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.

M1/M2-macrophage Polarization-based Hepatotoxicity in d-galactosamine-induced Acute Liver Injury in Rats.

d-galactosamine (d-GalN) is a well-known hepatotoxic agent that causes liver injury. Conversely, hepatic macrophages play a crucial role in maintaining liver tissue integrity. Macrophage functions were investigated in hepatic lesions induced by a single intraperitoneal injection of d-GalN (800 mg/kg body weight [BW]) in 6-week-old F344 rats. Blood and liver samples were examined at 8 hr and on 1, 2, 3, and 5 days postsingle injection (PSI). Hepatic lesions consisting of degeneration/sporadic foci of coagulation necrosis, inflammatory cell reaction, and reparative fibrosis were seen on PSI days 1 and 2, reflected by significantly increased serum levels of aspartate transaminase and alanine transaminase and upregulation of CD68 M1 (tumor necrosis factor-α, interleukin [IL]-6, and interferon-γ) and CD163 M2 (transforming growth factor-ß1, IL-10, monocyte chemoattractant protein-1, and IL-4) macrophage-related factors. Double immunofluorescence staining on PSI day 2 demonstrated that 82% of hepatic macrophages expressed of CD163/CD68 simultaneously; 65-75% of MHC class II macrophages showed co-expression of CD163 or CD68 and 95% CD204-expressing macrophages reacted to CD163 or CD68. These findings showed that both M1- and M2-macrophages contributed to the development of hepatic lesions induced by d-GalN and provided information about macrophage activation, indicating the importance of analysis of macrophage phenotypes for hepatotoxicity based on M1/M2-polarization.

Macrophage Populations and Expression of Regulatory Inflammatory Factors in Hepatic Macrophage-depleted Rat Livers under Lipopolysaccharide (LPS) Treatment.

To investigate the significance of the appearance of hepatic macrophages and expression of inflammatory factors in normal and macrophage-depleted livers, hepatic macrophages were depleted with liposome (Lipo)-encapsulated clodronate (CLD; 50 mg/kg, i.v.) followed by lipopolysaccharide (LPS) administration (0.1 mg/kg, i.p.) in F344 rats (CLD + LPS). Vehicle control rats (Lipo + LPS) received empty-Lipo before LPS. The low dose of LPS did not result in microscopic changes in the liver in either treatment group but did modulate M1 and M2 macrophage activity in Lipo + LPS rats without altering repopulating hepatic macrophages in CLD + LPS rats. LPS treatment in Lipo + LPS rats dramatically increased the M1 (IL-1ß, IL-6, TNF-α, and MCP-1) but not M2 macrophage-related factors (IL-4 and CSF-1) compared to CLD + LPS rats. In the CLD + LPS rats, the M2 macrophage-related factors IL-4 and CSF-1 were elevated. In conclusion, low-dose LPS activated hepatic macrophages in rat livers without causing liver injury or stimulating repopulating hepatic macrophages. These data suggest that LPS may alter the liver microenvironment by modulating M1 or M2 macrophage-related inflammatory mediators and macrophage-based hepatotoxicity.

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.

Effects of imidacloprid-contaminated feed exposure on spermatogenic cells and Leydig cells in testes of adult male rabbits (Oryctolagus cuniculus).

This research was undertaken to assess the results of repeated exposure to the insecticide; imidacloprid (IMI)-contaminated feed on testicular tissue, spermatogenic cell population, Leydig cell number, and sperm morphology in adult male rabbits (n = 24). The treatment groups received IMI (Bildor® 100 mg/L water spray on green grass)-contaminated green grass without wash (n = 8, not-washed-feed rabbit group) and after wash (n = 8, washed-feed rabbit group) once daily for two weeks on an alternate day basis. The rest of the rabbits, as control, received a normal pesticide-free standard feed. During the exposure time, there was no evident toxic symptom found on regular monitoring of IMI-treated rabbits. Histopathologically, the thickness of tunica albuginea of testes reduced significantly with loosely arranged connective tissues in IMI-treated rabbits. Within the testes, the bizarre-shaped seminiferous tubules were seen with increased lumen diameter in IMI-treated rabbits. The spermatogenic cells were disorganized and detached from the basement membrane in seminiferous tubules of IMI-exposed testes of rabbits. The spermatogenic cell population decreased significantly (P < 0.05) in IMI-treated rabbits compared to control rabbits. Leydig cell number decreased significantly (P < 0.05) in IMI-treated rabbits. A high percentage of morphologically abnormal spermatozoa was seen in IMI-treated rabbits. The degree of the histopathological changes was more prominent in the testes of IMI-exposed not-washed-feed rabbits. The results showed that insecticide-IMI has toxicological effects on testicular tissues, mainly spermatogenic and Leydig cell population of adult rabbits which may cause infertility. A short running title: Effect of imidacloprid on testicular tissue of rabbits.

Effects of exposure to imidacloprid contaminated feed on the visceral organs of adult male rabbits (Oryctolagus cuniculus).

The best-known and often used systemic, broad-spectrum neonicotinoid pesticide is imidacloprid (IMI). This study was carried out on adult male rabbits (n = 12) to assess the residual effects of exposure to IMI-contaminated diet on the liver, lung, heart, and kidney. Pesticide-exposed rabbits (n = 6) received IMI contaminated green grass (Bildor® 0.5 ml (100 mg)/L water) every alternative day once daily for up to 15 days. The remaining rabbits were fed a standard diet free of pesticides as a control. During routine monitoring of the rabbits throughout the experiment, there were no apparent toxic symptoms identified. On days 16, after deep anesthesia blood and visceral organs were collected. The levels of hepatic serum aspartate transaminase and alanine transaminase were considerably elevated in IMI-exposed rabbits (p ≤ 0.05). Thin layer chromatography revealed that the residue of IMI was at the detectable level in the liver and stomach. Histopathologically, the liver revealed coagulation necrosis with granulomatous inflammation and congestion in portal areas with dilated and congested central veins. The lungs showed congestion of blood vessels and granulomatous inflammation around the terminal bronchiole. Accumulations of inflammatory cells were observed in the cortico-medullary junction in the kidney. The heart showed necrosis and infiltration of mononuclear cells within the cardiac muscles. The findings of the current study emphasize that IMI-contaminated feed exposure causes toxicity into the cellular level of different visceral organs of adult male rabbits and it may also cause the similar toxic effects of the other mammals specially the occupationally exposed persons.

Draft genome sequence of multidrug-resistant Escherichia coli MAHK_SCM_BAU_30A strain isolated from a subclinical mastitis cow in Bangladesh.

This study announces the sequence of a multidrug-resistant Escherichia coli MAHK_SCM_BAU_30A strain isolated from bovine subclinical mastitis milk in 2022 in Bangladesh. Our assembled genome had a length of 4,884,948 bp, three plasmids, two CRISPR arrays, five prophages, 51 predicted antibiotic resistance, and 72 predicted virulence factor genes.

Electrical conductivity and total dissolved solid of raw milk for the detection of bovine subclinical mastitis.

Background and Aim: Bovine subclinical mastitis (SCM) is highly prevalent among dairy cattle. A cross-sectional study was conducted in Bangladesh to evaluate the performance of electric conductivity (EC) and total dissolved solids (TDS) tests for the detection of SCM. Materials and Methods: We randomly selected 108 milk samples from cows of different breeds in the primary milk-producing region of Pabna and Sirajgonj districts of Bangladesh. Samples were subjected to the California mastitis test (CMT), white side test (WST), electric conductivity (EC), TDS, and culture. A cow was considered positive for SCM if it tested positive in CMT, WST, and culture, whereas a cow was considered negative for SCM if it tested negative in all three methods. These gold standards have been used to evaluate the performance of the EC and TDS tests. The optimal EC and TDS cutoff values for the detection of SCM were determined using the "optimal cutoff" function in R version 4.3.1. Results: The optimal EC cutoff value for SCM detection was found to be 6159 µS/cm or 6.16 mS/cm. A positive likelihood ratio (LR+) of 31.2 and an area under the curve (AUC) of 0.905 were obtained for this cutoff value. The optimal cutoff value for TDS was 3100 mg/L of milk, which resulted in a positive LR+ of 45.5 and an AUC of 0.924. Conclusion: To the best of our knowledge, this is the first study to evaluate the performance of EC and TDS tests in detecting SCM in Bangladesh. These results suggest that EC and TDS tests, which are inexpensive, rapid, and easy to conduct, can effectively detect SCM at the farm level.

Pathological study and molecular detection of zoonotic diseases in small ruminants at slaughterhouses in Mymensingh, Bangladesh.

Background and Aim: Slaughterhouses act as a significant public health hotspot in developing countries like Bangladesh. The study aimed to investigate small ruminants at slaughterhouses for pathological study and molecular detection of important zoonotic diseases. Materials and Methods: A total of 75 goats and 14 sheep were investigated from June 2019 to January 2020 at different slaughterhouses in Mymensingh division, Bangladesh. The targeted diseases were tuberculosis (TB), listeriosis, Q fever, brucellosis, anthrax, toxoplasmosis, hydatidosis, and linguatulosis. The tentative diagnosis was made based on gross and histopathological lesions. Polymerase chain reaction (PCR) was performed to confirm the causal agents of zoonotic diseases using disease-specific primers. Results: Grossly, caseous nodule formation in the visceral organs; enlarged and calcifications of mesenteric lymph nodes (MLNs); hydatid cyst formation in the liver were the predominant lesions observed. Histopathologically, granuloma, caseous necrosis, and calcifications admixed with acid-fast bacteria in the MLNs, liver, spleen, and kidney were seen as suggestive of infectivity due to TB. Septic lymphadenitis mixed with rod-shaped bacteria, doughnut granuloma, fibroplasia accompanied by eosinophils and lymphocytic infiltration in MLNs, and portal granuloma were observed in listeriosis, Q fever, linguatulosis, and toxoplasmosis suspected cases, respectively. The PCR amplified Mycobacterium tuberculosis complex (372 bp), Mycobacterium bovis (600 bp), Listeria monocytogenes (517 bp), Toxoplasma gondii (512 bp), and Coxiella burnetii (687 bp) species-specific amplicons. In addition, linguatulosis and hydatidosis were identified in six and three goats, respectively. Brucellosis and anthrax were not detected in any cases. The slaughterhouse samples were also found to harbor the coexistence of different zoonotic pathogens. Conclusion: Deadly infectious zoonotic diseases in goats and sheep at slaughterhouses may cause widespread public health risks. As a result, more intensive monitoring and epidemiological surveys are required to successfully prevent and control zoonotic diseases.

Identification of peste des petits ruminants virus along with co-infecting diseases of goats in Bangladesh.

Objective: Peste des petits ruminants (PPR) virus is the main infectious cause of goat mortality in Bangladesh, and co-infection may make diseases more severe. This study aimed to detect PPR and co-infecting diseases in goats. Materials and Methods: One hundred goats suspected to be infected with the PPR virus were collected from various areas of Mymensingh district, Bangladesh. A systemic post-mortem examination was carried out on PPR-suspected goats. Lungs, spleen, and lymph nodes (pre-scapular) were used for ribonucleic acid extraction, whereas lungs and mesenteric lymph nodes were used for deoxyribonucleic acid extraction. Seven-pair primer sets were used for molecular detection of pathogens specific for PPR, goat pox, contagious ecthyma (Orf), foot and mouth disease (FMD) virus, Klebsiella sp., and Mycobacterium sp. Reverse transcriptase-polymerase chain reaction (RT-PCR) or polymerase chain reaction (PCR) were used to find the exact cause. Results: Out of 100 PPR-suspected goats examined, 55 goats were confirmed as PPR-detected by RT-PCR. Among the 55 PPR-positive goats, 2 were co-infected with goat pox, 2 with tuberculosis, 10 with Klebsiella sp. infection, and 6 with FMD as detected by PCR and RT-PCR. Moreover, 12 goats were co-infected with PPRV and fascioliasis. Conclusion: About 58% of PPR virus-infected goats were co-infected with other organisms. There is a need to design technology to detect the state of co-infectivity at its early onset and future preventive and therapeutic strategies for co-infecting diseases. This is the first study in Bangladesh to describe co-infecting diseases of goats along with PPR.

Fascioliasis may promote tuberculous infectivity in small ruminants.

Fascioliasis and bovine tuberculosis (TB) are global impediments to livestock development. We investigated the co-infectivity of fascioliasis and TB in small ruminants at slaughter. A total of 84 goats and 16 sheep were investigated from different slaughter houses in Mymensingh city, Bangladesh from June 2019 to February 2020. Grossly, acute fascioliasis was characterized by hemorrhagic tracts in the liver and chronic fascioliasis with biliary cirrhosis and pipe-stem liver. Grossly, seven goats and two sheep were associated with the acute and sixty goats and seven sheep were associated with the chronic phase of fascioliasis. Five goats' livers showed both the acute and chronic phases of fascioliasis. In TB, granulomas with central core of caseous necrosis were seen in the lymph nodes (21), livers (10) and lungs (01) of goats or in the lymph nodes (03) and liver (01) of sheep. Histopathologically, biliary cirrhosis was seen in fascioliasis and granuloma, caseous necrosis and calcification in TB. In co-infection revealed granuloma (TB) with acid-fast bacilli and widespread biliary cirrhosis in the livers of goats (14) and sheep (02). The fragments of the 16S rRNA gene (372 bp, M. tuberculosis complex) and MPB83 gene (600 bp, M. bovis) were detected in the lymph nodes, livers and lungs using polymerase chain reaction. This study showed the existence of co-infectivity of Fasciola and M. bovis in goats and sheep in Bangladesh. Chronic fascioliasis may be associated with establishing tuberculous infection in small ruminants. Therefore, extremely zoonotic bovine TB control programs require active management of fascioliasis.

Immunophenotypic analysis of the distribution of hepatic macrophages, lymphocytes and hepatic stellate cells in the adult rat liver.

The liver consists of parenchymal hepatocytes and non-parenchymal cells. Non-parenchymal cells, Kupffer cells, hepatic stellate cells and cholangiocytes have crucial roles in liver homeostasis and liver pathology. To establish baseline data, this study investigated immunohistochemically the distribution of non-parenchymal cells in perivenular areas (PV), periportal areas (PP) and Glisson's sheath (GS) of adult rat liver. Liver tissues were collected from the left lateral lobe of rats. CD163-positive macrophages were seen along the sinusoid of PV and PP areas, indicating Kupffer cells. Double immunofluorescence showed, Kupffer cells partly co-expressed CD68 and MHC class II antigens in the liver. The numbers of Kupffer cells were significantly high in PP areas as compared with PV or GS areas. CD68-positive exudative macrophages were highly localized in PP and GS areas and a comparatively low PV area. MHC class II-positive dendritic cells (activated macrophages) were localized mainly in GS. Granzyme B-positive NK cells were mainly localized in the Glisson's sheath. CD3-positive T cells and CD20-positive B cells were distributed along the sinusoids of the PP and PV areas of hepatic lobules. Vimentin and glial fibrillary acidic protein (GFAP)-positive hepatic stellate cells were localized along sinusoids in the hepatic lobules of the liver. Cholangiocytes reacting to cytokeratin 19 were seen on interlobular bile ducts in Glisson's sheath of the liver. This study shows that heterogeneous macrophage populations, liver-resident lymphocytes and hepatic stellate cells localized in PP and PV areas or GS areas of the liver with cells specific patterns.

Properties of macrophages and lymphocytes appearing in rat renal fibrosis followed by repeated injection of cisplatin.

Properties of macrophages and lymphocytes appearing in renal fibrosis remains to be investigated. F344 rats were injected once a week with cisplatin (2 mg/kg body weight) for 8 weeks and examined at post-final injection weeks 1, 3, 6, 9, and 12. Rats developed progressive renal fibrosis at weeks 1 to 6 as fibrosis-progress phase, and subsequent amelioration at weeks 9 and 12. CD68+ M1-macrophages and major histocompatibility complex (MHC) class II+ macrophages remarkably increased persistently, whereas CD163+ M2-macrophages slightly increased. MHC class II+/CD68+ and MHC class II+/CD163+ macrophages were present, indicating that MHC class II+ macrophages might have both functions of M1- and M2-macrophages. In the fibrosis-progress phase, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ for M1-factors, and transforming growth factor (TGF)-ß1 and IL-10 for M2-factors tended to increase; tissue injury by M1 and fibrosis by M2 might have occurred simultaneously. Lots of CD4+ and CD8+ T cells appeared in close relation with MHC class II+ macrophages, and mainly CD4+ T cells formed aggregations. In the lymphocyte aggregates collected by laser microdissection, expression of IL-17A (for Th17 cells) and forkhead box P3 (FoxP3) (for Treg) significantly increased at weeks 1 and 6, respectively; presumably, Th17 cells might be involved in tissue injury, whereas Treg might be related to fibrosis amelioration. These results suggested that macrophages and T cells may contribute interrelatedly to renal fibrosis.

Effect of formaldehyde and urea contaminated feed exposure into the liver of young and adult pigeons (Columba livia).

BACKGROUND AND AIM: Nowadays, toxic chemical contaminants in food are a major food safety problem in Bangladesh. Among toxic food contaminants, formalin is used to preserve fruit, vegetables, and fish, where urea is used for the whitening of rice and puffed rice. The purpose of this study was to determine the biochemical and histopathological effects on the liver of young and adult pigeons after exposure to formalin and urea contaminated feed. MATERIALS AND METHODS: A total of 15 young and 15 adult pigeons were divided into control group, formaldehyde exposed group (2.5 mL formalin/kg feed), and urea exposed (1 g/kg feed) group. Each group consisted of five pigeons. After the experimentation procedures, the blood samples were collected for biochemical study, and the liver tissue was collected for histomorphological study. The statistical analysis was performed using the Student's t-test, and p<0.05 was considered as statistically significant. RESULTS: The aspartate transaminase serum hepatic enzyme was significantly increased in both formalin and urea exposed young and adult pigeons than the control pigeons. In control pigeons, parenchymal hepatocytes and non-parenchymal cells are regularly arranged. However, histological observation of the liver of formalin and urea exposed young, and adult pigeons showed coagulation necrosis with infiltration of many inflammatory cells around the central and portal veins. The necrotic areas are more extensive with massive infiltration of inflammatory cells in the liver of formalin-treated pigeons than the urea treated pigeons. CONCLUSION: The present study results show that low concentrations of formalin and urea in feed induced liver lesions in pigeons in different extents and indicate that exposure to toxic chemicals may affect homeostasis of the liver and cause liver injury or act as a co-factor for liver disease.

Zoonotic diseases appeared to be a major hurdle to successful deer farming in Bangladesh.

BACKGROUND AND AIM: Due to the diversified lifestyle and fancy ecology associated with Chitra deer (Axis axis), deer farming has become popular in Bangladesh. Diseases may be the common constrain of successful deer farming. This study aims to investigate the pathological, bacteriological, and nucleic acid based technologies to identify specific causes of morbidity and mortality of captive deer. MATERIALS AND METHODS: Two deer farms and a park deer (designated as farm A, B, and C) entailing 87, 54, and 20 deer, respectively, showed illness and death constitute the study materials. A total of 42 deer died during this investigation. Following death, routine post-mortem examination, histopathology, impression smear staining, isolation, and identification of bacteria were carried out. Polymerase chain reaction (PCR) and reverse transcription PCR were carried out to safeguard the etiology. RESULTS: Clinically, farm A and B showed the acute phase of illness and park deer showed chronic illness. Case fatality rates were 90%, 92%, and 100% in farms A, B, and C deer, respectively. Pasteurella multocida and Streptococcus pneumoniae were identified from the visceral organs of farm A deer. Farm B deer was infected with Clostridium perfringens type A. Park deer was infected with Mycobacterium tuberculosis and hydatid cyst. CONCLUSION: The infectivity in farm A deer was due to stress as induced by punishing weather. The infectivity in farm B deer was due to feeding a higher volume of protein in the diet. The park C deer may optate infection from companion man and animals living around. The diseases of captive deer identified mainly were zoonotic. It needs extensive veterinary services and specialized technologies to identify these diseases, monitor the infectivity and eliminate the public health important diseases at early onset.

Attenuation of thioacetamide-induced hepatocellular injury by short-term repeated injections associated with down-regulation of metabolic enzymes and relationship with MHC class II-presenting cells.

The liver is the primary organ participating in the metabolism of xenobiotics and is therefore an important target in the safety assessment of drugs, chemicals and environmental toxins. Drug-induced liver injury (DILI) has recently become widely recognized in human medicine as an adverse event. The progression of DILI often involves "damage-associated molecular patterns" (DAMPs) of gene and protein expression such as high-mobility group boxes (HMGBs), S100 proteins and heat shock proteins (Hsp). DAMPs are released from injured or necrotic cells and are bound to Toll-like receptors (TLRs) and modulate inflammatory reactions. Previously, in thioacetamide (TAA; 300mg/kg body weight, single injection)-induced rat liver, we demonstrated that the expressions of DAMPs, TLR4 and major histocompatibility complex (MHC) class II were simultaneously increased, accompanied with progression of hepatocellular injury and inflammation. Here we investigated the association of DILI and DAMPs, TLRs and MHC class II by using rat livers repeated injections with TAA (100mg/kg body weight, once, three times). Two days after TAA single injection, centrilobular hepatocellular necrosis with infiltration of mononuclear cells was observed, being paralleled with increase in serum levels of aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP). However, two days after duplicate and triplicate injections, only mild degenerative change of hepatocytes and slight infiltration of mononuclear cells were seen in the affected centrilobular area. Serum levels of AST, ALT and ALP were also decreased to the same levels of control. mRNA expressions of DAMPs (HMGBs, S100A4 and Hsp 70-2), TLR4 and MHC class II tended to be increased only on single injection, although the number of MHC class II-positive cells in the centrilobular area was still increased on each examination point. The analysis of enzymes (CYP2E1 and Flavin monooxygenase (FMO) 3), which metabolize TAA in hepatocytes, showed a significant decrease in FMO3 on the duplicate and triplicate injections. Autophagy and regulatory T cells were not significantly changed for the attenuation of hepatocyte injury. Collectively, these results suggest that hepatocytes may adapt accumulation of the toxicant by changing their enzyme functions; furthermore, MHC class II cells, which still showed increased number in the duplicate and triplicate injections, may be related with protection from the toxicant.

The kinetics of damage-associated molecular patterns (DAMPs) and toll-like receptors during thioacetamide-induced acute liver injury in rats.

Drug-induced liver injury (DILI) is a common problem in human medicine and it is a major reason to withdraw marketed drugs. However, the mechanism of DILI is still less known. Damage-associated molecular patterns (DAMPs), such as high-mobility group boxes (HMGBs), S100 proteins and heat shock proteins (HSPs), are released from injured or necrotic cells, bind to toll-like receptors (TLRs) and modulate inflammatory reactions. Here we investigated the kinetics of DAMPs, TLRs and MHC class II in a rat model of DILI with thioacetamide (TAA). After TAA administration, extensive necrosis was observed on days 1 and 2, followed by infiltration of inflammatory cells on day 3. The levels of serum liver enzymes also peaked on day 1. Expression of HMGB-1, -2 and S100A4 peaked on day 2. TLR-4 was up-regulated on day 3. The number of MHC class II-positive macrophages increased until day 2. These results suggest that HMGB-1, -2 and S100A4 are associated with hepatocellular necrosis and that DAMPs may activate TLR-4 and MHC class II during TAA-induced liver injury. Our data would contribute to the elucidation of the mechanism of DILI.

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.