Recombinant mycobacterial DNA-binding protein 1 with post-translational modifications boosts IFN-gamma production from BCG-vaccinated individuals' blood cells in combination with CpG-DNA.

Tuberculosis remains a large health threat, despite the availability of the tuberculosis vaccine, BCG. As BCG efficacy gradually decreases from adolescence, BCG-Prime and antigen-booster may be an efficient strategy to confer vaccine efficacy. Mycobacterial DNA-binding protein 1 (MDP1, namely Rv2986c, hupB or HU) is a major Mycobacterium tuberculosis protein that induces vaccine-efficacy by co-administration with CpG DNA. To produce MDP1 for booster-vaccine use, we have created recombinant MDP1 produced in both Escherichia coli (eMDP1) and Mycolicibacterium smegmatis (mMDP1), an avirulent rapid-growing mycobacteria. We tested their immunogenicity by checking interferon (IFN)-gamma production by stimulated peripheral blood cells derived from BCG-vaccinated individuals. Similar to native M. tuberculosis MDP1, we observed that most lysin resides in the C-terminal half of mMDP1 are highly methylated. In contrast, eMDP1 had less post-translational modifications and IFN-gamma stimulation. mMDP1 stimulated the highest amount of IFN-gamma production among the examined native M. tuberculosis proteins including immunodominant MPT32 and Antigen 85 complex. MDP1-mediated IFN-gamma production was more strongly enhanced when combined with a new type of CpG DNA G9.1 than any other tested CpG DNAs. Taken together, these results suggest that the combination of mMDP1 and G9.1 possess high potential use for human booster vaccine against tuberculosis.

Triterpenoid saponin from Panax ginseng increases the sensitivity of methicillin-resistant Staphylococcus aureus to ß-lactam and aminoglycoside antibiotics.

The triterpenoid saponins, ginsenosides, are the major bioactive compound of red ginseng and can exert various physiological activities. In the present study, we examined whether red ginseng extract (RGE) exerts antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). RGE had no bactericidal activity, at least in the range of dissolvable concentration. However, RGE reduced 0.03-0.25-fold the minimum inhibitory concentration (MIC) values of ß-lactam antibiotics (oxacillin, ampicillin, carbenicillin, and cefazolin) and aminoglycoside antibiotics (kanamycin and gentamicin) against the two laboratory strains of MRSA. Moreover, the fractional inhibitory concentration index indicated the synergistic activity of RGE with each of the antibiotics. RGE also increased the kanamycin sensitivity of 15 MRSA strains isolated from human volunteers and increased the ampicillin sensitivity of five MRSA strains isolated from dairy cows diagnosed with bovine mastitis. In contrast, RGE did not alter the MIC values of fosfomycin, tetracycline, and erythromycin, suggesting that RGE acts selectively. In contrast, Triton X-100, which was reported to reduce the MIC value of ß-lactam antibiotics to MRSA by increasing membrane permeability, reduced the MIC values of fosfomycin and tetracycline. These results indicate that RGE increases the bactericidal effect of antibiotics via a mechanism different from that used by Triton X-100. We found that ginsenoside Rg3 (Rg3), a component of RGE, was an essential compound that exhibits synergy activity with antibiotics. Furthermore, the non-natural compound K, which contains a common protopanaxadiol aglycon moiety with Rg3, also showed synergistic activity with antibiotics. Thus, Rg3 and compound K are potentially new antibiotic adjuvants against MRSA.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant organism that is prevalent worldwide. Therefore, the research and development of new agents against MRSA are required. We first found that ginsenoside Rg3 (Rg3) in red ginseng, made from the roots of Panax ginseng C. A. Meyer, increased the sensitivity of ß-lactam antibiotics and aminoglycoside antibiotics to MRSA. Furthermore, we identified that compound K, an unnatural ginsenoside analog, also increased the sensitivity of antibiotics to MRSA, similar to Rg3. By contrast, neither Rg3 nor compound K increased the sensitivity of fosfomycin, tetracycline, and erythromycin to MRSA, suggesting that these act selectively. In the present study, the natural compound Rg3 and its structural isomer, compound K, are potentially new antibiotic adjuvants against MRSA. Currently, multiple antibiotics are used to treat MRSA, but the use of these adjuvants is expected to enable the treatment of MRSA with a single antibiotic and low concentrations of antibiotics.

Association between diet quality and risk of stunting among school-aged children in Schistosoma mansoni endemic area of western Kenya: a cross-sectional study.

BACKGROUND: Healthy eating habits are essential for improving nutritional status and strengthening immunity against infectious diseases. This study examined the relationship between diet quality and stunting in school-aged children in an infectious disease-endemic area of western Kenya. METHODS: This cross-sectional study included 260 school-aged children (age 9-17 years) enrolled in primary schools in Mbita Sub-county, western Kenya. The nutritional status was assessed using anthropometric measurements. Dietary intake was measured using food frequency questionnaires and evaluated using the Food Pyramid (FP) score, which indicates adherence to the Kenyan food-based dietary guideline. Information on the children's age, sex, maternal education, and household wealth index was collected using a household-based questionnaire. Infections with the predominant parasites, such as Schistosoma (S.) mansoni, were detected via microscopy. The trend associations of the FP score with food group intake were examined to characterize the dietary intake of this population. Logistic regression analysis was performed to investigate the relationship between stunting and FP score tertiles, adjusted for sociodemographic and economic indicators and parasitic infection status. RESULTS: Among the studied schoolchildren, 15.0% exhibited stunting, while 76.2% were infected with S. mansoni. The mean FP score was 25.6 out of 50 points. A higher FP score was characterized by a high intake of roots and tubers, dairy products, pulses, and fruits and a low intake of cereals and animal-source foods. The analysis revealed a trend: a lower risk of stunting was evident in groups with elevated FP scores (p for trend = 0.065). However, these trend associations were observable among subjects with either negative or light S. mansoni infection (p for trend = 0.016). CONCLUSIONS: A higher quality diet, as evaluated by FP scores, was associated with a low risk of stunting among school-aged children. Notably, this association seemed to weaken in the presence of a high burden of S. mansoni infection. It highlights the importance of enhancing dietary quality through the promotion of diverse nutrient-dense foods alongside effective S. mansoni infection control for improved growth. This study contributes fundamental knowledge for understanding the diet-malnutrition relationship in areas endemic for S. mansoni infection.

Escherichia coli-derived outer-membrane vesicles induce immune activation and progression of cirrhosis in mice and humans.

BACKGROUND AND AIMS: Decompensated cirrhosis with fibrosis progression causes portal hypertension followed by an oedematous intestinal tract. These conditions weaken the barrier function against bacteria in the intestinal tract, a condition called leaky gut, resulting in invasion by bacteria and bacterial components. Here, we investigated the role of outer-membrane vesicles (OMVs) of Escherichia coli, which is the representative pathogenic gut-derived bacteria in patients with cirrhosis in the pathogenesis of cirrhosis. METHODS: We investigated the involvement of OMVs in humans using human serum and ascites samples and also investigated the involvement of OMVs from E. coli in mice using mouse liver-derived cells and a mouse cirrhosis model. RESULTS: In vitro, OMVs induced inflammatory responses to macrophages and neutrophils, including the upregulation of C-type lectin domain family 4 member E (Clec4e), and induced the suppression of albumin production in hepatocytes but had a relatively little direct effect on hepatic stellate cells. In a mouse cirrhosis model, administration of OMVs led to increased liver inflammation, especially affecting the activation of macrophages, worsening fibrosis and decreasing albumin production. Albumin administration weakened these inflammatory changes. In addition, multiple antibodies against bacterial components were increased with a progressing Child-Pugh grade, and OMVs were detected in ascites of patients with decompensated cirrhosis. CONCLUSIONS: In conclusion, OMVs induce inflammation, fibrosis and suppression of albumin production, affecting the pathogenesis of cirrhosis. We believe that our study paves the way for the future prevention and treatment of cirrhosis.

Escherichia coli-Derived Outer Membrane Vesicles Relay Inflammatory Responses to Macrophage-Derived Exosomes.

Extracellular vesicles are considered to be an inflammatory factor in several acute and chronic inflammatory diseases. The present study shows that exosomes from macrophages (Mφ) infected with live Escherichia coli induced secretion of proinflammatory factors by uninfected Mφ. Inflammatory responses induced by exosomes derived from Mφ infected with heat-inactivated E. coli or lipopolysaccharide were significantly weaker than those elicited by outer membrane vesicles (OMVs) released from live E. coli. Proteome analysis of exosomes from Mφ infected with live or heat-inactivated E. coli revealed that E. coli proteins OmpA, GroL1, DegP, CirA, and FepA are candidate triggers of exosome-mediated inflammatory responses. OMVs from a cirA-deleted strain suppressed exosome-mediated inflammatory responses by uninfected Mφ. The C terminus of the CirA protein (residues 158 to 633), which was relayed from E. coli-derived OMV to Mφ-derived exosomes, promoted exosome-mediated inflammatory responses by uninfected Mφ. These results suggest an alternative mechanism by which extracellular vesicles from E. coli OMV-elicited Mφ transmit proinflammatory responses to uninfected Mφ. IMPORTANCE Recently, extracellular membrane vesicles (EVs) were regarded as drivers that carry cargo such as proteins, lipids, metabolites, RNA, and DNA for intracellular signaling transduction. Mammalian cells release various types of EVs, including microvesicles shed from the plasma membrane, exosomes from endosomes, apoptotic bodies, and others. EVs have been reported to mediate inflammatory signals between mammalian cells. In addition, bacteria are also known to release EVs to carry various bacterial factors. In this study, we show that bacterial EVs lead host mammalian cells to release stimulatory EVs that enhance inflammatory responses. Our results provide a novel example that bacterial EVs transduce biological signals to mammalian EVs.

Interference of flagellar rotation up-regulates the expression of small RNA contributing to Bordetella pertussis infection.

Bacterial small RNAs (sRNAs) posttranscriptionally regulate gene expressions involved in various biological processes, including pathogenicity. Our previous study identified sRNAs, the expression of which was up-regulated in Bordetella pertussis, the causative agent of whooping cough, upon tracheal colonization of the bacteria; however, their roles in bacterial infection remain unknown. Here, we found that one sRNA, Bpr4, contributes to B. pertussis infection by posttranscriptionally up-regulating filamentous hemagglutinin (FHA), a major adhesin of the bacteria. Bpr4 bound to the 5' untranslated region of fhaB mRNA encoding FHA and inhibited its degradation mediated by RNaseE. Our results demonstrated that Bpr4 up-regulation was triggered by the interference of flagellar rotation, which caused the disengagement of MotA, a flagellar stator. Subsequently, MotA activated a diguanylate cyclase to generate cyclic di-GMP, which plays a role in Bpr4 up-regulation through the RisK/RisA two-component system. Our findings indicate that a flagellum-triggered sensory system contributes to B. pertussis infection.

Effect of Porcine Colostral Exosomes on T Cells in the Peripheral Blood of Suckling Piglets.

Growing evidence indicates that porcine colostral exosomes may contribute to the healthy development of piglets. Here, we evaluated in vitro the effect of porcine milk-derived exosomes, in particular colostral exosomes, on T cells in the peripheral blood of suckling piglets. A total of seven sows and thirteen suckling piglets were used. Peripheral blood mononuclear cells (PBMCs) from suckling piglets were cultured with or without milk-derived exosomes (control). Using flow cytometry, the proportion of each T cell subset in cultured PBMCs was analyzed three days post-incubation. PBMCs cultured with porcine colostral exosomes had a higher proportion of CD3+CD4-CD8+ T cells (cytotoxic T cells; Tc) than the control. However, exosomes induced no increase in the Tc cell population in PBMC whose endocytosis was inhibited. We further measured the concentrations of cytokines in the culture supernatant. Exosome-treated PBMCs had a higher cytokine IL-2 concentration than the control. The present study demonstrated that porcine colostral exosomes could increase the Tc cell proportion in the peripheral blood of suckling piglets, with the underlying mechanism believed to be the stimulation of IL-2 production in PBMCs via endocytosis. Moreover, our results suggested that porcine colostral exosomes were involved in the development of cellular immunity in suckling piglets.

Suppression of the doxorubicin response by hypoxia-inducible factor-1α is strictly dependent on oxygen concentrations under hypoxic conditions.

Hypoxia-inducible factor-1α (HIF-1α) and p53 are involved in anticancer drug resistance under hypoxic conditions. Here, we found that the cytotoxicity of anticancer drugs (doxorubicin, gemcitabine, and cisplatin) was lower at 1% O2 than at 5% O2. We examined the effects of these drugs on HIF-1α and p53 expression under different hypoxic oxygen concentrations. At 5% O2, the drugs decreased HIF-1α expression and increased p53 levels. At 1% O2, the drugs increased HIF-1α expression but did not alter p53 levels. When the HIF-1α protein was stabilized by DMOG under normoxic conditions, doxorubicin did not increase the level of p53 expression. These results show that the maintenance of HIF-1α expression blocked doxorubicin-dependent increases in p53 expression. We hypothesized the mechanism of HIF-1α protein translation might be different between at 5% and at 1% O2, because many reports indicate that the same mechanism of HIF-1α protein stabilization occurs under hypoxic conditions, such as 5% and 1% O2. The level of phosphorylated-4E-BP1, which causes translation of HIF-1α, was higher at 1% O2 than at 5% O2. Our results suggest that the sensitivity of tumor cells to anticancer drugs is dependent oxygen concentrations under hypoxic conditions, and involves 4E-BP1-dependent stabilization of the HIF-1α protein.

A Comparative Study on Egg Yolk IgY Production with Different Adjuvants and their Inhibitory Effects on Staphylococcus aureus.

Objectives: Atopic dermatitis (AD) is one of the most common skin disorders in infants and children and is often aggravated by increased Staphylococcus aureus (S. aureus) colonization. An inhibitory effect of a specific egg yolk antibody (IgY) on S. aureus growth was demonstrated in this study. Furthermore, the effects of water- or oil-based adjuvants on the preparation of anti-S. aureus IgY and hen immunization were compared. Methods: Hens were immunized intramuscularly with formalin-killed S. aureus mixed with either a water-soluble polysaccharide λ-carrageenan, oil-based Freund's complete adjuvant (FCA), or Freund's incomplete adjuvant (FIA). Anti-S. aureus IgYs (FIA-IgY, FCA/FIA-IgY, and λCarra-IgY) were purified from the egg yolk of immunized hen eggs, and the activity of the IgY against S. aureus antigen was measured by ELISA. The proportion of each IgY that was absorbed by S. aureus was also determined. Then, the effect of purified anti-S. aureus IgY on S. aureus growth inhibition was investigated in vitro. Results: The yolk of eggs and purified FIA-IgY from the FIA group showed the highest antibody activity, followed by FCA/FIA-IgY and λCarra-IgY. The proportion of each IgY that was absorbed by S. aureus antigen was as follows: FIA-IgY (18.1%), FCA/FIA-IgY (12.9%), and λCarra-IgY (7.0%). Only FIA-IgY significantly inhibited S. aureus growth in liquid medium. Conclusion: A specific IgY that was produced using the FIA adjutant inhibited S. aureus growth. Although water-soluble λ-carrageenan showed an adjuvant effect on anti-S. aureus IgY induction in egg yolk, but did not inhibit S. aureus growth. The use of the oil adjuvant FIA was necessary in the preparation of anti-S. aureus IgY as a treatment for AD symptoms.

Adduct Formation of Delamanid with NAD in Mycobacteria.

Delamanid (DLM), a nitro-dihydroimidazooxazole derivative currently approved for pulmonary multidrug-resistant tuberculosis (TB) therapy, is a prodrug activated by mycobacterial 7,8-didemethyl-8-hydroxy 5-deazaflavin electron transfer coenzyme (F420)-dependent nitroreductase (Ddn). Despite inhibiting the biosynthesis of a subclass of mycolic acids, the active DLM metabolite remained unknown. Comparative liquid chromatography-mass spectrometry (LC-MS) analysis of DLM metabolites revealed covalent binding of reduced DLM with a nicotinamide ring of NAD derivatives (oxidized form) in DLM-treated Mycobacterium tuberculosis var. Bacille de Calmette et Guérin. Isoniazid-resistant mutations in the type II NADH dehydrogenase gene (ndh) showed a higher intracellular NADH/NAD ratio and cross-resistance to DLM, which were restored by complementation of the mutants with wild-type ndh Our data demonstrated for the first time the adduct formation of reduced DLM with NAD in mycobacterial cells and its importance in the action of DLM.

Effects of orally active hypoxia inducible factor alpha prolyl hydroxylase inhibitor, FG4592 on renal fibrogenic potential in mouse unilateral ureteral obstruction model.

Orally active hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors that stabilize HIF protein and stimulate the production of erythropoietin have been approved to treat renal anemia. Our previous report suggested that HIF-1α dependent fibrogenic mechanisms are operating at the early onset of renal fibrosis and its contribution declines with the progression in mouse unilateral ureteral obstruction (UUO) model. The aim of the study is to evaluate the renal fibrogenic potential of FG4592, a recently approved orally active HIF prolyl hydroxylase inhibitor in mouse UUO model. Male C57BL/6J mice orally given FG-4592 (12.5 mg/kg/day and 50 mg/kg/day) were subjected to UUO. Neither dose of FG-4592 affected renal fibrosis or macrophage infiltration. FG-4592 had no effects on increased mRNA of collagen I, collagen III or transforming growth factor-ß1. At 3 days after UUO, higher dose of FG-4592 potentiated the increased mRNA expression of profibrogenic molecules, plasminogen activator inhibitor 1 (Pai-1) and connective tissue growth factor (Ctgf) but such potentiation disappeared at 7 days after UUO. It is suggested that FG-4592 used in the present study had little effects on renal fibrosis even though high dose of FG-4592 used in the present study transiently potentiated gene expression of Pai-1 and Ctgf in the UUO kidney.

CD4+ T Responses Other Than Th1 Type Are Preferentially Induced by Latency-Associated Antigens in the State of Latent Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis (M. tuberculosis) produces a diverse range of antigenic proteins in its dormant phase. The cytokine profiles of CD4+ T cell responses, especially subsets other than Th1 type (non-Th1 type), against these latency-associated M. tuberculosis antigens such as α-crystallin (Acr), heparin-binding hemagglutinin (HBHA), and mycobacterial DNA-binding protein 1 (MDP-1) remain elusive in relation to the clinical stage of M. tuberculosis infection. In the present study, peripheral blood mononuclear cells (PBMCs) collected from different stages of M. tuberculosis-infected cases and control PBMCs were stimulated with these antigens and ESAT-6/CFP-10. Cytokine profiles of CD4+ T cells were evaluated by intracellular cytokine staining using multicolor flow cytometry. Our results demonstrate that Th1 cytokine responses were predominant after TB onset independent of the type of antigen stimulation. On the contrary, non-Th1 cytokine responses were preferentially induced by latency-associated M. tuberculosis antigens, specifically IL-10 response against Acr in latent M. tuberculosis infection. From these results, we surmise a shift in the CD4+ T cell response from mixed non-Th1 to Th1 dominant type during TB progression.

Bordet-Gengou agar medium supplemented with albumin-containing biologics for cultivation of bordetellae.

Bordetella pertussis, B. parapertussis, and B. bronchiseptica cause respiratory infections in mammals, including humans, and are generally cultivated on Bordet-Gengou (BG) agar plates in laboratories. The medium requires animal blood as a supplement for better bacterial growth. However, using blood is problematic, as its constant supply is occasionally difficult because of the limited shelf-life. This study proposes modified BG agar plates supplemented with bovine serum albumin and fetal bovine serum as a simple and convenient medium that confers sufficient growth of bordetellae.

Metabolic adaptation to glycolysis is a basic defense mechanism of macrophages for Mycobacterium tuberculosis infection.

Macrophages are major components of tuberculosis (TB) granulomas and are responsible for host defenses against the intracellular pathogen, Mycobacterium tuberculosis. We herein showed the strong expression of hypoxia-inducible factor-1α (HIF-1α) in TB granulomas and more rapid death of HIF-1α-conditional knockout mice than wild-type (WT) mice after M. tuberculosis infection. Although interferon-γ (IFN-γ) is a critical host-protective cytokine against intracellular pathogens, HIF-1-deficient macrophages permitted M. tuberculosis growth even after activation with IFN-γ. These results prompted us to investigate the role of HIF-1α in host defenses against infection. We found that the expression of lactate dehydrogenase-A (LDH-A) was controlled by HIF-1α in M. tuberculosis-infected macrophages IFN-γ independently. LDH-A is an enzyme that converts pyruvate to lactate and we found that the intracellular level of pyruvate in HIF-1α-deficient bone marrow-derived macrophages (BMDMs) was significantly higher than in WT BMDMs. Intracellular bacillus replication was enhanced by an increase in intracellular pyruvate concentrations, which were decreased by LDH-A. Mycobacteria in phagosomes took up exogenous pyruvate more efficiently than glucose, and used it as the feasible carbon source for intracellular growth. These results demonstrate that HIF-1α prevents the hijacking of pyruvate in macrophages, making it a fundamental host-protective mechanism against M. tuberculosis.

Serum antibody profiles in individuals with latent Mycobacterium tuberculosis infection.

One-third of the world's humans has latent tuberculosis infection (LTBI), representing a large pool of potentially active TB. Recent LTBI carries a higher risk of disease progression than remote LTBI. Recent studies suggest important roles of antibodies in TB pathology, prompting us to investigate serum antibody profiles in a cohort with LTBI. In this single-center prospective observational study, we analyzed IgG-antibody concentrations against five major Mycobacterium tuberculosis (Mtb) antigens (including 6 kDa early secretory antigenic target (ESAT6), CFP10, and antigen 85A, which are expressed mainly in the growth phase; and mycobacterial DNA-binding protein 1 (MDP1) and alpha-crystallin like protein (Acr), which are expressed in the dormant phases) in individuals with recent (n=13) or remote (n=12) LTBI, no Mtb infection (n=19), or active TB (n=15). Antibody titers against ESAT6 and MDP1 were significantly higher in individuals with recent LTBI than in those with no Mtb infection or remote LTBI. All pairwise antibody titers against these five major antigens were significantly correlated throughout the stages of Mtb infection. Five individuals with recent LTBI had significantly higher antibody titers against ESAT6 (P = 0.03), Ag85A (P = 0.048), Acr (P = 0.057), and MDP1 (P = 0.0001) than in individuals with remote LTBI; they were also outside the normal range (+2 SDs). One of these individuals was diagnosed with active pulmonary TB at 18-month follow-up examination. These findings indicated that concentrations of antibodies against both multiplying and dormant Mtb are higher in recent LTBI and that individuals with markedly higher antibody titers may be appropriate candidates for prophylactic therapy.

Histidine and arginine modulate intestinal cell restitution via transforming growth factor-ß1.

Intestinal wound healing depends on the precise balance of restitution, proliferation, and differentiation of intestinal epithelial cells (IECs). In a previous study, we revealed that IEC proliferation was suppressed under histidine deficiency. However, the role of histidine in cell restitution is poorly understood. Meanwhile, addition of arginine to basal medium enhanced IEC restitution after wounding. However, there are no reports on whether histidine or arginine deficiency influences IEC restitution. We examined the roles of histidine and arginine in IEC restitution using the rat intestinal epithelial cell-6 (IEC-6) cell line. In the present study, the cell restitution in medium lacking histidine (ΔHis) or arginine (ΔArg) was most greatly decreased among media lacking each of the 20 intravital amino acids, compared with that in medium containing all 20 intravital amino acids (Full). TGF-ß1 is a known repair factor for cell restitution. The TGF-ß1 extracellular concentration and Tgf-ß1 mRNA level were decreased in ΔHis or ΔArg. Supplementation of 10 µM histidine to ΔHis or 50 µM arginine to ΔArg recovered the decreases in both cell restitution and TGF-ß1 extracellular concentration. Phosphorylation of Smad2, a signaling molecule for the TGF-ß pathway, was decreased in ΔHis or ΔArg. Additionally, the phosphorylation of mammalian target of rapamycin, p70 ribosomal protein S6 kinase and extracellular signal-regulated kinase were decreased in ΔHis or ΔArg. The present findings suggested that deletion of histidine or arginine led to a decrease in IEC restitution through a decrease in TGF-ß1. We revealed that histidine and arginine play important roles in IEC restitution.

A dipeptidyl peptidase-4 (DPP-4) inhibitor, linagliptin, attenuates cardiac dysfunction after myocardial infarction independently of DPP-4.

Dipeptidyl peptidase-4 (DPP-4) inhibitors not only improve impaired glucose tolerance in diabetes, but also have pleiotropic extra-pancreatic effects such as preconditioning effect for myocardial ischemia-reperfusion injury. Here, we investigated the anti-remodeling effects of linagliptin, a DPP-4 inhibitor, by use of DPP-4-deficient rats. After the induction of myocardial infarction (MI), Fischer 344 rats with inactivating mutation of DPP-4 were orally administrated with a DPP-4 inhibitor, linagliptin (5 mg kg-1·day-1), or vehicle in drinking water for 4 weeks. Linagliptin did not affect hemodynamic status, body weight, and infarct size. In echocardiography, linagliptin tended to improve left ventricular (LV) systolic function, and significantly improved LV diastolic function, surprisingly. Interstitial fibrosis in marginal region and macrophage infiltration were significantly lower in the linagliptin group than those in the vehicle group. Fibrosis-related gene expressions, such as collagen I and transforming growth factor-ß1 (TGF-ß1), and inflammation-related expressions, such as macrophage chemotactic protein 1 and matrix metalloproteinase-2 (MMP-2), were significantly suppressed in marginal area of the linagliptin-treated rats compared with the vehicle rats. The TGF-ß1 and MMP-2 protein levels were attenuated by linagliptin in DPP-4-deficient cardiac fibroblasts. Linagliptin can attenuate MI-induced cardiac remodeling via a DPP-4-independent pathway.

High-density lipoprotein suppresses tumor necrosis factor alpha production by mycobacteria-infected human macrophages.

Immune responses to parasitic pathogens are affected by the host physiological condition. High-density lipoprotein (HDL) and low-density lipoprotein (LDL) are transporters of lipids between the liver and peripheral tissues, and modulate pro-inflammatory immune responses. Pathogenic mycobacteria are parasitic intracellular bacteria that can survive within macrophages for a long period. Macrophage function is thus key for host defense against mycobacteria. These basic facts suggest possible effects of HDL and LDL on mycobacterial diseases, which have not been elucidated so far. In this study, we found that HDL and not LDL enhanced mycobacterial infections in human macrophages. Nevertheless, we observed that HDL remarkably suppressed production of tumor necrosis factor alpha (TNF-α) upon mycobacterial infections. TNF-α is a critical host-protective cytokine against mycobacterial diseases. We proved that toll-like receptor (TLR)-2 is responsible for TNF-α production by human macrophages infected with mycobacteria. Subsequent analysis showed that HDL downregulates TLR2 expression and suppresses its intracellular signaling pathways. This report demonstrates for the first time the substantial action of HDL in mycobacterial infections to human macrophages.

Role of hypoxia-inducible factor-1 in the development of renal fibrosis in mouse obstructed kidney: Special references to HIF-1 dependent gene expression of profibrogenic molecules.

The aim of the study is to clarify the role of hypoxia-inducible factor-1 (HIF-1) in the development of renal fibrosis in mouse obstructive nephropathy. We used mice with floxed HIF-1α alleles and tamoxifen-inducible Cre/ERT2 recombinase under ubiquitin C promoter to induce global HIF-1α deletion. Following tamoxifen administration, mice were subjected to unilateral ureteral obstruction (UUO). At 3, 7 and 14 days after UUO, renal gene expression profiles and interstitial fibrosis were assessed. HIF-1 dependent up-regulation of prolyl hydroxylase 3 and glucose transporter-1 was observed in the obstructed kidney at 3 and 7 days but not at 14 days after UUO. Various factors promoting fibrosis were up-regulated during the development of fibrosis. HIF-1 dependent gene expression of profibrotic molecules, plasminogen activator inhibitor 1, connective tissue growth factor, lysyl oxidase like 2 and transglutaminase 2 was observed in the obstructed kidney but such HIF-1 dependency was limited to the early onset of renal fibrosis. Global HIF-1 deletion tended to attenuate interstitial collagen I deposition at 3 days but had no effects thereafter. It is suggested that HIF-1 dependent profibrogenic mechanisms are operating at the early onset of renal fibrosis but its contribution declines with the progression in mouse UUO model.

S-allyl-glutathione improves experimental liver fibrosis by regulating Kupffer cell activation in rats.

S-allyl-glutathione (SAG) is one of the metabolites of diallyl sulfide (DAS), a component of garlic. DAS has shown preventative effects on carcinogenesis in animal models. However, whether synthetic SAG can improve liver fibrosis has not been investigated. We examined the potential preventive effects of SAG on acute and chronic models of liver fibrosis by chronic carbon tetrachloride (CCl4) administration. SAG inhibited liver fibrogenesis induced by CCl4 in a dose-dependent manner and reduced heat shock protein-47 (HSP47), a collagen-specific chaperone, and other fibrosis markers. In fibrosis regression models, after administration of either CCl4 for 9 wk or dimethyl nitrosamine (DMN) for 6 wk, SAG markedly accelerated fibrolysis in both models. In the regression stage of DMN-treated liver, SAG normalized the ratio of M2 phenotype (expression of mannose receptor) in Kupffer cells (KCs). Consistent with these results, the culture supernatants of SAG-treated M2-phenotype KCs inhibited collagen-α1(I) chain (COL1A1) mRNA expression in primary culture-activated rat hepatic stellate cells (HSCs). However, SAG did not directly inhibit HSC activation. In an acute model of CCl4 single injection, SAG inhibited hepatic injury dose dependently consistent with the inhibited the elevation of the bilirubin and ALT levels. These findings suggest that SAG could improve the fibrogenic and fibrolysis cascade via the regulation of excess activated and polarized KCs. SAG may also serve as a preventive and therapeutic agent in fibrosis of other organs for which current clinical therapy is unavailable. NEW & NOTEWORTHY S-allyl-glutathione (SAG) is a metabolite of diallyl sulfide, a component of garlic. SAG increased hepatic glutathione levels and GSH-to-GSSG ratio in normal rats. SAG treatment before or after liver fibrosis from chronic CCl4 administration improved liver fibrosis and regression. SAG decreased heat shock protein-47 (HSP47), a collagen-specific chaperone, and other fibrosis markers in CCl4-treated livers. SAG-treated Kupffer cell conditioned medium also inhibited collagen-α1(I) chain (COL1A1) mRNA expression and other markers in primary culture hepatic stellate cells.