surface. Microvilli were also formed at the luminal surface of intracytoplasmic inclusions. In the interstitial tissue of the portal tract, tumor cells were closely associated with fibroblasts. However, no junctional specializations were seen between them. The present study demonstrated that human colon carcinoma cell line LM-H3 formed desmosomes with hepatocytes soon after invasion of the hepatic cell plate, suggesting the regulatory role of an interaction with hepatocytes in the growth of metastatic foci within the liver parenchyma.

Background/Aims: We recently demonstrated prion as a new marker for hepatic stellate cell activation in rats. Here, we have examined prion expression in normal and diseased human livers. Methods: Prion expression was examined at protein level by immunohistochemistry and at mRNA level by in situ hybridization. Results: While normal livers were negative for prion, all liver specimens but one from patients with chronic liver disease were positively stained. In chronic hepatitis, prion protein expression was found not only in the sinusoidal lining cells within the lobules but also in mesenchymal cells in expanded portal tracts. In alcoholic liver disease, prion-positive cells were found mainly in the areas of alcoholic hepatitis. Immunoelectronmicroscopy revealed that prion-positive cells were activated stellate cells. In situ hybridization demonstrated that the distribution of prion mRNA is similar to that of prion protein. In chronic hepatitis, the number of prion-positive cells correlated with the grade of activity but not with the stage of fibrosis. In alcoholic liver disease, levels of prion protein expression were significantly increased in the presence of alcoholic hepatitis. Conclusion: Prion as a novel marker of activated stellate cells correlates well with disease activity in human chronic liver diseases.

We previously found that a small dose (2 μg per mouse) of staphylococcal enterotoxin B (SEB) induced early emerging unresponsiveness in intrahepatic-lymphocyte populations (IHLs). The purpose of this study was to reveal the inducing role of accessory cells involved in IHLs in this phenomenon. IHLs prepared at 3 to 24 hours after SEB injection failed to proliferate in response not only to SEB but also to SEA, representing ligand-nonspecific unresponsiveness, whereas spleen cells (SPCs) and mesenteric lymph-node cells showed transient proliferation. Unresponsiveness in IHLs was related to a deficit of their accessory cell function as measured by coculture of irradiated IHLs and antigen-specific, type 1 T-helper (Th1) clone cells. High levels of nitrite were detected in the culture supernatant. Supplement ofNG -monomethyl-l -arginine lowered nitrite levels and concurrently restored the proliferative response of Th1 cells, indicating the involvement of nitric oxide in suppression. Adherent cells prepared from IHLs well reproduced these results. As shown by flow cytometry, Mac-1high Ia+ cells, which mainly included F4/80+ cells (macrophages) and a minor population of CD11c+ cells (dendritic cells), increased in proportion in IHLs but not in SPCs at 6 to 24 hours. Depletion of Mac-1high cells from IHLs with antibody-coated magnetic beads recovered the proliferative response. Depleted Mac-1high cells had a monocytoid appearance. In immunostained sections, Kupffer cells came to highly express both Mac-1 and Ia at 12 hours. These results indicate that Mac-1highIa+ adherent cells, largely Kupffer cells activated by SEB, nonspecifically suppress the proliferation of Th1 cells via nitric oxide production before manifestation of ligand-specific unresponsiveness. (Hepatology 2000;32:507-513.)

By using a selective ROCK inhibitor Y-27632, the role of Rho-ROCK signaling in the function of hepatic stellate cells in culture was studied. Stellate cells maintained the "star-like" configuration of the quiescent stage in the presence of Y-27632, while the expression of smooth muscle alpha-actin and PDGF receptor beta was not affected by the agent. Serum-stimulated migration of the cells was significantly suppressed by Y-27632. The contraction of stellate cells induced by 5 nM endothelin-1 was attenuated by the agent in a dose-dependent manner. Formation of F-actin stress fibers and phosphorylation of myosin light chain was apparently reduced by Y-27632 even under the stimulation with endothelin-1. On the other hand, ex vivo liver perfusion experiment revealed that endothelin-1 (2 nM)-induced increase of portal vein constriction was almost completely inhibited by 20 microM Y-27632 with a concomitant improvement of hepatocyte degeneration. These results suggest that ROCK is one of the key regulators of stellate cell motility and that the clinical application of ROCK inhibitors such as Y-27632 should be considered in the reduction of portal hypertension in liver fibrosis and cirrhosis.

Dendritic cells (DCs) are widely distributed in the airways and can serve as potent antigen-presenting cells. To clarify their involvement in the cell-mediated immune responses of the lung, we immunohistochemically investigated their distribution and kinetics during pulmonary delayed-type hypersensitivity (DTH) reactions induced in sensitized mice by intratracheal instillation of hapten. Cellular infiltrate appeared around the bronchiole and its accompanying blood vessel at 12 h after elicitation and progressively expanded by 48 h. As quantitated by computer-assisted morphometry, I-A(+) DCs and CD4(+) Th cells significantly increased in number around the bronchiole to a maximum at 24 h, whereas F4/80(+) macrophages were predominantly accumulated around the accompanying vessel with a peak at 48 h. Serial-section analysis revealed that DCs were colocalized with Th cells in the inflamed peribronchiolar tissue. Immunoelectron microscopy demonstrated that DCs found inside and around the capillaries and venules of peribronchiolar interstitium displayed round forms, indicating their emigration from here, while those situated far from the microvessels were elongated, often in close apposition to the lymphocytes. Mitosis of DCs was rarely seen. The present results suggest that peribronchiolar accumulation of DCs resulting from accelerated influx of blood-borne immature DCs and the interaction with T cells at the application site may play inducing roles in the development of pulmonary DTH reactions by enhancing the recruitment of macrophages.

Langerhans cells (LCs) and dendritic epidermal T cells (DETCs) constitute the skin immune system. To demonstrate the kinetics of in vivo activation of murine LCs and DETCs in the elicitation phase of contact hypersensitivity, we measured the cell area positively stained for I-A and gammadeltaT-cell receptor (or Thy-1.2), respectively, under a fluorescence microscope at various time intervals after topical application of dinitrofluorobenzene. The fluorescence-positive area of LCs increased in parallel with that of DETCs at 1 h and 24 h, indicating the biphasic activation of LCs and DETCs. Early activation was hapten-specific and often exhibited close LC-to-DETC apposition. Experiments with in vivo administration of neutralizing anticytokine antibodies revealed that none of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta were involved in the induction of early activation of LCs and DETCs, while TNF-alpha and IL-1beta mediated late activation of LCs, and IFN-gamma and IL-1beta mediated that of DETCs. Our results indicate that LCs and DETCs are synchronously and biphasically activated in the epidermis during the elicitation phase of contact hypersensitivity and suggest that different mechanisms may control early and late activation.

The hepatic sinusoids are preferentially supplied with portal venous blood and equipped with fenestrated endothelial cells that are distinct from capillary endothelial cells. We previously observed in rats that sinusoidal capillarization proceeded concurrently with arterial blood supply during hepatocarcinogenesis. This study aimed to clarify the inducing role of arterialization in sinusoidal capillarization by investigating phenotypical, morphological and functional alterations to sinusoidal endothelial cells (SECs) in arterialized rat livers induced by portal branch ligation. At one week, after massive hepatic necrosis following ligation, the livers were restored to their normal architecture without causing post-necrotic fibrosis. At 12-21 weeks, they exhibited a normal histology except for mild pericellular fibrosis which developed along sinusoids or between adjacent hepatocytes. SECs expressed factor VIII-related antigen and showed a decrease in the number of fenestrae and porosity, still lacking any basement membrane but further retaining the functional capacity for carmine dye uptake. Stellate cells, while occasionally associated with large amounts of collagen bundles, contained many lipid droplets and expressed no alpha-smooth muscle actin, indicating a quiescent property. Kupffer cells were commonly found within the sinusoids. The present results indicate that arterialization of the liver induces a partial (but not complete) transition of SECs into capillary-type endothelial cells, suggesting that arterialization might be one of the factors which induce sinusoidal capillarization in the development of hepatocellular carcinoma.

Structural adaptations in the liver to constantly receive and release a large volume of circulating blood at low pressure are present at many levels; alteration of these structures can modify flow and perturb pressure gradients. Liver growth multiplies the lobule number by a factor of 4-5 after birth. Lobule configuration conforms with observations in space division, each unit being bordered by planes; curvature will impede expansibility and retractability among units. Lobular organization with hepatocytic plates and sinusoids, being radial centrally and reticular peripherally, maximizes its reversible distensibility. Resistance sites in the portal, sinusoidal, and hepatic system are subject to species variations; real portal sphincters are photographed in the frog. Small venules are demonstrably resistive. In endothelin-1-induced rat portal hypertension, the distal segment of preterminal portal venules constricts most intensely, whereas the terminal portal venules and sinusoids are flaccid. Their pericytes and arachnocytes (stellate cells, Ito cells, retinol-storing cells), respectively, possess no effective contractile machinery. In the dog, the initial sublobular veins react with venoconstriction to many stimulations. Well-developed musculature in hepatic veins, as in man and pig, can regulate flow by junctional constriction. These histoarchitectonics provide hepatic hemodynamics with high capacitance and high compliance properties. The hepatic artery supplies oxygenated blood to five stromal compartments: peribiliary vascular plexus, portal tract interstitium, portal vein vasa vasorum, hepatic capsule, and central-sublobular-hepatic vein vasa vasorum. Its role as the nutrient vessel to the veins is established, but what influence it may have in the pathophysiology of portal hypertension awaits clarification.

Suppression subtractive hybridization was used to clone genes associated with the activation of hepatic stellate cells and 13 genes were found to be dominantly expressed in activated stellate cells. Among them, one was identical to the 421-837th base pairs of cDNA sequence reported for rat prion-related protein (PrP). In cultured stellate cells, PrP mRNA expression increased in a time-dependent manner in parallel with smooth muscle (SM) α-actin mRNA expression. In situ hybridization demonstrated that PrP mRNA was localized in and around the fibrous septa of carbon tetrachloride (CCl 4 )-treated liver. Cellular PrP (PrP c ) was produced by culture-activated stellate cells, and immunohistochemically detected in the fibrous septa of CCl 4 -damaged liver and sinusoidal linings of common bile duct-ligated liver, consistent with the localization of SM α-actin. Immunoelectron microscopy revealed that PrP c resided on the plasma membrane of stellate cells. These results indicate that PrP expression is closely related to stellate cell activation associated with fibrogenic stimuli.

Langerhans cells (LCs) are activated in the epidermis by external and internal stimuli, such as antigens and cytokines, respectively. To reveal the morphologic and functional properties of in vivo-activated LCs during inflammation, we injected the streptococcal preparation OK-432 intradermally into the earskin of mice and performed time-course analyses by immunofluorescence and electron microscopy. Cellular infiltrate appeared in the dermis at 6 h after OK-432 injection and had progressively extended to the dermoepidermal junction at 12 and 24 h. Immunostaining for class II antigen revealed that LCs were enlarged and extended long dendrites during inflammation. Acidic compartments such as lysosomes and multivesicular bodies also increased in number and Golgi apparatuses developed as demonstrated by electron microscopy and morphometric analysis. Birbeck granules, although not showing numerical changes, were translocated from the Golgi area to the subplasmalemmal area. After epicutaneous application of cationic ferritin, LCs often contained endosomes as the result of engulfment by the cytoplasmic projections. The present results indicate that nonspecifically induced dermal inflammation is capable of inducing activation of LCs in vivo, and that in vivo-activated LCs have the capacity for active endocytosis and intracellular digestion or processing.

Concanavalin A (Con A) stimulation induces T-cell activation-associated hepatic injury. This study is designed to show the involvement of microcirculatory disturbance in the pathogenesis. Con A administration led to prominent intrasinusoidal hemostasis, which consisted of erythrocyte agglutination, lymphocyte/neutrophil sticking to endothelial cells, and platelet aggregation and degranulation, resulting in a marked decrease in the intrahepatic blood flow and elevation of portal perfusion pressure. After hemostasis, confluent hepatic necrosis occurred within the congested area of liver parenchyma. Reduction in the extent of hemostasis by the treatment with heparin (thrombin inhibitor) or cyproheptadine (serotonin inhibitor) decreased hepatic injury. Pretreatment with either anti-tumor necrosis factor alfa (TNF-alpha) or anti-interferon gamma (IFN-gamma) monoclonal antibody (MAb) moderately decreased hemostasis and hepatic injury, whereas combined use of two MAbs almost perfectly protected mice from these disorders. Complete obliteration of hemostasis and hepatic injury was also accomplished by the pretreatment with FK506 which suppressed TNF-alpha and IFN-gamma production. Intrasinusoidal accumulation of leukocytes and platelets was, however, not blocked by FK506, indicating that Con A activities other than the stimulation of cytokine production are responsible for this event. The administration of anti-CD3 MAb, a T-cell stimulant without agglutination activities, which elevated plasma cytokine levels in a comparable degree without inducing prominent leukocyte infiltration, did not induce hepatic congestion and injury. These findings indicate that the agglutination activities of Con A and T-cell activation mediated TNF-alpha/IFN-gamma production are both required for the induction of intrasinusoidal hemostasis, which is indispensable for the development of hepatic injury.

Tumor metastasis can be prevented by inhibiting angiogenesis. In the present study, we have demonstrated that the angiogenesis inhibitor TNP-470 also suppresses the development of primary hepatic nodules. Hepatocarcinogenesis was performed by the feeding of 2-acetylaminofluorene to hepatectomized rats during 8-14 weeks of age. Predominantly arterial-to-portal circulation and sinusoidal capillarization were determined by the staining of nodules with arterially infused ink and immunostaining for factor VIII-related antigen, respectively. Intraperitoneal administration of 30 mg/kg b.w. of TNP-470 twice a week significantly reduced the number of hepatic nodules. Among the nodules, hyperplastic nodules stained with ink, atypical hyperplastic nodules and hepatocellular carcinoma, all of which possess structurally altered sinusoidal endothelial cells or capillary-type endothelial cells, were dramatically decreased in number. Suppression was observed equally in nodules of all sizes. TNP-470 was more effective when administered during 8-20 weeks than during 14-26 weeks. In contrast, ink-non-stained hyperplastic nodules, which have normal sinusoidal endothelial cells, were not affected at all. The present results indicate that TNP-470 suppresses the development of primary hepatic nodules whose microvessels are capillaries or transitional forms from sinusoids to capillaries.

A high-dose bolus regimen for interleukin(IL)-2 administration to cancer patients frequently causes serious side-effects in which various organs are involved. In order to reveal the mechanism of toxicities associated with this regimen, we compared the augmenting effect of high-dose IL-2 on murine organ-associated lymphocytes between neoplastic and non-neoplastic states. Intraperitoneal administration of IL-2 at a dose of 10(5) JRU (Japanese Reference Units) twice daily for 3 days led to the death of all the syngeneic MH134-hepatoma- or X5563-myeloma-bearing mice, whereas it had no lethal effect on non-tumor-bearing mice. Histological and morphometric analyses demonstrated that tumor-bearing mice displayed more extensive infiltration of large granular lymphocytes and agranular lymphocytes in the liver and lungs than did the non-tumor-bearing mice. Large granular lymphocytes had the ultrastructural characteristics of lymphokine-activated killer cells. Lymphocytes often underwent extravasation into the interstitial space and exhibited local proliferation without causing any direct injury to apposed parenchymal cells. Flow-cytometric analysis of hepatic mononuclear cells demonstrated that IL-2-receptor-beta (IL-2R beta)-bearing lymphocytes, i.e., natural killer cells and intermediate CD3 cells, were increased in number in the neoplastic state before the IL-2 injection. The present study indicates that the tumor-bearing state increases the number of organ-associated IL-2R beta + lymphocytes, which are then greatly amplified by the challenge of high-dose IL-2, leading to the functional disturbance of organs. We have further demonstrated here that an intermittent low-dose IL-2 regimen has a potential therapeutic effect on tumor regression without causing lethal side-effects.

It is reported that some bacteria or bacterial components cause thymic atrophy via the apoptotic process. The present study demonstrated for the first time in vivo induction of apoptosis in the mouse thymus by mycobacterial cord factor (CF) (trehalose 6,6'-dimycolate). When 300 microg of purified CF from Mycobacterium tuberculosis was intravenously administered to BALB/c mice in the form of water-in-oil-in-water (w/o/w) emulsion, thymic atrophy and pulmonary granulomas were induced with a peak on day 7, whereas, in the form of liposomes, CF induced thymic atrophy on days 14 to 21 in parallel with the development of hepatic granulomas. Thymic atrophy resulted from the depletion of cortical lymphocytes via apoptosis as revealed by DNA fragmentation and karyorrhectic changes. In contrast, mycobacterial sulfatide (2,3,6,6'-tetraacyl trehalose 2'-sulfate) caused neither thymic atrophy nor granuloma formation. Compared to lipopolysaccharide-induced thymocyte apoptosis, CF (w/o/w)-induced thymocyte apoptosis developed more slowly, reached a maximum later, and lasted longer but was less intense. Although serum tumor necrosis factor alpha (TNF-alpha) levels in CF-treated mice were not significantly elevated, administration of anti-TNF-alpha antibody almost completely inhibited thymic atrophy and granuloma formation. Serum corticosterone levels were only slightly elevated by CF administration. The present results indicate that mycobacterial CF induces thymic atrophy via apoptosis, which is closely linked with granuloma formation.

We analyzed the mycolic acid composition of trehalose 6,6'-dimycolate (TDM) obtained from Mycobacterium, using thin layer chromatography, gas chromatography and gas chromatography-mass spectrometry. Utilizing TDM, whose structure was confirmed, granuloma formation in mice was investigated. All TDM used exhibited considerable granuloma formation activity in the lung and spleen. In particular, TDM from M. bovis showed the greatest activity and toxicity among mycobacterial TDM. We therefore discussed the relationship between the chemical structure and granuloma-forming activity of TDM, especially in relation to the structure of mycolic acid in TDM.

We investigated here the kinetics of natural killer (NK) cells and extrathymic T cells, which include intermediate CD3 cells and gamma delta T cells, in the cord factor-induced granulomatous inflammation of the lungs and liver. In Balb/c mice, pulmonary inflammation elevated the proportion of NK cells and that of extrathymic T cells to mononuclear cells in the lungs. C3H/He mice exhibited shorter-term inflammation of the lungs than Balb/c mice and accordingly showed a smaller increase in the proportions of pulmonary NK cells and intermediate CD3 cells. In the liver of Balb/c mice, hepatic NK cells increased as well with the granulomatous changes, while intermediate CD3 cells exhibited a transient decrease before they increased. The present study has demonstrated that granulomatous inflammation is accompanied by the increase of lung-associated NK cells and extrathymic T cells and that there exists a difference between these two mouse strains in the induction of these lymphocyte subsets by cord factor.

There are three mycolic acid homologues with C22-, C24- and C26-alpha-units in Mycobacterium. In order to reveal the composition and distribution of these homologues in each subclass and molecular species of mycolic acids and to compare them with the composition of constitutive non-polar fatty acids (free and bound forms), we have separated non-polar fatty acids and each subclass of mycolic acids from 21 mycobacterial species by thin-layer chromatography, and analyzed non-polar fatty acid methyl esters by gas chromatography (GC) and the cleavage products of methyl mycolate by pyrolysis GC. We further performed mass chromatographic analysis of trimethylsilyl (TMS) ether derivatives of mycolic acid methyl esters by monitoring [B-29]+ ions (loss of CHO from the alpha-branched-chain structure of mycolic acids) of m/z 426, 454 and 482 which are attributed to C22-, C24- and C26-alpha-units of TMS ether derivatives of methyl mycolates, respectively, (Kaneda, K. et al, J. Clin. Microbiol. 24: 1060-1070, 1986). By pyrolysis GC, C22:0, C24:0 and C26:0 fatty acid methyl esters generated by the C2-C3 cleavage of C22-, C24- and C26-alpha-unit-containing mycolic acid methyl esters, respectively, were detected. Their proportion was almost the same among subclasses of mycolic acids in every Mycobacterium and also similar to the proportion of constitutive non-polar C22:0, C24:0 and C26:0 fatty acids. By mass chromatography, the composition and distribution of C22- and C24-alpha-unit-containing homologues were revealed to be similar between alpha- and alpha'-mycolic acids in every Mycobacterium.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoptosis was originally defined as shrinkage necrosis, as a distinct mode of cellular death from coagulative necrosis in the rat liver lobes after portal branch ligation. To reveal the functional role of the apoptosis of hepatocytes in the volume reduction and recovery of normal hepatic architecture in portal-deprived liver lobes, we ligated the portal vein branch supplying the left and median liver lobes in Wistar rats. The liver lobes were perfusion-fixed with glutaraldehyde solution via the proximal site of the ligated portion of the portal vein on Days 1, 2, 4, 7 and 14 after operation and examined light and electron microscopically. On Day 2 after ligation, massive necrosis of hepatocytes occurred in the central to intermediate zones of the liver lobule, and apoptotic hepatocytes were observed in the boundary region between necrotic and normal areas. Such necrotic area-associated apoptosis of hepatocytes was most frequent on Day 2, declining thereafter. The sequential changes of the cell organelles in apoptotic hepatocytes were distinct from those in necrotic hepatocytes. On Day 7, when necrotic areas had almost disappeared the apoptosis of hepatocytes occurred mostly between intact hepatocytes in the "combined hepatic cell cords" which included no obvious sinusoidal lumen between hepatic cell cords. Such necrotic area-nonassociated apoptosis began to increase in frequency on Day 4, reached a peak on Day 7 and was gone by Day 14, when normal hepatic architecture was recovered. The present study suggests that both necrotic area-associated and non-associated apoptosis of hepatocytes may be induced by mild ischemia and contribute in part to the volume reduction of ligated liver lobes. It further reveals that the necrotic area-non-associated apoptosis of hepatocytes plays a role in reconstructing the architecture of hepatic cell cords after necrotic hepatocytes have undergone dissolution in the liver lobule.