Salinity negatively correlates with the production and immunity of chicken: A molecular insight for food security and safety issues.

Salinity intrusion into the freshwater system due to climate change and anthropogenic activities is a growing global concern, which has made humans and domesticated animals more susceptible to diseases, resulting in less productivity. However, the effects of salinity on domesticated and wild birds, especially in terms of production and immunity, have not been fully elucidated yet. Therefore, this study was designed to examine the effects of salinity on the production and immunity of birds and the mechanisms by which immunity is compromised. Broiler chicks were subjected to different concentrations of salty water (control = normal water, treatment = 5 g/L, treatment = 10 g/L, and treatment = 15 g/L). The collected blood and organs from different groups of broilers were biochemically and histopathologically examined. Birds in salt-treated groups consumed significantly less feed than the control group, while the feed conversion ratio (FCR) was significantly higher. Body weight gain was significantly lower in salt-treated groups compared to control. Serum analysis revealed a lower systemic antibody titer in the salt-treated groups compared to the control. Primary lymphoid organs (thymus and bursa of Fabricius) were reduced in size in the salt-treated group due to cellular migration and depletion from these organs. Importantly, most of the parenchyma of lymphoid organs was replaced with fibrotic tissue. Gut microbes, Escherichia coli (E. coli) and Salmonella spp., from salt-treated groups, showed less viability but developed antibiotic resistance. Levels of salinity were significantly and negatively correlated with feed intake, body weight gain, antibody titer, lymphoid organ size, and viable count of gut microbes, while FCR, fibrosis of lymphoid organs, and antibiotic resistance were significant positively correlated. In conclusion, increased salinity is a possible threat to food security and safety as it decreases body weight gain, reduces immunity, and influences the development of multi-drug resistance in gut microbes.

Acetaminophen-Induced Rat Hepatotoxicity Based on M1/M2-Macrophage Polarization, in Possible Relation to Damage-Associated Molecular Patterns and Autophagy.

Overdose of acetaminophen (APAP), an antipyretic drug, is an important cause of liver injury. However, the mechanism in the rat model remains undetermined. We analyzed APAP-induced hepatotoxicity using rats based on M1/M2-macrophage functions in relation to damage-associated molecular patterns (DAMPs) and autophagy. Liver samples from six-week-old rats injected with APAP (1000 mg/kg BW, ip, once) after 15 h fasting were collected at hour 10, and on days 1, 2, 3, and 5. Liver lesions consisting of coagulation necrosis and inflammation were seen in the affected centrilobular area on days 1 and 2, and then, recovered with reparative fibrosis by day 5. Liver exudative enzymes increased transiently on day 1. CD68+ M1-macrophages increased significantly on days 1 and 2 with increased mRNAs of M1-related cytokines such as IFN-g and TNF-α, whereas CD163+ M2-macrophages appeared later on days 2 and 3. Macrophages reacting to MHC class II and Iba1 showed M1-type polarization, and CD204+ macrophages tended to be polarized toward M2-type. At hour 10, interestingly, HMGB1 (representative DAMPs) and its related signals, TLR-9 and MyD88, as well as LC3B+ autophagosomes began to increase. Collectively, the pathogenesis of rat APAP hepatotoxicity, which is the first, detailed report for a rat model, might be influenced by macrophage functions of M1 type for tissue injury/inflammation and M2-type for anti-inflammatory/fibrosis; particularly, M1-type may function in relation to DAMPs and autophagy. Understanding the interplayed mechanisms would provide new insight into hepato-pathogenesis and contribute to the possible development of therapeutic strategies.

Attenuation of alpha-naphthylisothiocyanate (ANIT)-induced biliary fibrosis by depletion of hepatic macrophages in rats.

Biliary fibrosis is a complex process in which macrophages and myofibroblasts may play central roles. We investigated biliary fibrosis lesions induced in the Glisson's sheath in rats by alpha-naphthylisothiocyanate (ANIT) administration under macrophage depletion. Hepatic macrophages were depleted in F344 rats with liposome-encapsulated clodronate (CLD) (10mL/kg body weight, i.v) followed by bile duct injury with ANIT (75mg/kg body weight, i.p) (ANIT+CLD group). Rats received empty-liposomes (Lipo) followed by ANIT, and served as control (ANIT+Lipo group). In both ANIT+Lipo and ANIT+CLD groups, ANIT-induced bile duct injury with inflammatory cell infiltration was seen on days 1-3, and subsequently reparative fibrosis occurred on days 5 and 7. In comparisons between the two groups, macrophages reacting to CD68, CD163, MHC class II and CD204 were less in numbers in ANIT+CLD group; the most sensitive immunophenotype was of CD163-positive. Furthermore, in ANIT+CLD group interstitial mesenchymal cells/myofibroblasts reacting to vimentin, desmin and α-smooth muscle actin were also less in grades and tended to be delayed in appearance. Interestingly, MCP-1, IFN-γ, IL-10, and TGF-ß1 mRNAs were significantly increased mainly on day 2 in ANIT+Lipo group, while the levels of these factors were prominently lower in ANIT+CLD group. Collectively, depletion of hepatic macrophages plays roles in attenuating biliary fibrogenesis by production of inflammatory factors. The present results indicated clearly importance of macrophage functions in the pathogenesis of biliary fibrosis.

Transient effects of empty liposomes on hepatic macrophage populations in rats.

Liposomes have been used as a vehicle for encapsulating chemicals or toxins in toxicological studies. We investigated the transient effects of empty liposomes on hepatic macrophages by applying a single intravenous injection at a dose of 10 ml/kg body weight in 6-week-old male F344 rats. One day after injection, the numbers of hepatic macrophages reacting to CD163, CD68, Iba-1, MHC class II, Gal-3 and CD204 were significantly increased in liposome-treated rats. CD163(+) Kupffer cells and CD68(+) macrophages with increased phagocytic activity in hepatic lobules were most sensitive. The histological architecture of the liver was not changed following liposome injection; however, hepatocytes showed increased proliferating activity, demonstrable with proliferation marker immunostaining and by an increase in gene profiles related to the cell cycle. In the liposome-treated rats, interestingly, AST and ALT values were significantly decreased, and MCP-1, IL-1ß and TGF-ß1 mRNAs were significantly increased. Collectively, the present study found that hepatic macrophages activated by liposomes can influence liver homeostasis. This information would be useful for background studies on liposomes.

Depletion of Hepatic Macrophages Aggravates Liver Lesions Induced in Rats by Thioacetamide (TAA).

Hepatic macrophages play crucial roles in hepatotoxicity. We investigated immunophenotypes of macrophages in liver injury induced in rats by thioacetamide (TAA; 300 mg/kg, intraperitoneal) after hepatic macrophage depletion; hepatic macrophages were depleted by liposomal clodronate (CLD; 10 ml/kg, i.v.) one day before TAA injection. Samples were obtained on post-TAA injection days 0, 1, 2, 3, 5, and 7. TAA injection induced coagulation necrosis of hepatocytes on days 1 through 3 and subsequent reparative fibrosis on days 5 and 7 in the centrilobular area, accompanied by increased numbers of M1 macrophages (expressing cluster of differentiation [CD]68 and major histocompatibility complex class II) and M2 macrophages (expressing CD163 and CD204) mainly on days 1 through 3. TAA + CLD treatment markedly decreased the numbers of M1 and M2 macrophages mainly on days 1 through 3; CD163(+) Kupffer cells were most sensitive to CLD depletion. In TAA + CLD-treated rats, interestingly, coagulation necrosis of hepatocytes was prolonged with more increased levels of hepatic enzymes (aspartate transaminase, alanine transaminase, and alkaline phosphatase) to TAA-treated rats; reparative fibrosis was incomplete and replaced by dystrophic calcification in the injured area, indicating the aggravated damage. Furthermore, in TAA + CLD-treated rats, inflammatory factors (monocyte chemoattractant protein [MCP]-1, interferon-γ, tumor necrosis factor-α, and interleukin-10) and fibrosis-related factors (transforming growth factor-ß1, matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-1) were decreased at messenger RNA levels, indicating abnormal macrophage functions. It was clearly demonstrated that hepatic macrophages have important roles in tissue damage and remodeling in hepatotoxicity.

Establishment and characterization of a transplantable tumor line (RMM) and cell line (RMM-C) from a malignant amelanotic melanoma in the F344 rat, with particular reference to galectin-3 expression in vivo and in vitro.

To investigate characteristics of malignant melanomas with various pathobiological features, a homotransplantable tumor line (RMM) was established from a spontaneous amelanotic melanoma found in the pinna of an aged F344 rat. RMM tumors were transplanted in syngeneic rats by serial subcutaneous implantation with 100% intake. The original and RMM tumors consisted of spindle-shaped cells arranged mainly in interlacing bundles. Immunohistochemically, the neoplastic cells were positive to PNL-2 (melanocytes), nestin (neuroectodermal stem cells), S-100 (neurogenic cells) and vimentin (mesenchymal cells). Electron microscopically, tumor cells possessed single membrane-bound pre-melanosomes. Further, a cell line (RMM-C) was induced from an RMM tumor. RMM-C cells and the induced tumors in syngeneic rats showed immunohistochemical reactions similar to the original and RMM tumors. Interestingly, serum level of galectin-3 expression was increased with growing RMM tumors, and the expression was influenced by TNF-α (increase) or TGF-ß1 (decrease), indicating a possible biomarker of amelanotic melanomas. The RMM tumors and RMM-C cell line could become useful tools for studies on the pathobiology, including tumor immunity, and development of therapeutic strategies against this malignancy. These tools are the first tumor lines of amelanotic melanomas in the rat.

Immunophenotypical characterization and influence on liver homeostasis of depleting and repopulating hepatic macrophages in rats injected with clodronate.

Hepatic macrophages (including Kupffer cells) play a crucial role in the homeostasis and act as mediators of inflammatory response in the liver. Hepatic macrophages were depleted in male F344 rats by a single intravenous injection of liposomal clodronate (CLD; 50mg/kg body weight), and immunophenotypical characteristics of depleting and repopulating macrophages were analyzed by different antibodies specific for macrophages. CD163(+) Kupffer cells were almost completely depleted on post-injection (PI) days 1-12. Macrophages reacting to CD68, Iba-1, and Gal-3 were drastically reduced in number on PI day 1 and then recovered gradually until PI day 12. MHC class II(+) and CD204(+) macrophages were moderately decreased during the observation period. Although hepatic macrophages detectable by different antibodies were reduced in varying degrees, Kupffer cells were the most susceptible to CLD. Liver situation influenced by depleted hepatic macrophages was also investigated. No marked histological changes were seen in the liver, but the proliferating activity of hepatocytes was significantly increased, supported by changes of gene profiles relating to cell proliferation on microarray analysis on PI day 1; the values of AST and ALT were significantly elevated; macrophage induction/activation factors (such as MCP-1, CSF-1, IL-6 and IL-4) were increased exclusively on PI day 1, whereas anti-inflammatory factors such as IL-10 and TGF-ß1 remained significantly decreased after macrophage depletion. The present study confirmed importance of hepatic macrophages in liver homeostasis. The condition of hepatic macrophages should be taken into consideration when chemicals capable of inhibiting macrophage functions are evaluated.

M1-/M2-macrophages contribute to the development of GST-P-positive preneoplastic lesions in chemically-induced rat cirrhosis.

Glutathione S-transferase placental form (GST-P) expression in hepatocyte foci is regarded as a preneoplastic change in rats. We aimed to reveal the contribution of polarized macrophages in development of GST-P-positive pseudolobules (PLs) in chemically-induced rat cirrhosis. F344 rats were injected with thioacetamide (100mg/kg BW, twice a week, intraperitoneally). Macrophage immunophenotypes and expression of M1-/M2-related factors were analyzed by immunohistochemistry, real-time RT-PCR and laser microdissection. GST-P-positive foci/clusters were clearly observed at post-first injection week 15. GST-P-positive PLs were distinguishable at weeks 20-32. Microarray analysis revealed upregulation of preneoplastic genes in GST-P-positive PLs at week 32. M1 (CD68(+), Iba1(+))-and M2 (CD163(+), CD204(+), Gal-3(+))-macrophages were greater in number in the GST-P-positive PLs, whereas MHC class II-positive (M1) macrophage number was fewer in the GST-P-positive PLs. Expression of both M1 (IFN-γ, IL-1ß, TNF-α, Iba1)- and M2 (IL-4, TGF-ß1, IL-10)-related factors were higher in GST-P-positive PLs. Our results showed that both M1- and M2-macrophage populations contribute to the development of hepatic preneoplastic lesions. MHC class II-positive macrophages may be related to anti-tumor progression, since their kinetics showed reverse pattern to other macrophage phenotypes.

Analysis of glial fibrillary acidic protein (GFAP)-expressing ductular cells in a rat liver cirrhosis model induced by repeated injections of thioacetamide (TAA).

Glial fibrillary acidic protein (GFAP), a type III intermediate filament protein, is expressed in hepatic stellate cells (HSCs), the principal fibrogenic cell type in the liver. Further, GFAP could be a marker for hepatic progenitor cells (HPCs). In this study, the participation of GFAP-expressing cells in HPC expansion/ductular reaction was investigated in a rat model of liver cirrhosis. Six-week-old male F344 rats were injected intraperitoneally with thioacetamide (100mg/kg BW, twice a week) and examined at post-first injection weeks 5, 10, 15, 20 and 25. Fibrosis-related proliferation of ductular cells was observed as demonstrated by CK19 immunostaining. Some of these cells were stained with GFAP. No co-staining was observed between CK19 and α-smooth muscle actin (α-SMA; myofibroblast marker). There were proliferating ductular cells stained with α-fetoprotein or ß-catenin; the ductular reaction was related to increased expression of hepatocarcinogenesis-related factors (Wnt2, Wnt4 and glypican-3). These results for the first time show the participation of GFAP-positive HPCs in ductular reaction in a chemically induced rodent model. Though the ductular cells were chaperoned by myofibroblasts, they show no direct evidence for epithelial to mesenchymal transition. These findings shed new light in understanding the roles of GFAP-expressing HPCs in liver cirrhosis and provide further evidence of interaction between newly-formed bile ductules and HSCs, suggesting that both cells could be in the common lineage of HPCs.

Immunohistochemical characterization of glial fibrillary acidic protein (GFAP)-expressing cells in a rat liver cirrhosis model induced by repeated injections of thioacetamide (TAA).

Hepatic stellate cells, the principal fibrogenic cell type in the liver, are known to express the astrocyte marker glial fibrillary acidic protein (GFAP). However, the exact role of GFAP-expressing cells in liver fibrosis remains to be elucidated. In this study, cellular properties of GFAP-expressing cells were investigated in a rat model of liver cirrhosis. Six-week-old male F344 rats were injected intraperitoneally with thioacetamide (100 mg/kg BW, twice a week) and examined at post first injection weeks 5, 10, 15, 20 and 25. Appearance of GFAP-expressing myofibroblasts peaked at week 15, associated with fibrosis progression. The majority of GFAP-expressing myofibroblasts co-expressed vimentin, desmin and alpha-smooth muscle actin. Some GFAP-positive myofibroblasts co-expressed nestin (neural stem cell marker), while a few co-expressed A3 (mesenchymal stem cell marker) and Thy-1 (immature mesenchymal cell marker). A few GFAP expressing cells underwent both mitosis and apoptosis. These results indicate that there is a dynamic participation of GFAP-expressing myofibroblasts in rat liver cirrhosis, and that they are mainly derived from hepatic stellate cells, and partly from cells in the stem cell lineage. These findings, which were shown for the first time in detail, would be useful to understand the role of GFAP-expressing myofibroblasts in the pathogenesis of chemically induced liver cirrhosis.

Participation of bone morphogenetic protein (BMP)-6 and osteopontin in cisplatin (CDDP)-induced rat renal fibrosis.

The significance of bone morphogenetic protein (BMP)-6 and osteopontin (OPN) in renal fibrosis is poorly understood. We investigated the expression of BMP-6 and OPN in cisplatin (CDDP; 6mg/kg, once, i.p.)-induced renal fibrosis in F344 rats, and their effects on kidney fibroblast (NRK-49F), mesenchymal pericyte (MT-9) and renal epithelial cell (NRK-52E) lines. Histopathologically, the CDDP injection showed desquamation of renal tubular epithelial cells at the cortico-medullary junction on days 1-3 that followed insufficient regeneration on days 5-9 and progressive interstitial fibrosis by day 35. In addition to TGF-ß1 (the most powerful fibrogenic factor), increase in mRNAs of BMP-6 and OPN was seen consistently after the injection. Immunohistochemically, BMP-6 was expressed both in the damaged renal epithelial cells and spindle-shaped myofibroblasts (expressing α-smooth muscle action [α-SMA]) in the fibrotic areas; OPN expression was seen exclusively in the injured renal epithelial cells. Treatment of BMP-6 or OPN increased α-SMA mRNA in MT-9 cells, similar to effects of TGF-ß1 on MT-9 and NRK-49F cells. Interestingly, TGF-ß1 addition increased BMP-6 and OPN mRNAs in NRK-52E cells. Collectively, it was found that BMP-6 and OPN considerably participate in progressive renal fibrosis through development of myofibroblasts, in relation with TGF-ß1.

M1- and M2-macrophage polarization in rat liver cirrhosis induced by thioacetamide (TAA), focusing on Iba1 and galectin-3.

INTRODUCTION: Resident and exudate macrophages play an important role in the development of liver cirrhosis. Ionized calcium binding adaptor molecule 1(+) (Iba1(+)) and galectin-3(+) (Gal-3(+)) macrophages regulate liver fibrosis probably through pro-inflammatory and pro-fibrotic factors. Macrophages show polarized functions in liver fibrosis; however, M1-/M2-polarization of Iba1(+) and Gal-3(+) macrophages remains obscured. This study investigated the M1-/M2-polarized properties of Iba1(+) and Gal-3(+) macrophages in chemical-induced liver cirrhosis. MATERIALS AND METHODS: Cirrhosis was induced in F344 rats by repeated injections of thioacetamide (100mg/kg BW, twice a week for 25 weeks). Liver samples were collected from post-first-injection (PFI) week 5 to 25. Macrophage immunophenotypes and myofibroblasts in the fibrous bridges (FBs) and pseudolobules (PLs) were analyzed by immunohistochemistry. Expressions of M1- and M2-related factors were analyzed with RT-PCR, separately in FBs and PLs. RESULTS: Activation of myofibroblasts was most pronounced in livers at week 15. CD68(+) (M1), CD204(+) (M2), Iba1(+) and Gal-3(+) macrophages in the FBs increased gradually and peaked at week 15, consistent with the upregulation of both M1-(MCP-1, IFN-γ, IL-1ß, IL-6, and TNF-α) and M2-(TGF-ß1, IL-4, and IL-10) related factors. Iba1(+) and Gal-3(+) macrophages showed both M1- and M2-immunophenotypes. CD163(+) macrophages showed a persistent increase, consistent with TGF-ß1 upregulation. MHC class II(+) macrophages increased in the developing fibrotic lesions, and then reduced in the advanced stage cirrhosis. CONCLUSION: Both M1- and M2-macrophage polarizations occur during development of liver cirrhosis. Iba1(+) and Gal-3(+) macrophages participate in liver cirrhosis through production of both M1- and M2-related factors.

Inflammatory regulation of iron metabolism during thioacetamide-induced acute liver injury in rats.

Systemic iron homeostasis is tightly regulated by the interaction between iron regulatory molecules, mainly produced by the liver. However, the molecular mechanisms of iron regulation in liver diseases remain to be elucidated. Here we analyzed the expression profiles of iron regulatory molecules during transient iron overload in a rat model of thioacetamide (TAA)-induced acute liver injury. After TAA treatment, mild hepatocellular degeneration and extensive necrosis were observed in the centrilobular region at hour 10 and on day 1, respectively. Serum iron increased transiently at hour 10 and on day 1, in contrast to hypoferremia in other rodent models of acute inflammation reported previously. Thereafter, up-regulation of hepcidin, a central regulator of systemic iron homeostasis, was observed in hepatocytes on day 2. Expression of transferrin receptor 1 and ferritin subunits increased to a peak on day 3, followed by increases in liver iron content and stainable iron on day 5, in parallel with regeneration of hepatocytes. Histopathological lesions and hepatocellular iron accumulation disappeared until day 10. The hepcidin induction was preceded by activation of IL6/STAT3 pathway, whereas other pathways known to induce hepcidin were down-regulated. IL6 was expressed by MHC class II-positive macrophages in the portal area, suggestive of dendritic cells. Our results suggest that IL6 released by portal macrophages may regulate hepatocyte hepcidin expression via STAT3 activation during transient iron overload in TAA-induced acute liver injury.

M1- and M2-macrophage polarization in thioacetamide (TAA)-induced rat liver lesions; a possible analysis for hepato-pathology.

"Classically activated macrophages (M1)" and "alternatively activated macrophages (M2)", which appear in injured tissues, control either inflammation or remodeling. The mechanism remains unclear. To clarify the M1-/M2-macrophage polarization in acute liver injury, M1- and M2-related factors were analysed in F344 rats by a single injection of TAA (300 mg/kg BW), and liver samples were collected on post injection (PI) hour 10 and days 1 to 10. Macrophage immunophenotypes were analyzed by single and double immunolabeling. M1-/M2-related factors were analyzed by real-time RT-PCR. On PI hour 10 (when centrilobular lesions were not still developed), expressions of IFN-γ, TNF-α, IL-1ß, and IL-6 for M1, and IL-4 for M2 were already increased, followed by increased expressions of IL-10 and TGF-ß1 for M2 on PI days 1-3 with development of centrilobular lesions and subsequent reparative fibrosis. On PI hour 10, CD204⁺ and MHC class II⁺ macrophages already increased in the intact periportal/Glisson's sheath regions, accompanied by an increased number of granzyme B⁺ NK cells. Reactive cells at PI hour 10 might produce M1-related factors. In addition to these macrophages, CD68⁺ and CD163⁺ macrophages, and CD3⁺ T cells appeared in the injured centrilobular region on PI days 1-3; there were macrophages reacting simultaneously to CD68/MHC class II, CD163/MHC class II, CD68/CD204, CD163/CD204, and MHC class II/CD204 in varying degrees. Although CD68⁺ and CD163⁺ macrophages are regarded as M1- and M2-types, respectively, the double labeling indicated that macrophage immunophenotypes are interchangeable in injured regions and subsequent fibrosis. An M1-/M2-macrophage paradigm would be useful to analyze hepatotoxicity and to understand the pathogenesis.

Characterization of glial fibrillary acidic protein (GFAP)-expressing hepatic stellate cells and myofibroblasts in thioacetamide (TAA)-induced rat liver injury.

Hepatic stellate cells (HSCs), which can express glial fibrillary acidic protein (GFAP) in normal rat livers, play important roles in hepatic fibrogenesis through the conversion into myofibroblasts (MFs). Cellular properties and possible derivation of GFAP-expressing MFs were investigated in thioacetamide (TAA)-induced rat liver injury and subsequent fibrosis. Seven-week-old male F344 rats were injected with TAA (300mg/kg BW, once, intraperitoneally), and were examined on post single injection (PSI) days 1-10 by the single and double immunolabeling with MF and stem cell marker antibodies. After hepatocyte injury in the perivenular areas on PSI days 1 and 2, the fibrotic lesion consisting of MF developed at a peak on PSI day 3, and then recovered gradually by PSI day 10. MFs expressed GFAP, and also showed co-expressions such cytoskeletons (MF markers) as vimentin, desmin and α-SMA in varying degrees. Besides MFs co-expressing vimentin/desmin, desmin/α-SMA or α-SMA/vimentin, some GFAP positive MFs co-expressed with nestin or A3 (both, stem cell markers), and there were also MFs co-expressing nestin/A3. However, there were no GFAP positive MFs co-expressing RECA-1 (endothelial marker) or Thy-1 (immature mesenchymal cell marker). GFAP positive MFs showed the proliferating activity, but they did not undergo apoptosis. However, α-SMA positive MFs underwent apoptosis. These findings indicate that HSCs can proliferate and then convert into MFs with co-expressing various cytoskeletons for MF markers, and that the converted MFs may be derived partly from the stem cell lineage. Additionally, well-differentiated MFs expressing α-SMA may disappear by apoptosis for healing. These findings shed some light on the pathogenesis of chemically induced hepatic fibrosis.

Immunohistochemical characterization of macrophages and myofibroblasts in fibrotic liver lesions due to Fasciola infection in cattle.

Histopathologically, fibrosis in Fasciola-infected cattle livers was characterized by inflammatory cell infiltration, such as eosinophils and macrophages, pseudo-lobule, pseudo-bile ducts and fibrotic bridges separating pseudo-lobules; the fibrotic lesions were developed in the Glisson's sheath. Pseudo-bile ducts consisting of epithelial cells reacted clearly to cytokeratin (CK) 19, indicating cholangiocyte origin. Immunophenotypes of macrophages and myofibroblasts were investigated in the fibrotic livers. Macrophages positive for CD68 (reflecting phagocytosis) and CD163 (representing proinflammatory cytokine production) were increased, and those for CD204 (implying lipid metabolism) and Iba-1 (a calcium-binding protein playing role in chemotaxis) decreased in fibrotic livers compared to control livers. Spindle-shaped myofibroblasts positive for vimentin, desmin and α-smooth muscle actin (α-SMA) increased in the peribiliary connective tissues, although the desmin-positive cells were fewer. In addition to the usefulness of these antibodies for macrophage detection in cattle livers, this study shows that macrophages with different immunophenotypes participate in Fasciola-infected cattle livers, in relation to development of myofibroblasts expressing mainly vimentin and α-SMA.

Slowly progressive cholangiofibrosis induced in rats by α-naphthylisothiocyanate (ANIT), with particular references to characteristics of macrophages and myofibroblasts.

A progressive cholangiofibrosis was developed as an animal model in 6-week-old male F344 rats by repeated intraperitoneal injections of α-naphthylisothiocyanate (ANIT) for 19 weeks; liver samples were examined at post-first injection (PFI) weeks 3, 7, 10, 13, 16 and 19, focusing on characteristics of macrophages and myofibroblasts by immunohistochemical analyses. In the affected Glisson's sheath consisting of inflammatory cell infiltrates, bile duct proliferation and advancing fibrosis, the number of macrophages reacting to OX6 (recognizing MHC class II) increased consistently (PFI weeks 3-19), suggesting a central role of antigen presenting cells in the biliary fibrosis; macrophages reacting to ED1 (CD68, reflecting phagocytic activity) and ED2 (CD163, relating to proinflammatory factor production) showed a significantly increased number at PFI weeks 7-19 and PFI weeks 13-19, respectively. Interestingly, macrophages positive for SRA-E5 (CD204, reflecting lipid metabolism) increased at PFI weeks 7-19, and the appearance was limited in the sinusoids around the affected Glisson's sheath. Myofibroblasts appearing in the affected Glisson's sheath reacted to vimentin and desmin at early (PFI weeks 3-7) and mid (PFI weeks 10-13) stages, and then they came to strongly express α-smooth muscle actin at late stage (PFI weeks 16-19). This study shows that macrophages exhibit heterogeneous properties depending on stages and locations; in association with such macrophage populations, myofibroblasts expressing various cytoskeletons participate in cholangiofibrosis. These characteristics would be useful in evaluating the pathogenesis of possible cholangio-toxicants.

Expressions of Iba1 and galectin-3 (Gal-3) in thioacetamide (TAA)-induced acute rat liver lesions.

Ionized calcium binding adaptor molecule 1 (Iba1) is associated with membrane ruffling and motility of cells. Galectin-3 (Gal-3) is a ß-galactoside binding animal lectin, and regulates fibrogenesis probably through transforming growth factor-ß1. To evaluate macrophage properties, expressions of Iba1 and Gal-3 were investigated, in relation to macrophages expressing CD68 (ED1; reflecting increased phagocytosis) and CD163 (ED2; implying proinflammatory factor productions) in centrilobular lesions induced in rat livers with thioacetamide (TAA; 300 mg/kg body weight, once intraperitoneally). In agreement with expression patterns of CD68(+) and CD163(+) macrophages, cells reacting to Iba1 and Gal-3 were increased in numbers on post-injection (PI) days 1-5, peaking on day 2; thereafter, the positive cells gradually decreased to control levels until PI days 7 and 10. The increased expressions of Iba1 and Gal-3 were confirmed at mRNA levels by the RT-PCR. Double immunofluorescence staining on PI days 2 and 3 demonstrated Iba1 expression in 15-46% of CD68(+) and CD163(+) macrophages, and Gal-3 expression in 65-82% of CD68(+) and CD163(+) macrophages; Gal-3 expression was observed in 84-93% of Iba1(+) cells. Interestingly, Gal-3 was also expressed in a small number of α-smooth muscle actin-positive myofibroblasts in fibrotic lesions developed in injured centrilobular areas. These findings indicate that macrophages with various functions can participate in development of liver lesions and resultant fibrosis. Besides CD68 and CD163, Iba1 and Gal-3 immunohistochemistry for macrophages would be useful to analyze the pathogenesis behind developing hepatotoxicity.

Novel cholix toxin variants, ADP-ribosylating toxins in Vibrio cholerae non-O1/non-O139 strains, and their pathogenicity.

Cholix toxin (ChxA) is a recently discovered exotoxin in Vibrio cholerae which has been characterized as a third member of the eukaryotic elongation factor 2-specific ADP-ribosyltransferase toxins, in addition to exotoxin A of Pseudomonas aeruginosa and diphtheria toxin of Corynebacterium diphtheriae. These toxins consist of three characteristic domains for receptor binding, translocation, and catalysis. However, there is little information about the prevalence of chxA and its genetic variations and pathogenic mechanisms. In this study, we screened the chxA gene in a large number (n = 765) of V. cholerae strains and observed its presence exclusively in non-O1/non-O139 strains (27.0%; 53 of 196) and not in O1 (n = 485) or O139 (n = 84). Sequencing of these 53 chxA genes generated 29 subtypes which were grouped into three clusters designated chxA I, chxA II, and chxA III. chxA I belongs to the prototype, while chxA II and chxA III are newly discovered variants. ChxA II and ChxA III had unique receptor binding and catalytic domains, respectively, in comparison to ChxA I. Recombinant ChxA I (rChxA I) and rChxA II but not rChxA III showed variable cytotoxic effects on different eukaryotic cells. Although rChxA II was more lethal to mice than rChxA I when injected intravenously, no enterotoxicity of any rChxA was observed in a rabbit ileal loop test. Hepatocytes showed coagulation necrosis in rChxA I- or rChxA II-treated mice, seemingly the major target for ChxA. The present study illustrates the potential of ChxA as an important virulence factor in non-O1/non-O139 V. cholerae, which may be associated with extraintestinal infections rather than enterotoxicity.

Immunohistochemical characterization of macrophages and myofibroblasts in α-Naphthylisothiocyanate (ANIT)--induced bile duct injury and subsequent fibrogenesis in rats.

To investigate pathogenesis of post-bile duct (BD) injury fibrosis, interlobular BD epithelial injury was induced in male F344 rats by a single IP injection of α-naphthylisothiocyanate (75 mg/kg body weight) and rats were observed for 12 days. On days 1 to 2, cholangiocytes were injured and desquamated. On days 3 to 5, the affected BD began to regenerate, showing positive staining for CK19 and vimentin. On days 5 to 9, fibrotic areas gradually developed around regenerating BD in Glisson's sheath. These consisted of cells positive for vimentin, desmin, and α-SMA; vimentin- and desmin-positive cells were increased in early stage (days 1-3), whereas α-SMA-positive cells appeared in mid (days 4-7) and late stages (days 8-12), although there were cells coexpressing these cytoskeletons. On day 12, BD regeneration almost completed, with reduced fibrosis. Macrophages positive for ED2 (CD163) increased transiently in early stage, whereas those reacting to ED1 (CD68), OX6 (MHC II), and SRA-E5 (CD204) showed a consistent increase throughout the experiment. Interestingly, OX6-positive cells were limited to Glisson's sheath, whereas SRA-E5-positive cells were seen exclusively along sinusoids of hepatic lobules. MCP-1 mRNA increased significantly in early stage. This study shows that macrophages exhibiting different immunophenotypes and distributions participate in post-BD injury fibrosis associated with myofibroblasts expressing various mesenchymal cytoskeletons.