A novel antibiotic class targeting the lipopolysaccharide transporter.

Carbapenem-resistant Acinetobacter baumannii (CRAB) has emerged as a major global pathogen with limited treatment options1. No new antibiotic chemical class with activity against A. baumannii has reached patients in over 50 years1. Here we report the identification and optimization of tethered macrocyclic peptide (MCP) antibiotics with potent antibacterial activity against CRAB. The mechanism of action of this molecule class involves blocking the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane, through inhibition of the LptB2FGC complex. A clinical candidate derived from the MCP class, zosurabalpin (RG6006), effectively treats highly drug-resistant contemporary isolates of CRAB both in vitro and in mouse models of infection, overcoming existing antibiotic resistance mechanisms. This chemical class represents a promising treatment paradigm for patients with invasive infections due to CRAB, for whom current treatment options are inadequate, and additionally identifies LptB2FGC as a tractable target for antimicrobial drug development.

Synthesis, Characterization, and in vivo Evaluation of a Novel Potent Autotaxin-Inhibitor.

The autotaxin-lysophosphatidic acid (ATX-LPA) signaling pathway plays a role in a variety of autoimmune diseases, such as rheumatoid arthritis or neurodegeneration. A link to the pathogenesis of glaucoma is suggested by an overactive ATX-LPA axis in aqueous humor samples of glaucoma patients. Analysis of such samples suggests that the ATX-LPA axis contributes to the fibrogenic activity and resistance to aqueous humor outflow through the trabecular meshwork. In order to inhibit or modulate this pathway, we developed a new series of ATX-inhibitors containing novel bicyclic and spirocyclic structural motifs. A potent lead compound (IC50 against ATX: 6 nM) with good in vivo PK, favorable in vitro property, and safety profile was generated. This compound leads to lowered LPA levels in vivo after oral administration. Hence, it was suitable for chronic oral treatment in two rodent models of glaucoma, the experimental autoimmune glaucoma (EAG) and the ischemia/reperfusion models. In the EAG model, rats were immunized with an optic nerve antigen homogenate, while controls received sodium chloride. Retinal ischemia/reperfusion (I/R) was induced by elevating the intraocular pressure (IOP) in one eye to 140 mmHg for 60 min, followed by reperfusion, while the other untreated eye served as control. Retinae and optic nerves were evaluated 28 days after EAG or 7 and 14 days after I/R induction. Oral treatment with the optimized ATX-inhibitor lead to reduced retinal ganglion cell (RGC) loss in both glaucoma models. In the optic nerve, the protective effect of ATX inhibition was less effective compared to the retina and only a trend to a weakened neurofilament distortion was detectable. Taken together, these results provide evidence that the dysregulation of the ATX-LPA axis in the aqueous humor of glaucoma patients, in addition to the postulated outflow impairment, might also contribute to RGC loss. The observation that ATX-inhibitor treatment in both glaucoma models did not result in significant IOP increases or decreases after oral treatment indicates that protection from RGC loss due to inhibition of the ATX-LPA axis is independent of an IOP lowering effect.

Proapoptotic effects of the chemokine, CXCL 10 are mediated by the noncognate receptor TLR4 in hepatocytes.

UNLABELLED: Aberrant expression of the chemokine CXC chemokine ligand (CXCL)10 has been linked to the severity of hepatitis C virus (HCV)-induced liver injury, but the underlying molecular mechanisms remain unclear. In this study, we describe a yet-unknown proapoptotic effect of CXCL10 in hepatocytes, which is not mediated through its cognate chemokine receptor, but the lipopolysaccharide receptor Toll-like receptor 4 (TLR4). To this end, we investigated the link of CXCL10 expression with apoptosis in HCV-infected patients and in murine liver injury models. Mice were treated with CXCL10 or neutralizing antibody to systematically analyze effects on hepatocellular apoptosis in vivo. Direct proapoptotic functions of CXCL10 on different liver cell types were evaluated in detail in vitro. The results showed that CXCL10 expression was positively correlated with liver cell apoptosis in humans and mice. Neutralization of CXCL10 ameliorated concanavalin A-induced tissue injury in vivo, which was strongly associated with reduced liver cell apoptosis. In vitro, CXCL10 mediated the apoptosis of hepatocytes involving TLR4, but not CXC chemokine receptor 3 signaling. Specifically, CXCL10 induced long-term protein kinase B and Jun N-terminal kinase activation, leading to hepatocyte apoptosis by caspase-8, caspase-3, and p21-activated kinase 2 cleavage. Accordingly, systemic application of CXCL10 led to TLR4-induced liver cell apoptosis in vivo. CONCLUSION: The results identify CXCL10 and its noncognate receptor, TLR4, as a proapoptotic signaling cascade during liver injury. Antagonism of the CXCL10/TLR4 pathway might be a therapeutic option in liver diseases associated with increased apoptosis.

Interference with oligomerization and glycosaminoglycan binding of the chemokine CCL5 improves experimental liver injury.

BACKGROUND: The chemokine CCL5 is involved in the recruitment of immune cells and a subsequent activation of hepatic stellate cells (HSC) after liver injury. We here investigate whether inhibition of CCL5 oligomerization and glycosaminoglycan binding by a mutated CCL5 protein ((44)AANA(47)-CCL5) has the potential to ameliorate liver cell injury and fibrosis in vivo. METHODOLOGY: Liver injury was induced in C57BL/6 mice by intraperitoneal injection of carbon tetrachloride (CCl(4)) in an acute and a chronic liver injury model. Simultaneously, mice received either (44)AANA(47)-CCL5 or vehicle. Liver cell necrosis and fibrosis was analyzed by histology, and measurement of serum transaminases and hydroxyproline. Intrahepatic mRNA expression of fibrosis and inflammation related genes were determined by quantitative RT-PCR and infiltration of immune cells was assessed by FACS analysis and immunocytochemistry. In vitro, HSC were stimulated with conditioned media of T-cell enriched splenocytes. PRINCIPAL FINDINGS: (44)AANA(47)-CCL5 treated mice displayed a significantly reduced degree of acute liver injury (liver cell necrosis, transaminases) and fibrosis (Sirus red positive area and hydroxyproline content) compared to vehicle treated mice. Ameliorated fibrosis by (44)AANA(47)-CCL5 was associated with a decreased expression of fibrosis related genes, decreased α-smoth muscle antigen (αSMA) and a reduction of infiltrating immune cells. In the acute model, (44)AANA(47)-CCL5 treated mice displayed a reduced immune cell infiltration and mRNA levels of TNF, IL-1 and CCL3 compared to vehicle treated mice. In vitro, conditioned medium of T-cell enriched splenocytes of (44)AANA(47)-CCL5 treated mice inhibited the chemotaxis and proliferation of HSC. CONCLUSIONS: The results provide evidence that inhibition of oligomerization and glycosaminoglycan binding of the chemokine CCL5 is a new therapeutic strategy for the treatment of acute and chronic liver injuries and represents an alternative to chemokine receptor antagonism.

The chemokine receptor CXCR3 limits injury after acute toxic liver damage.

Although acute liver failure is a rare disease, its presence is associated with high morbidity and mortality in affected patients. While a contribution of the immune system to the outcome of toxic liver failure is anticipated, functionally relevant immune cell receptors for liver cell damage need to be better defined. We here investigate the relevance of the chemokine receptor CXCR3, which is important for hepatic immune cell infiltration, in a model of experimental acute liver failure. Liver injury was induced by a single intraperitoneal injection of carbon tetrachloride (CCl(4)) in CXCR3(-/-), CCR1(-/-), CCR5(-/-) and wild-type mice. In this model, CXCR3(-/-) mice displayed augmented liver damage compared with all other mouse strains as assessed by liver histology and serum transaminases 24 and 72 h after injury. Phenotypically, CXCR3(-/-) mice had significantly reduced intrahepatic NK and NKT cells after injury at all investigated time points (all P<0.05), but strongly elevated expression levels of IL1-ß, TNF-α and IFN-γ. In line with a functional role of innate immune cells, wild-type mice depleted for NK cells with an anti-ASIALO GM1 antibody before liver injury also displayed increased liver injury after CCl(4) challenge. CXCR3(-/-) and NK cell-depleted mice show reduced apoptotic liver cells (TUNEL assay), but more necrotic hepatocytes. Functionally, the augmented liver cell necrosis in CXCR3(-/-) and NK cell-depleted mice was associated with increased expression of high mobility group 1 (HMGB1) protein and a consecutive enhanced infiltration of neutrophils into the liver. In conclusion, the results demonstrate a primarily unexpected beneficial role of CXCR3 in acute toxic liver injury. These findings should be taken into account when planning trials with CXCR3 antagonists.

Chemokine Cxcl9 attenuates liver fibrosis-associated angiogenesis in mice.

UNLABELLED: Recent data suggest that the chemokine receptor CXCR3 is functionally involved in fibroproliferative disorders, including liver fibrosis. Neoangiogenesis is an important pathophysiological feature of liver scarring, but a functional role of angiostatic CXCR3 chemokines in this process is unclear. We therefore investigated neoangiogenesis in carbon tetrachloride (CCl(4))-induced liver fibrosis in Cxcr3(-/-) and wildtype mice by histological, molecular, and functional imaging methods. Furthermore, we assessed the direct role of vascular endothelial growth factor (VEGF) overexpression on liver angiogenesis and the fibroproliferative response using a Tet-inducible bitransgenic mouse model. The feasibility of attenuation of angiogenesis and associated liver fibrosis by therapeutic treatment with the angiostatic chemokine Cxcl9 was systematically analyzed in vitro and in vivo. The results demonstrate that fibrosis progression in Cxcr3(-/-) mice was strongly linked to enhanced neoangiogenesis and VEGF/VEGFR2 expression compared with wildtype littermates. Systemic VEGF overexpression led to a fibrogenic response within the liver and was associated with a significantly increased Cxcl9 expression. In vitro, Cxcl9 displayed strong antiproliferative and antimigratory effects on VEGF-stimulated endothelial cells and stellate cells by way of reduced VEGFR2 (KDR), phospholipase Cγ (PLCγ), and extracellular signal-regulated kinase (ERK) phosphorylation, identifying this chemokine as a direct counter-regulatory molecule of VEGF signaling within the liver. Accordingly, systemic administration of Cxcl9 led to a strong attenuation of neoangiogenesis and experimental liver fibrosis in vivo. CONCLUSION: The results identify direct angiostatic and antifibrotic effects of the Cxcr3 ligand Cxcl9 in a model of experimental liver fibrosis. The amelioration of liver damage by systemic application of Cxcl9 might offer a novel therapeutic approach for chronic liver diseases associated with increased neoangiogenesis.

A 7-gene signature of the recipient predicts the progression of fibrosis after liver transplantation for hepatitis C virus infection.

Fibrosis recurrence after liver transplantation (LT) for hepatitis C virus (HCV) is a universal event and strongly determines a patient's prognosis. The recipient risk factors for fibrosis recurrence are still poorly defined. Here we assess a genetic risk score as a predictor of fibrosis after LT. The cirrhosis risk score (CRS), which comprises allele variants in 7 genes (adaptor-related protein complex 3 S2, aquaporin 2, antizyme inhibitor 1, degenerative spermatocyte homolog 1 lipid desaturase, syntaxin binding protein 5-like, toll-like receptor 4, and transient receptor potential cation channel M5), was calculated for 137 patients who underwent LT for HCV infection and experienced HCV reinfection of the graft. The patients were stratified into 3 CRS categories: <0.5, 0.5 to 0.7, and >0.7. All patients underwent protocol biopsy after LT (median follow-up = 5 years), and liver fibrosis was assessed according to the Desmet and Scheuer score. The data were analyzed with univariate and multivariate analyses. The results showed that the highest CRS category was strongly associated with the presence of F2 or F3 fibrosis in protocol biopsy samples 1, 3, and 5 years after LT (P = 0.006, P = 0.001, and P = 0.02, respectively). Overall, 75.0% of the patients with a CRS > 0.7 developed at least F2 fibrosis, whereas 51.5% developed F3 fibrosis during follow-up. The predictive value of the CRS for fibrosis progression was independent of known clinical risk factors, including the age of the donor, the sex of the recipient, and the occurrence of acute rejection. A Kaplan-Meier analysis confirmed the prognostic value of the CRS with respect to the recurrence of severe liver fibrosis in HCV-infected patients after LT (log rank = 6.23, P = 0.03). In conclusion, the genetic signature of the recipient predicts the likelihood of severe liver fibrosis in the graft after HCV recurrence. The CRS might help with early clinical decision making (eg, the selection of patients for antiviral therapy after LT).

Macrophage migration inhibitory factor (MIF) exerts antifibrotic effects in experimental liver fibrosis via CD74.

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that has been implicated in various inflammatory diseases. Chronic inflammation is a mainstay of liver fibrosis, a leading cause of morbidity worldwide, but the role of MIF in liver scarring has not yet been elucidated. Here we have uncovered an unexpected antifibrotic role for MIF. Mice genetically deleted in Mif (Mif(-/-)) showed strongly increased fibrosis in two models of chronic liver injury. Pronounced liver fibrosis in Mif(-/-) mice was associated with alterations in fibrosis-relevant genes, but not by a changed intrahepatic immune cell infiltration. Next, a direct impact of MIF on hepatic stellate cells (HSC) was assessed in vitro. Although MIF alone had only marginal effects on HSCs, it markedly inhibited PDGF-induced migration and proliferation of these cells. The inhibitory effects of MIF were mediated by CD74, which we detected as the most abundant known MIF receptor on HSCs. MIF promoted the phosphorylation of AMP-activated protein kinase (AMPK) in a CD74-dependent manner and, in turn, inhibition of AMPK reversed the inhibition of PDGF-induced HSC activation by MIF. The pivotal role of CD74 in MIF-mediated antifibrotic properties was further supported by augmented liver scarring of Cd74(-/-) mice. Moreover, mice treated with recombinant MIF displayed a reduced fibrogenic response in vivo. In conclusion, we describe a previously unexplored antifibrotic function of MIF that is mediated by the CD74/AMPK signaling pathway in HSCs. The results imply MIF and CD74 as targets for treatment of liver diseases.

A Duffy antigen receptor for chemokines (DARC) polymorphism that determines pro-fibrotic chemokine serum concentrations is not directly associated with severity of hepatitis C infection.

Genetic host factors influence the progression of hepatitis C infection (HCV). Chemokines play important roles in HCV-induced liver fibrosis. Recently, a single nucleotide polymorphism in the Duffy antigen receptor for chemokines (DARC) was identified which strongly determines the serum concentrations of pivotal pro-fibrotic chemokines, including CCL2. We here tested the hypothesis that this genetic variant (rs12075 A/G) is a risk factor for liver fibrosis in HCV infection. Overall, 880 patients with HCV from three cohorts and 108 controls were genotyped for rs12075. Although serum CCL2 levels were associated with early liver fibrosis, rs12075 itself was not associated with HCV infection or the severity of liver disease in any of the cohorts. The lack of association was evident in qualitative and quantitative analysis despite sufficient statistical power. We conclude that gene variations that strongly determine serum concentrations of chemokines are not necessarily risk markers of the disease traits in which these molecules play pathophysiological roles.

Antagonism of the chemokine Ccl5 ameliorates experimental liver fibrosis in mice.

Activation of hepatic stellate cells in response to chronic inflammation represents a crucial step in the development of liver fibrosis. However, the molecules involved in the interaction between immune cells and stellate cells remain obscure. Herein, we identify the chemokine CCL5 (also known as RANTES), which is induced in murine and human liver after injury, as a central mediator of this interaction. First, we showed in patients with liver fibrosis that CCL5 haplotypes and intrahepatic CCL5 mRNA expression were associated with severe liver fibrosis. Consistent with this, we detected Ccl5 mRNA and CCL5 protein in 2 mouse models of liver fibrosis, induced by either injection of carbon tetrachloride (CCl4) or feeding on a methionine and choline-deficient (MCD) diet. In these models, Ccl5-/- mice exhibited decreased hepatic fibrosis, with reduced stellate cell activation and immune cell infiltration. Transplantation of Ccl5-deficient bone marrow into WT recipients attenuated liver fibrosis, identifying infiltrating hematopoietic cells as the main source of Ccl5. We then showed that treatment with the CCL5 receptor antagonist Met-CCL5 inhibited cultured stellate cell migration, proliferation, and chemokine and collagen secretion. Importantly, in vivo administration of Met-CCL5 greatly ameliorated liver fibrosis in mice and was able to accelerate fibrosis regression. Our results define a successful therapeutic approach to reduce experimental liver fibrosis by antagonizing Ccl5 receptors.

CXC chemokine ligand 4 (Cxcl4) is a platelet-derived mediator of experimental liver fibrosis.

UNLABELLED: Liver fibrosis is a major cause of morbidity and mortality worldwide. Platelets are involved in liver damage, but the underlying molecular mechanisms remain elusive. Here, we investigate the platelet-derived chemokine (C-X-C motif) ligand 4 (CXCL4) as a molecular mediator of fibrotic liver damage. Serum concentrations and intrahepatic messenger RNA of CXCL4 were measured in patients with chronic liver diseases and mice after toxic liver injury. Platelet aggregation in early fibrosis was determined by electron microscopy in patients and by immunohistochemistry in mice. Cxcl4(-/-) and wild-type mice were subjected to two models of chronic liver injury (CCl(4) and thioacetamide). The fibrotic phenotype was analyzed by histological, biochemical, and molecular analyses. Intrahepatic infiltration of immune cells was investigated by fluorescence-activated cell sorting, and stellate cells were stimulated with recombinant Cxcl4 in vitro. The results showed that patients with advanced hepatitis C virus-induced fibrosis or nonalcoholic steatohepatitis had increased serum levels and intrahepatic CXCL4 messenger RNA concentrations. Platelets were found directly adjacent to collagen fibrils. The CCl(4) and thioacetamide treatment led to an increase of hepatic Cxcl4 levels, platelet activation, and aggregation in early fibrosis in mice. Accordingly, genetic deletion of Cxcl4 in mice significantly reduced histological and biochemical liver damage in vivo, which was accompanied by changes in the expression of fibrosis-related genes (Timp-1 [tissue inhibitor of matrix metalloproteinase 1], Mmp9 [matrix metalloproteinase 9], Tgf-beta [transforming growth factor beta], IL10 [interleukin 10]). Functionally, Cxcl4(-/-) mice showed a strongly decreased infiltration of neutrophils (Ly6G) and CD8(+) T cells into the liver. In vitro, recombinant murine Cxcl4 stimulated the proliferation, chemotaxis, and chemokine expression of hepatic stellate cells. CONCLUSION: The results underscore an important role of platelets in chronic liver damage and imply a new target for antifibrotic therapies.

The chemokine scavenging receptor D6 limits acute toxic liver injury in vivo.

The chemokine decoy receptor D6 is a promiscuous chemokine receptor lacking classical signaling functions. It negatively regulates inflammation by targeting CC chemokines to cellular internalization and degradation. Here we analyze the function of D6 in acute CCl(4)-induced liver damage in constitutive D6(-/-) and wild-type mice. The degree of liver injury was assessed by liver histology, serum transaminases, IL-6, and TNFalpha mRNA expression. Protein levels of D6 ligands (CCL2, CCL3, CCL5) and the non-D6-ligand CXCL9 within the livers were determined by ELISAs. The intrahepatic infiltration of immune cells was characterized by FACS. Genetic deletion of D6 led to prolonged liver damage after acute CCl(4) administration. The augmented liver damage in D6(-/-) mice was associated with increased protein levels of intrahepatic inflammatory chemokines CCL2, CCL3, and CCL5 after 48 h, whereas CXCL9 was not different between knockout and wild-type mice. Functionally, increased intra-hepatic CC chemokine concentrations led to increased infiltration of CD45(+) leukocytes, which were mainly identified as T and NK cells. In conclusion, the chemokine scavenger receptor D6 has a non-redundant role in acute toxic liver injury in vivo. These results support the importance of post-translational chemokine regulation and describe a new mechanism of immune modulation within the liver.

Antifibrotic effects of CXCL9 and its receptor CXCR3 in livers of mice and humans.

BACKGROUND & AIMS: Fibrosis is the hallmark of chronic liver diseases, yet many aspects of its mechanism remain to be defined. Chemokines are ubiquitous chemotactic molecules that mediate many acute and chronic inflammatory conditions, and CXC chemokine genes colocalize with a locus previously shown to include fibrogenic genes. We investigated the roles of the chemokine CXCL9 and its receptor CXCR3 in liver fibrosis. METHODS: The effects of CXCL variants on fibrogenesis were analyzed using samples from patients with hepatitis C virus infection and by induction of fibrosis in CXCR3(-/-) and wild-type mice. In mice, intrahepatic immune cell subsets were investigated and interferon gamma messenger RNA levels were measured at baseline and after injury. Human serum CXCL9 levels were measured and correlated with CXCL9 variant and fibrosis severity. The effects of stimulation with CXCL9 were investigated on human hepatic stellate cells (LX-2). RESULTS: Specific CXCL9 variants were associated with liver fibrosis in mice and humans; CXCL9 serum concentrations correlated with genotypes and levels of fibrosis in patients. In contrast to other chemokines, CXCL9 exerted antifibrotic effects in vitro, suppressing collagen production in LX-2 cells. CXCR3(-/-) mice had increased liver fibrosis; progression was associated with decreased numbers of intrahepatic interferon gamma-positive T cells and reduced interferon gamma messenger RNA, indicating that CXCL9-CXCR3 regulates Th1-associated immune pathways. CONCLUSIONS: This is the first description of a chemokine-based antifibrotic pathway in the liver; antifibrotic therapies might be developed to modulate CXC chemokine levels.

A functional variation in CHI3L1 is associated with severity of liver fibrosis and YKL-40 serum levels in chronic hepatitis C infection.

BACKGROUND/AIMS: YKL-40 is a chitinase-like protein involved in matrix remodelling and a non-invasive fibrosis marker. We assessed whether a functional promoter polymorphism in CHI3L1, encoding YKL-40, is associated with HCV-induced liver fibrosis and influences YKL-40 serum concentrations. METHODS: The CHI3L1 -131G-->C promoter polymorphism was genotyped in two cohorts of HCV infected patients (n=440) by 5'-endonuclease assays. Histological fibrosis scores and YKL-40 serum levels (ELISA) were associated with CHI3L1 -131G-->C by quantitative and qualitative genetic analyses and corrected by multivariate analysis. RESULTS: CHI3L1 -131G-->C genotype was strongly associated with the stage of liver fibrosis in the screening (n=265, P=0.001) and validation cohort (n=175, P=0.009). Homozygous carriers of the G allele were protected from severe fibrosis (F3/F4). This association was confirmed after correction for age and gender. Functionally, the G allele was associated with reduced serum levels of YKL-40 in HCV infected patients (P=0.002). CONCLUSIONS: The CHI3L1 promoter polymorphism -131G-->C determines YKL-40 serum levels and is associated with the severity of HCV-induced liver fibrosis. These results suggest a functional role of YKL-40 in liver fibrogenesis and should be taken into account when using YKL-40 as a non-invasive fibrosis marker.

Genetic variations of the chemokine scavenger receptor D6 are associated with liver inflammation in chronic hepatitis C.

Chronic hepatitis C (HCV) represents one of the most common chronic infections worldwide and is a major indication for liver transplantation. Liver inflammation is the main predictor of advanced fibrosis in HCV. Inflammatory cells are recruited to the liver by chemokines. Recently, a novel class of chemokine receptors has been characterized that lack signaling functions and are termed scavenger receptors. We determine here whether genetic variations of the scavenger receptor D6 contribute to the grade of liver inflammation in HCV. Four haplotype tagging single nucleotide polymorphisms (SNPs) were identified from HapMap that cover the genetic information of D6 (CCBP2). Among these SNPs, rs4683336 was associated with liver inflammation in qualitative (p = 0.003) and quantitative (p = 0.0086) genotype analysis. This association was confirmed in an independent cohort of HCV-infected patients (p = 0.006 for qualitative and p = 0.0046 for quantitative analysis, respectively). Furthermore, the haplotype that is tagged by marker rs4683336 was significantly correlated with liver inflammation when compared with the most common D6 haplotype (p = 0.014). The importance of genetic variations in D6 was supported through the demonstration of an association of D6 mRNA expression with histologic inflammation in liver biopsies and a considerable range of D6 mRNA expression in isolated human hepatocytes. In conclusion, we demonstrate that variations in a chemokine scavenging receptor are significantly correlated with clinical inflammatory phenotypes such as HCV infection.

The fractalkine receptor CX3CR1 is involved in liver fibrosis due to chronic hepatitis C infection.

BACKGROUND/AIMS: The chemokine receptor CX3CR1 and its specific ligand fractalkine (CX3CL1) are known to modulate inflammatory and fibroproliferative diseases. Here we investigate the role of CX3CR1/fractalkine in HCV-induced liver fibrosis. METHODS: A genotype analysis of CX3CR1 variants was performed in 211 HCV-infected patients. Hepatic expression of CX3CR1 was studied in HCV-infected livers and isolated liver cell populations by RT-PCR and immunohistochemistry. The effects of fractalkine on mRNA expression of profibrogenic genes were determined in isolated hepatic stellate cells (HSC) and CX3CR1 genotypes were related to intrahepatic TIMP-1 mRNA levels. RESULTS: The intrahepatic mRNA expression of CX3CR1 correlates with the stage of HCV-induced liver fibrosis (P=0.03). The CX3CR1 coding variant V249I is associated with advanced liver fibrosis, independent of the T280M variant (P=0.009). CX3CR1 is present on primary HSC and fractalkine leads to a suppression of tissue inhibitor of metalloproteinase (TIMP)-1 mRNA in HSC (P=0.03). Furthermore, CX3CR1 genotypes are associated with TIMP-1 mRNA expression in HCV-infected liver (P=0.03). CONCLUSIONS: The results identify the fractalkine receptor CX3CR1 as susceptibility a gene for hepatic fibrosis in HCV infection. The modulation of TIMP-1 expression by fractalkine and CX3CR1 genotypes provides functional support for the observed genotype-phenotype association.

Five novel Kitasatospora species from soil: Kitasatospora arboriphila sp. nov., K. gansuensis sp. nov., K. nipponensis sp. nov., K. paranensis sp. nov. and K. terrestris sp. nov.

A polyphasic study was carried out to establish the taxonomic positions of six strains isolated from diverse soil samples and provisionally assigned to the genus Kitasatospora. The isolates were found to have chemical and morphological properties consistent with their classification as Kitasatospora strains. Direct 16S rRNA gene sequence data confirmed the taxonomic position of the strains following the generation of phylogenetic trees by using three tree-making algorithms. Five of the isolates were considered to merit species status using complementary genotypic and phenotypic data. These organisms were designated Kitasatospora arboriphila sp. nov. (HKI 0189(T)=2291-120(T)=DSM 44785(T)=NCIMB 13973(T)), Kitasatospora gansuensis sp. nov. (HKI 0314(T)=2050-015(T)=DSM 44786(T)=NCIMB 13974(T)), Kitasatospora nipponensis sp. nov. (HKI 0315(T)=2148-013(T)=DSM 44787(T)=NCIMB 13975(T)), Kitasatospora paranensis sp. nov. (HKI 0190(T)=2292-041(T)=DSM 44788(T)=NCIMB 13976(T)) and Kitasatospora terrestris sp. nov. (HKI 0186(T)=2293-012(T)=DSM 44789(T)=NCIMB 13977(T)). The remaining organism, isolate HKI 0316 (=2122-022=DSM 44790=NCIMB 13978), was considered to be a strain of Kitasatospora kifunensis on the basis of 16S rRNA gene sequence, DNA-DNA relatedness and phenotypic data.

Occurrence and non-detectability of maytansinoids in individual plants of the genera Maytenus and Putterlickia.

Individual plants belonging to different species of the family Celastraceae collected from their natural habitats in South Africa (Putterlickia verrucosa (E. Meyer ex Sonder) Szyszyl., Putterlickia pyracantha (L.) Szyszyl., Putterlickia retrospinosa van Wyk and Mostert) and Brazil (Maytenus ilicifolia Mart. ex Reiss., Maytenus evonymoides Reiss., Maytenus aquifolia Mart.) were investigated for the presence of maytansinoids and of maytansine, an ansamycin of high cytotoxic activity. Maytansinoids were not detectable in plants grown in Brazil. Analysis of plants growing in South Africa, however, showed clearly that maytansinoids were present in some individual plants but were not detectable in others. Molecular biological analysis of a Putterlickia verrucosa cell culture gave no evidence for the presence of the aminohydroxybenzoate synthase gene which is unique to the biosynthesis of aminohydroxybenzoate, a precursor of the ansamycins including maytansinoids. Moreover, this gene was not detectable in DNA extracted from the aerial parts of Putterlickia plants. In contrast, observations indicate that this gene may be present in microbes of the rhizosphere of Putterlickia plants. Our observations are discussed with respect to the possibility that the roots of Putterlickia plants may be associated with microorganisms which are responsible for the biosynthesis of maytansine or maytansinoids.

New and bioactive compounds from Streptomyces strains residing in the wood of Celastraceae.

Wood from three different plants of the Celastraceae growing in their natural habitats in Brazil (Maytenus aquifolia Mart.) and South Africa [Putterlickia retrospinosa van Wyk and Mostert, P. verrucosa (E. Meyer ex Sonder) Szyszyl.] was established as a source of endophytic bacteria using a medium selective for actinomycetes. Two isolates were identified as Streptomyces setonii and S. sampsonii whereas two others were not assignable to any of the known Streptomyces species. They were preliminarily named Streptomyces Q21 and Streptomyces MaB-QuH-8. The latter strain produces a new chloropyrrol and chlorinated anthracyclinone. The chloropyrrol showed high activity against a series of multiresistent bacteria and mycobacteria.