Deciphering and prediction of transcriptome dynamics under fluctuating field conditions.

Determining the drivers of gene expression patterns is more straightforward in laboratory conditions than in the complex fluctuating environments where organisms typically live. We gathered transcriptome data from the leaves of rice plants in a paddy field along with the corresponding meteorological data and used them to develop statistical models for the endogenous and external influences on gene expression. Our results indicate that the transcriptome dynamics are predominantly governed by endogenous diurnal rhythms, ambient temperature, plant age, and solar radiation. The data revealed diurnal gates for environmental stimuli to influence transcription and pointed to relative influences exerted by circadian and environmental factors on different metabolic genes. The model also generated predictions for the influence of changing temperatures on transcriptome dynamics. We anticipate that our models will help translate the knowledge amassed in laboratories to problems in agriculture and that our approach to deciphering the transcriptome fluctuations in complex environments will be applicable to other organisms.

JAK inhibition during the early phase of SARS-CoV-2 infection worsens kidney injury by suppressing endogenous antiviral activity in mice.

Coronavirus disease 2019 (COVID-19) induces respiratory dysfunction as well as kidney injury. Although the kidney is considered a target organ of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and affected by the COVID-19-induced cytokine storm, the mechanisms of renal reaction in SARS-CoV-2 infection are unknown. In this study, a murine COVID-19 model was induced by nasal infection with mouse-adapted SARS-CoV-2 (MA10). MA10 infection induced body weight loss along with lung inflammation in mice 4 days after infection. Serum creatinine levels and the urinary albumin/creatinine ratio increased on day 4 after MA10 infection. Measurement of the urinary neutrophil gelatinase-associated lipocalin/creatinine ratio and hematoxylin and eosin staining revealed tubular damage in MA10-infected murine kidneys, indicating kidney injury in the murine COVID-19 model. Interferon (IFN)-γ and interleukin-6 upregulation in the sera of MA10-infected mice, along with the absence of MA10 in the kidneys, implied that the kidneys were affected by the MA10 infection-induced cytokine storm rather than by direct MA10 infection of the kidneys. RNA-sequencing analysis revealed that antiviral genes, such as the IFN/Janus kinase (JAK) pathway, were upregulated in MA10-infected kidneys. Upon administration of the JAK inhibitor baricitinib on days 1-3 after MA10 infection, an antiviral pathway was suppressed, and MA10 was detected more frequently in the kidneys. Notably, JAK inhibition upregulated the hypoxia response and exaggerated kidney injury. These results suggest that endogenous antiviral activity protects against SARS-CoV-2-induced kidney injury in the early phase of infection, providing valuable insights into the pathogenesis of COVID-19-associated nephropathy.NEW & NOTEWORTHY Patients frequently present with acute kidney injury or abnormal urinary findings after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Here, we investigated how the kidneys respond during SARS-CoV-2 infection using a murine coronavirus disease 2019 (COVID-19) model and showed that Janus kinase-mediated endogenous antiviral activity protects against kidney injury in the early phase of SARS-CoV-2 infection. These findings provide valuable insights into the renal pathophysiology of COVID-19.

In vivo transfection of cytokine genes into tumor cells using a synthetic vehicle promotes antitumor immune responses in a visceral tumor model.

The tumor microenvironment (TME) strongly affects the clinical outcomes of immunotherapy. This study aimed to activate the antitumor immune response by manipulating the TME by transfecting genes encoding relevant cytokines into tumor cells using a synthetic vehicle, which is designed to target tumor cells and promote the expression of transfected genes. Lung tumors were formed by injecting CT26.WT intravenously into BALB/c mice. Upon intravenous injection of the green fluorescent protein-coding plasmid encapsulated in the vehicle, 14.2% tumor-specific expression was observed. Transfection of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and CD40 ligand (L)-plasmid combination and interferon gamma (IFNγ) and CD40L-plasmid combination showed 45.5% and 54.5% complete remission (CR), respectively, on day 60; alternate treatments with both the plasmid combinations elicited 66.7% CR, while the control animals died within 48 days. Immune status analysis revealed that the density of dendritic cells significantly increased in tumors, particularly after GM-CSF- and CD40L-gene transfection, while that of regulatory T cells significantly decreased. The proportion of activated killer cells and antitumoral macrophages significantly increased, specifically after IFNγ and CD40L transfection. Furthermore, the level of the immune escape molecule programmed death ligand-1 decreased in tumors after transfecting these cytokine genes. As a result, tumor cell-specific transfection of these cytokine genes by the synthetic vehicle significantly promotes antitumor immune responses in the TME, a key aim for visceral tumor therapy.

Difference in the Mechanism of Iron Overload-Enhanced Acute Hepatotoxicity Induced by Thioacetamide and Carbon Tetrachloride in Rats.

Iron overload has been recognized as a risk factor for liver disease; however, little is known about its pathological role in the modification of liver injury. The purpose of this study is to investigate the influence of iron overload on liver injury induced by two hepatotoxicants with different pathogenesis in rats. Rats were fed a control (Cont), 0.8% high-iron (0.8% Fe), or 1% high-iron diet (1% Fe) for 4 weeks and were then administered with saline, thioacetamide (TAA), or carbon tetrachloride (CCl4). Hepatic and systemic iron overload were seen in the 0.8% and 1% Fe groups. Twenty-four hours after administration, hepatocellular necrosis induced by TAA and hepatocellular necrosis, degeneration, and vacuolation induced by CCl4, as well as serum transaminase values, were exacerbated in the 0.8% and 1% Fe groups compared to the Cont group. On the other hand, microvesicular vacuolation induced by CCl4 was decreased in 0.8% and 1% Fe groups. Hepatocellular DNA damage was increased by iron overload in both models, whereas a synergistic effect of oxidative stress by excess iron and hepatotoxicant was only present in the CCl4 model. The data showed that dietary iron overload exacerbates TAA- and CCl4-induced acute liver injury with different mechanisms.

Deficiency of interleukin-19 exacerbates acute lung injury induced by intratracheal treatment of hydrochloric acid.

Interleukin (IL-19) belongs to the IL-10 family of cytokines and plays diverse roles in inflammation, cell development, viral responses, and lipid metabolism. Acute lung injury (ALI) is a severe respiratory condition associated with various diseases, including severe pneumonia, sepsis, and trauma, lacking established treatments. However, the role of IL-19 in acute inflammation of the lungs is unknown. We reported the impact of IL-19 functional deficiency in mice crossed with an ALI model using HCl. Lungs damages, neutrophil infiltration, and pulmonary edema induced by HCl were significantly worse in IL-19 knockout (KO) mice than in wild-type (WT) mice. mRNA expression levels of C-X-C motif chemokine ligand 1 (CXCL1) and IL-6 in the lungs were significantly higher in IL-19 KO mice than in WT mice. Little apoptosis was detected in lung injury in WT mice, whereas apoptosis was observed in exacerbated area of lung injury in IL-19 KO mice. These results are the first to show that IL-19 is involved in acute inflammation of the lungs, suggesting a novel molecular mechanism in acute respiratory failures. If it can be shown that neutrophils have IL-19 receptors and that IL-19 acts directly on them, it would be a novel drug target.

High genome heterozygosity revealed vegetative propagation over the sea in Moso bamboo.

BACKGROUND: Moso bamboo (Phyllostachys edulis) is a typical East Asian bamboo that does not flower for > 60 years and propagates without seed reproduction. Thus, Moso bamboo can be propagated vegetatively, possibly resulting in highly heterozygous genetic inheritance. Recently, a draft genome of Moso bamboo was reported, followed by whole genome single nucleotide polymorphisms (SNP) analysis, which showed that the genome of Moso bamboo in China has regional characteristics. Moso bamboo in Japan is thought to have been introduced from China over the sea in 1736. However, it is unclear where and how Moso bamboo was introduced in Japan from China. Here, based on detailed analysis of heterozygosity in genome diversity, we estimate the spread of genome diversity and its pedigree of Moso bamboo. RESULTS: We sequenced the whole genome of Moso bamboo in Japan and compared them with data reported previously from 15 regions of China. Only 4.1 million loci (0.37% of the analyzed genomic region) were identified as polymorphic loci. We next narrowed down the number of polymorphic loci using several filters and extracted more reliable SNPs. Among the 414,952 high-quality SNPs, 319,431 (77%) loci were identified as heterozygous common to all tested samples. The result suggested that all tested samples were clones via vegetative reproduction. Somatic mutations may accumulate in a heterozygous manner within a single clone. We examined common heterozygous loci between samples from Japan and elsewhere, from which we inferred that an individual closely related to the sample from Fujian, China, was introduced to Japan across the sea without seed reproduction. In addition, we collected 16 samples from four nearby bamboo forests in Japan and performed SNP and insertion/deletion analyses using a genotyping by sequencing (GBS) method. The results suggested that a small number of somatic mutations would spread within and between bamboo groves. CONCLUSIONS: High heterozygosity in the genome-wide diversity of Moso bamboo implies the vegetative propagation of Moso bamboo from China to Japan, the pedigree of Moso bamboo in Japan, and becomes a useful marker to approach the spread of genome diversity in clonal plants.

Absence of Increased Susceptibility to Acetaminophen-Induced Liver Injury in a Diet-Induced NAFLD Mouse Model.

Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease and its influence on drug-induced liver injury (DILI) is not fully understood. We investigated whether NAFLD can influence acetaminophen (APAP [N-acetyl-p-aminophenol])-induced hepatotoxicity in a diet-induced obese (DIO) mouse model of NAFLD. The male C57BL/6NTac DIO mice, fed a high-fat diet for more than 12 weeks, developed obesity, hyperinsulinemia, impaired glucose tolerance, and hepatomegaly with hepatic steatosis, similar to human NAFLD. In the acute toxicity study after a single dose of APAP (150 mg/kg), compared with control lean mice, the DIO mice had decreased serum transaminase levels and less severe hepatocellular injury. The DIO mice also had altered expression of genes related to APAP metabolism. Chronic APAP exposure for 26 weeks did not predispose the DIO mice with NAFLD to more severe hepatotoxicity compared with the lean mice. These results suggested that the C57BL/6NTac DIO mouse model appears to be more tolerant to APAP-induced hepatotoxicity than lean mice, potentially related to altered xenobiotic metabolizing capacity in the fatty liver. Further mechanistic studies with APAP and other drugs in NAFLD animal models are necessary to investigate the mechanism of altered susceptibility to intrinsic DILI in some human NAFLD patients.

The effect of lipopolysaccharide on liver homeostasis and diseases based on the mutual interaction of macrophages, autophagy, and damage-associated molecular patterns in male F344/DuCrlCrlj rats.

Lipopolysaccharide (LPS) has dose-dependent biphasic functions (cell protective versus cell toxic). To clarify the different effects of LPS on liver homeostasis or liver diseases, comparisons were made between low and high doses of LPS, in terms of the mutual relation of hepatic macrophages, autophagy, and damage-associated molecular patterns (DAMPs) in male F344/DuCrlCrlj rats. Rats injected with low dose (0.1 mg/kg) or high dose (2.0 mg/kg) of LPS were examined at 6, 10, and 24 hours following single injections. Histologically, focal hepatocellular necrosis was occasionally present in high-dose animals, whereas there were no significant changes in low-dose animals. In low-dose animals, Kupffer cells reacting to CD163 and CD204 were hypertrophic and regarded as M2 macrophages, which promote resolution of inflammation and tissue repair, whereas in high-dose animals, infiltration of M1 macrophages expressing CD68 and major histocompatibility complex class II, which enhance cell injury, was seen. Hepatocytes with high-mobility-group box-1 (HMGB1) (one of DAMPs)-positive cytoplasmic granules appeared more frequently in high-dose animals than in low-dose animals, indicating the translocation of nuclear HMGB1 into the cytoplasm. However, although light-chain 3 beta-positive autophagosomes in hepatocytes increased in both doses, abnormally vacuolated autophagosomes were only seen in injured hepatocytes in the high-dose group, indicating possible extracellular release of HMGB1, which might result in cell injury and inflammation. These findings suggested that low-dose LPS induced a favorable mutual relationship among hepatic macrophages, autophagy, and DAMPs leading to cytoprotection of hepatocytes, whereas failures of the relationship in high-dose LPS caused hepatocyte injury.

Macrophage pathology in hepatotoxicity.

The liver is the most important organ that metabolizes and detoxifies chemicals taken into the body. Therefore, there is always a risk of liver damage owing to the toxic effects of chemicals. The mechanisms of hepatotoxicity have been studied extensively and deeply based on toxic effects of chemicals themselves. However, it is important to note that liver damage is variously modified by the patho-biological reactions evoked mainly via macrophages. Macrophages appearing in hepatotoxicity are evaluated by the M1/M2 polarization; M1 macrophages promote tissue injury/inflammation, whereas M2 macrophages show anti-inflammatory action including reparative fibrosis. The "portal vein-liver barrier" regulated by Kupffer cells and dendritic cells in and around the Glisson's sheath may be related to the initiation of hepatotoxicity. In addition, Kupffer cells exhibit the two-sides of functions (that is, M1 or M2 macrophage-like functions), depending on microenvironmental conditions which may be raised in part by gut microbiota-derived lipopolysaccharide. Furthermore, damage-associated molecular patterns (DAMPs) (in particular, HMGB1) and autophagy (which degrades DAMPs) also play roles in the polarity of M1/M2 macrophages. The mutual relation of "DAMPs (HMGB-1)-autophagy-M1/M2 macrophage polarization" as the patho-biological reaction should be taken into consideration in hepatotoxicity evaluation.

Analyses of damage-associated molecular patterns, particularly biglycan, in cisplatin-induced rat progressive renal fibrosis.

Damage-associated molecular patterns (DAMPs) and their receptors (TLR-2 and -4) may play important roles in renal fibrosis, of which the pathogenesis is complicated. We used rat renal lesions induced by a single intraperitoneal injection of cisplatin at 6 mg/kg body weight; consisting of tissue damage of renal tubules on days 1 and 3, further damage and regeneration with inflammation mainly on days 5 and 7, and interstitial fibrosis on days 9, 12, 15, and 20. Microarray analyses on days 5 (the commencement of inflammation) and 9 (the commencement of interstitial fibrosis) showed that DAMPs increased by more than two-fold relative to control included common extra-cellular matrix (ECM) components such as laminin (Lamc2) and fibronectin, and heat shock protein family, as well as fibrinogen, although it was limited analysis; Lamc2, an element of basement membrane, may be regarded as an indicator for damaged renal tubules. In the real-time RT-PCR analyses, TLR-2 significantly increased transiently on day 1, whereas TLR-4 significantly increased on days 9 and 15, almost in agreement with the increased biglycan (a small leucine-rich proteoglycan as ubiquitous ECM component). As M1/M2 macrophages participated in renal lesions, such as inflammation and fibrosis, presumably, TLR-4, which may be expressed in immune cells, could play crucial roles in the formation of renal lesions in association with biglycan.

What is going on with the hormonal control of flowering in plants?

Molecular genetic studies using Arabidopsis thaliana as a model system have overwhelmingly revealed many important molecular mechanisms underlying the control of various biological events, including floral induction in plants. The major genetic pathways of flowering have been characterized in-depth, and include the photoperiod, vernalization, autonomous and gibberellin pathways. In recent years, novel flowering pathways are increasingly being identified. These include age, thermosensory, sugar, stress and hormonal signals to control floral transition. Among them, hormonal control of flowering except the gibberellin pathway is not formally considered a major flowering pathway per se, due to relatively weak and often pleiotropic genetic effects, complex phenotypic variations, including some controversial ones. However, a number of recent studies have suggested that various stress signals may be mediated by hormonal regulation of flowering. In view of molecular diversity in plant kingdoms, this review begins with an assessment of photoperiodic flowering, not in A. thaliana, but in rice (Oryza sativa); rice is a staple crop for human consumption worldwide, and is a model system of short-day plants, cereals and breeding crops. The rice flowering pathway is then compared with that of A. thaliana. This review then aims to update our knowledge on hormonal control of flowering, and integrate it into the entire flowering gene network.

Reloading DNA History in Rice Domestication.

Although crop domestication is a prehistoric event, DNA (or genome) sequences of modern cultivars and the accession lines of wild relatives contain information regarding the history of crop domestication and the breeding process. Accordingly, with plentiful genomic data, many new findings have been obtained concerning the crop domestication process, for which various (some controversial) interpretations exist. Since approximately 20 years ago, dozens of quantitative trait genes (QTGs) related to the domestication process have been cloned from several crops including rice, a global staple food. However, the determination of how and when these QTGs were involved in rice domestication requires a precise understanding of the DNA code. In addition to the identification of domestication-related QTGs, large-scale rice genome analysis based on short-read Illumina data (but with shallow depth) including more than 1,000 rice cultivars and hundreds of wild rice (or Oryza rufipogon) lines, along with extensive genome analysis including more than 3,000 cultivars with sufficient Illumina data, has been reported. From these data, the genome-wide changes during rice domestication have been explained. However, these genome-wide changes were not interpreted based on QTG changes for domestication-related traits during rice domestication. In addition, a substantial gap remains between the archeological hypothesis based on ancient relics and findings from DNA variations among current cultivars. Thus, this review reconsiders the present status of rice domestication research from a biologist's perspective.

Possible Cytoprotection of Low Dose Lipopolysaccharide in Rat Thioacetamide-Induced Liver Lesions, Focusing on the Analyses of Hepatic Macrophages and Autophagy.

Lipopolysaccharide (LPS) may influence hepatic macrophages and autophagy. We evaluated the potential participation of macrophages and autophagosomes in thioacetamide (TAA)-induced rat liver injury under pretreatment of a low dose LPS (0.1 mg/kg BW, intraperitoneally; nonhepatotoxic dose). F344 rats were pretreated with LPS (LPS + TAA) or saline (TAA alone) at 24 hours before TAA injection (100 mg/kg BW, intraperitoneally); rats were examined on Days 0 (controls), 1, 2, and 3 after TAA injection. Data were compared between TAA alone and LPS + TAA rats. LPS pretreatment significantly reduced TAA-induced hepatic lesion (centrilobular necrosis with inflammation) on Days 1 and 2, being reflected by declined hepatic enzyme values and decreased number of apoptotic cells. LC3B-immunoreacting autophagosomes (as cytoplasmic fine granules) were significantly increased on Days 1 and 2 in hepatocytes of LPS + TAA rats. In LPS + TAA rats, hepatic macrophages reacting to CD68, CD163, and MHC class II mainly on Day 2 and mRNA levels of macrophage-related factors (MCP-1, IL-1ß, and IL-4) on Day 1 were significantly decreased. Collectively, the low-dose LPS pretreatment might act as cytoprotection against TAA-induced hepatotoxicity through increased autophagosomes and decreased hepatic macrophages, although the dose/time-dependent cytoprotection of LPS should be further investigated at molecular levels.

High Ambient Temperatures Inhibit Ghd7-Mediated Flowering Repression in Rice.

The anticipation of changing seasons is crucial for reproduction in plants. Despite the broad cultivation area, the effects of ambient temperature on photoperiodic flowering are largely unknown in rice. Here, we first examined flowering time under four distinct conditions: short-day or long-day and high or low temperature, using cultivars, nearly isogenic lines, and mutants in rice. We also examined gene expression patterns of key flowering-time genes using the same lines under various conditions including temporal dynamics after light pulses. In addition to delayed flowering because of low growth rates, we found that photoperiodic flowering is clearly enhanced by both Hd1 and Ghd7 genes under low-temperature conditions in rice. We also revealed that PhyB can control Ghd7 repressor activity as a temperature sensor to inhibit Ehd1, Hd3a and RFT1 at lower temperatures, likely through a post-transcriptional regulation, despite inductive photoperiod conditions. Furthermore, we found that rapid reduction of Ghd7 messenger RNA (mRNA) under high-temperature conditions can lead to mRNA increase in a rice florigen gene, RFT1. Thus, multiple temperature-sensing mechanisms can affect photoperiodic flowering in rice. The rising of ambient temperatures in early summer likely contributes to the inhibition of Ghd7 repressor activity, resulting in the appropriate floral induction of rice in temperate climates.

Oleamide rescues tibialis anterior muscle atrophy of mice housed in small cages.

Skeletal muscle atrophy causes decreased physical activity and increased risk of metabolic diseases. We investigated the effects of oleamide (cis-9,10-octadecanamide) treatment on skeletal muscle health. The plasma concentration of endogenous oleamide was approximately 30 nm in male ddY mice under normal physiological conditions. When the stable isotope-labelled oleamide was orally administered to male ddY mice (50 mg/kg), the plasma concentration of exogenous oleamide reached approximately 170 nm after 1 h. Male ddY mice were housed in small cages (one-sixth of normal size) to enforce sedentary behaviour and orally administered oleamide (50 mg/kg per d) for 4 weeks. Housing in small cages decreased tibialis anterior (TA) muscle mass and the cross-sectional area of the myofibres in TA muscle. Dietary oleamide alleviated the decreases in TA muscle and resulted in plasma oleamide concentration of approximately 120 nm in mice housed in small cages. Housing in small cages had no influence on the phosphorylation levels of Akt serine/threonine kinase (Akt), mechanistic target of rapamycin (mTOR) and ribosomal protein S6 kinase (p70S6K) in TA muscle; nevertheless, oleamide increased the phosphorylation levels of the proteins. Housing in small cages increased the expression of microtubule-associated protein 1 light chain 3 (LC3)-II and sequestosome 1 (p62), but not LC3-I, in TA muscle, and oleamide reduced LC3-I, LC3-II and p62 expression levels. In C2C12 myotubes, oleamide increased myotube diameter at ≥100 nm. Furthermore, the mTOR inhibitor, Torin 1, suppressed oleamide-induced increases in myotube diameter and protein synthesis. These results indicate that dietary oleamide rescued TA muscle atrophy in mice housed in small cages, possibly by activating the phosphoinositide 3-kinase/Akt/mTOR signalling pathway and restoring autophagy flux.

Appearance of Heterogeneous Macrophages During Development of Isoproterenol-Induced Rat Myocardial Fibrosis.

Macrophages appearing in lesions are polarized toward M1 (for inflammation) and M2 (for anti-inflammation/fibrosis) types. We analyzed immunophenotypes of macrophages appearing in myocardial lesion in rats injected once with isoproterenol (10 mg/kg body weight). Inflammation following myocardial necrosis on day 1 was seen with a peak on days 3 and 5, and thereafter, reparative fibrosis developed on days 7 to 28. CD68+ M1 macrophages were seen in the early stages of injury and inflammatory on days 1 to 7, and thereafter, CD163+ M2 macrophages increased in the late stages of fibrosis on days 7 to 28. There was the polarization of M1 and M2 macrophages. The kinetics of macrophages reacting to Iba-1 and Galectin-3 was similar to that of M1 macrophages, indicating that Iba1- and Gal-3-positive macrophages might have functions of M1 type. Double immunofluorescence revealed that CD204- and MHC class II-positive macrophages are polarized toward M1 and M2 types, respectively. CCR2 messenger RNA expression is transiently elevated on day 1. Since CCR2 is a marker of blood monocytes, M1 macrophages might be recruited from blood monocytes. Collectively, macrophages expressing heterogeneous immunophenotypes participate in myocardial fibrosis. These findings would be useful for understanding the pathogenesis of myocardial fibrosis and analyzing myocardial toxicity.

Characterization of Immature Myofibroblasts of Stellate Cell or Mesenchymal Cell Origin in D-Galactosamine-Induced Liver Injury in Rats.

Lesions of D-galactosamine (D-GalN)-induced hepatotoxicity resemble those of human acute viral hepatitis. This study investigated hepatic mesenchymal cells including hepatic stellate cells (HSCs) and myofibroblasts in D-GalN-induced hepatotoxicity. Rats, injected with D-GalN (800 mg/kg body weight, once, intraperitoneally) were examined on post single injection (PSI) at 8 hours and days 1 to 5. Lesions consisting of hepatocyte necrosis and reparative fibrosis were present diffusely or focally within the hepatic lobules on PSI days 1 and 2, and then the injury recovered on PSI days 3 and 5. Myofibroblasts expressing vimentin, desmin, and α-smooth muscle actin (α-SMA) were present in the lesions. Double immunofluorescence showed that myofibroblasts reacted simultaneously to vimentin/α-SMA, desmin/α-SMA, and desmin/vimentin; furthermore, myofibroblasts reacting to vimentin, desmin, and α-SMA also co-expressed glial fibrillary acidic protein (GFAP), a marker of HSCs. Additionally, GFAP-expressing myofibroblasts reacted to nestin and A3 (both are markers of immature mesenchymal cells). Cells reacting to Thy-1, a marker for immature mesenchymal cells, also appeared in fibrotic lesions. In agreement with the myofibroblastic appearance, mRNAs of fibrosis-related factors (TGF-ß1, PDGF-ß, TNF-α, Timp2, and Mmp2) increased mainly on PSI days 1 and 2. Myofibroblasts with expression of various cytoskeletal proteins were present in diffuse or focal hepatic lesions, and they might be derived partly from immature HSCs and from immature mesenchymal cells.

Analyses of hemorrhagic diathesis in high-iron diet-fed rats.

Iron overload has been well recognized to cause oxidant-mediated cellular/tissue injury; however, little is known about the effects of iron overload on the blood coagulation system. We encountered an unexpected bleeding tendency in rats fed a high-iron diet in a set of studies using iron-modified diets. In this study, we investigated the mechanism of hemorrhagic diathesis induced by dietary iron overload in rats. Six-week-old F344/DuCrlCrlj male rats were fed a standard (containing 0.02% iron) or a high-iron diet (containing 1% iron) for 6 weeks and were then sampled for hematological, blood biochemical, coagulation, and pathological examinations. Serum and liver iron levels increased in rats fed the high-iron diet (Fe group) and serum transferrin was almost saturated with iron. However, serum transaminase levels did not increase. Moreover, plasma prothrombin time and activated partial thromboplastin time were significantly prolonged, regardless of the presence of hemorrhage. The activity of clotting factors II and VII (vitamin K-dependent coagulation factors) decreased significantly, whereas that of factor VIII was unaltered. Blood platelet levels were not influenced by dietary iron overload, suggesting that the bleeding tendency in iron-overloaded rats is caused by secondary hemostasis impairment. In addition, hemorrhage was observed in multiple organs in rats fed diets containing more than 0.8% iron. Our results suggest that iron overload can increase the susceptibility of coagulation abnormalities caused by latent vitamin K insufficiency.

Participation of Somatic Stem Cells, Recognized by a Unique A3 Antibody, in Mucosal Epithelial Regeneration in Dextran Sulfate Sodium (DSS)-Induced Rat Colonic Lesions.

A3, generated as a monoclonal antibody against rat malignant fibrous histiocytoma cells, recognizes somatic stem cells in rats. We analyzed the distribution of A3-positive cells in dextran sulfate sodium (DSS)-induced colonic lesions consisting of regenerating mucosa and fibrosis. Male 6-week-old F344 rats were administered 5% DSS in drinking water for 5 to 7 days, and lesions at recovery stage were also examined. In untreated control adult colons, A3-positive cells are localized around the crypts where stem cell niche is formed. Histopathologically, in colons of DSS-administered rats, mucosal atrophy, inflammatory cell infiltration, and fibrosis were observed in the lamina propria; thereafter, mucosal epithelia were desquamated, and crypts were decreased gradually with decrease in surrounding A3-positive cells. At the early recovery stage, crypts showed regeneration with reappearance of A3-positive cells. Interestingly, A3-positive cells aggregated in desquamated mucosa surface of fibrosis. Aggregated A3-positive cells coexpressed with vimentin, Thy-1, and partly CK19 but did not react simultaneously with α-SMA. Likely, aggregated A3-positive cells may be rescue cells with nature of both mesenchymal and epithelial cells to maintain self-renewal after injury in the colon. A3 antibody would become a useful tool to investigate the participation of stem cells in rat colonic lesions.

Characterization of Macrophages and Myofibroblasts Appearing in Dibutyltin Dichloride-Induced Rat Pancreatic Fibrosis.

Macrophages and myofibroblasts are important in fibrogenesis. The cellular characteristics in pancreatic fibrosis remain to be investigated. Pancreatic fibrosis was induced in F344 rats by a single intravenous injection of dibutyltin dichloride. Histopathologically, the induced pancreatic fibrosis was divided into 3 grades (1+, 2+, and 3+), based on collagen deposition. Immunohistochemically, CD68-expressing M1 macrophages increased with grade and CD163-expressing M2 macrophages also increased later than M1 macrophage appearance. Double immunofluorescence showed that there were macrophages coexpressing CD68 and CD163, suggesting a possible shift from M1 to M2 types; similarly, increased major histocompatibility complex class II- and CD204-expressing macrophages were polarized toward M1 and M2 types, respectively. These findings indicated the participation of M1- and M2-polarized macrophages. Mesenchymal cells staining positive for vimentin, desmin, and α-smooth muscle actin (α-SMA) increased with grade. There were mesenchymal cells coexpressing vimentin/α-SMA, desmin/α-SMA, and glial fibrillary acidic protein (GFAP)/α-SMA; Thy-1-expressing immature mesenchymal cells also increased in fibrotic lesions. Because α-SMA expression is a reliable marker for myofibroblasts, α-SMA-expressing pancreatic myofibroblasts might be originated from GFAP-expressing pancreatic stellate cells or Thy-1-expressing immature mesenchymal cells; the myofibroblasts could simultaneously express cytoskeletal proteins such as vimentin and desmin. The present findings would provide useful information for analyses based on features of macrophages and myofibroblasts in chemically induced pancreatic fibrosis.