Circulating TNF-RII, IP-10 and HGF are associated with severity of COVID-19 in oncologic patients.

The COVID-19 patients showed hyperinflammatory response depending on the severity of the disease but little have been reported about this response in oncologic patients that also were infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sixty-five circulating cytokines/chemokines were quantified in 15 oncologic patients, just after SARS-CoV-2 infection and fourteen days later, and their levels were compared in patients who required hospitalisation by COVID-19 versus non-hospitalised patients. A higher median age of 72 years (range 61-83) in oncologic patients after SARS-CoV-2 infection was associated with hospitalisation requirement by COVID-19 versus a median age of 49 years (20-75) observed in the non-hospitalised oncologic patients (p = 0.008). Moreover, oncologic patients at metastatic stage or with lung cancer were significantly associated with hospitalisation by COVID-19 (p = 0.044). None of these hospitalised patients required ICU treatment. Higher basal levels of tumour necrosis factor receptor II (TNF-RII), interferon-γ (IFNγ)-induced protein 10 (IP-10) and hepatocyte growth factor (HGF) in plasma were significantly observed in oncologic patients who required hospitalisation by COVID-19. Higher TNF-RII, IP-10 and HGF levels after the SARS-CoV-2 infection in oncologic patients could be used as biomarkers of COVID-19 severity associated with hospitalisation requirements.

Point-of-care biomarker assay for rapid multiplexed detection of CRP and IP-10.

Rapid and accurate measurements of immune protein markers are essential for diagnosis and treatment in all clinical settings. The recent pandemic has revealed a stark need for developing new tools and assays that could be rapidly used in diverse settings and provide useful information to clinicians. Here, we describe the development and test application of a novel one-step CRP/IP-10 duplex assay for the LightDeck platform capable of delivering reproducible and accurate measurements in under eight minutes. We used the optimized assay to measure CRP and IP-10 levels in human blood and serum samples from healthy, SARS-CoV-2 (COVID-19) positive, and influenza-like illness (ILI) presenting patients. Our results agreed with previously published analyte levels and enabled us to make statistically significant comparisons relevant to multiple clinical parameters. Our duplex assay is a simple and powerful tool for aiding prognostic decision-making in diverse settings.

Variation in Leishmania chemokine suppression driven by diversification of the GP63 virulence factor.

Leishmaniasis is a neglected tropical disease with diverse outcomes ranging from self-healing lesions, to progressive non-healing lesions, to metastatic spread and destruction of mucous membranes. Although resolution of cutaneous leishmaniasis is a classic example of type-1 immunity leading to self-healing lesions, an excess of type-1 related inflammation can contribute to immunopathology and metastatic spread. Leishmania genetic diversity can contribute to variation in polarization and robustness of the immune response through differences in both pathogen sensing by the host and immune evasion by the parasite. In this study, we observed a difference in parasite chemokine suppression between the Leishmania (L.) subgenus and the Viannia (V.) subgenus, which is associated with severe immune-mediated pathology such as mucocutaneous leishmaniasis. While Leishmania (L.) subgenus parasites utilize the virulence factor and metalloprotease glycoprotein-63 (gp63) to suppress the type-1 associated host chemokine CXCL10, L. (V.) panamensis did not suppress CXCL10. To understand the molecular basis for the inter-species variation in chemokine suppression, we used in silico modeling to identify a putative CXCL10-binding site on GP63. The putative CXCL10 binding site is in a region of gp63 under significant positive selection, and it varies from the L. major wild-type sequence in all gp63 alleles identified in the L. (V.) panamensis reference genome. Mutating wild-type L. (L.) major gp63 to the L. (V.) panamensis sequence at the putative binding site impaired cleavage of CXCL10 but not a non-specific protease substrate. Notably, Viannia clinical isolates confirmed that L. (V.) panamensis primarily encodes non-CXCL10-cleaving gp63 alleles. In contrast, L. (V.) braziliensis has an intermediate level of activity, consistent with this species having more equal proportions of both alleles. Our results demonstrate how parasite genetic diversity can contribute to variation in immune responses to Leishmania spp. infection that may play critical roles in the outcome of infection.

A novel method to produce synthetic murine CXCL10 for efficient screening of functional variants.

Antitumor immune responses depend on the infiltration of solid tumors by effector T cells, a process guided by chemokines. In particular, the chemokine CXCL10 has been shown to play a critical role in mediating recruitment of CXCR3 + cytolytic T and NK cells in tumors, though its use as a therapeutic agent has not been widely explored. One of the limitations is due to the rapid inactivation of CXCL10 by dipeptidyl peptidase 4 (DPP4), a broadly expressed enzyme that is active in plasma and other bodily fluids. In the present study, we describe a novel method to produce synthetic CXCL10 that is resistant to DPP4 N-terminal truncation. Using a Fmoc solid-phase peptide synthesis approach, synthetic murine WT CXCL10 was produced, showing similar biochemical and biological properties to the recombinant protein. This synthesis method supported production of natural (amino acid substitution, insertion or deletion) and non-natural (chemical modifications) variants of CXCL10. In association with a functional screening cascade that assessed DPP4-mediated cleavage, CXCR3 signaling potency and chemotactic activity, we successfully generated 20 murine CXCL10 variants. Among those, two non-natural variants with N-methylated Leu3 (MeLeu3) and a reduced amide bond between Pro2 and Leu3 (rLeu3), respectively, showed resistance to DPP4 truncation but decreased CXCR3 signaling and chemotactic activity. Interestingly, MeLeu3 and rLeu3 CXCL10 behaved as DPP4 inhibitors, preventing the truncation of WT CXCL10. This study highlights the potential of using Fmoc solid-phase chemistry in association with biochemical and biological characterization to rapidly identify CXCL10 variants with desired properties. These novel methods unlock the opportunity to develop DPP4 resistant CXCL10 variants, as well as other chemokine substrates, while maintaining chemotactic properties.

Activation of the Legionella pneumophila LegK7 Effector Kinase by the Host MOB1 Protein.

Legionella pneumophila infects alveolar macrophages and can cause life-threatening pneumonia in humans. Upon internalization into the host cell, L. pneumophila injects numerous effector proteins into the host cytoplasm as a part of its pathogenesis. LegK7 is an effector kinase of L. pneumophila that functionally mimics the eukaryotic Mst kinase and phosphorylates the host MOB1 protein to exploit the Hippo pathway. To elucidate the LegK7 activation mechanism, we determined the apo structure of LegK7 in an inactive form and performed a comparative analysis of LegK7 structures. LegK7 is a non-RD kinase that contains an activation segment that is ordered, irrespective of stimulation, through a unique ß-hairpin-containing segment, and it does not require phosphorylation of the activation segment for activation. Instead, bacterial LegK7 becomes an active kinase via its heterologous molecular interaction with the host MOB1 protein. MOB1 binding triggers reorientation of the two lobes of the kinase domain, as well as a structural change in the interlobe hinge region in LegK7, consequently reshaping the LegK7 structure into an ATP binding-compatible closed conformation. Furthermore, we reveal that LegK7 is an atypical kinase that contains an N-terminal capping domain and a hydrophilic interlobe linker motif, which play key roles in the MOB1-induced activation of LegK7.

Computational Study of C-X-C Chemokine Receptor (CXCR)3 Binding with Its Natural Agonists Chemokine (C-X-C Motif) Ligand (CXCL)9, 10 and 11 and with Synthetic Antagonists: Insights of Receptor Activation towards Drug Design for Vitiligo.

Vitiligo is a hypopigmentary skin pathology resulting from the death of melanocytes due to the activity of CD8+ cytotoxic lymphocytes and overexpression of chemokines. These include CXCL9, CXCL10, and CXCL11 and its receptor CXCR3, both in peripheral cells of the immune system and in the skin of patients diagnosed with vitiligo. The three-dimensional structure of CXCR3 and CXCL9 has not been reported experimentally; thus, homology modeling and molecular dynamics could be useful for the study of this chemotaxis-promoter axis. In this work, a homology model of CXCR3 and CXCL9 and the structure of the CXCR3/Gαi/0ßγ complex with post-translational modifications of CXCR3 are reported for the study of the interaction of chemokines with CXCR3 through all-atom (AA-MD) and coarse-grained molecular dynamics (CG-MD) simulations. AA-MD and CG-MD simulations showed the first activation step of the CXCR3 receptor with all chemokines and the second activation step in the CXCR3-CXCL10 complex through a decrease in the distance between the chemokine and the transmembrane region of CXCR3 and the separation of the ßγ complex from the α subunit in the G-protein. Additionally, a general protein-ligand interaction model was calculated, based on known antagonists binding to CXCR3. These results contribute to understanding the activation mechanism of CXCR3 and the design of new molecules that inhibit chemokine binding or antagonize the receptor, provoking a decrease of chemotaxis caused by the CXCR3/chemokines axis.

Proteolytic chemokine cleavage as a regulator of lymphocytic infiltration in solid tumors.

In the past decade, immune-based therapies such as monoclonal antibodies against tumor epitopes or immune checkpoint inhibitors have become an integral part of contemporary cancer treatment in many entities. However, a fundamental prerequisite for the success of such therapies is a sufficient trafficking of tumor-infiltrating lymphocytes into the tumor microenvironment. This infiltration is facilitated by chemokines, a group of about 50 small proteins capable of chemotactically guiding leukocytes. Proteolytic inactivation of chemokines leading to an impaired infiltration of immune effector cells appears to be an efficient immune escape mechanism of solid cancers.The CXCR3 and CX3CR1 chemokine receptor ligands CXCL9-11 and CX3CL1, respectively, are mainly responsible for the tumor-suppressive lymphocytic infiltration into the tumor micromilieu. Their structure explains the biochemical basis of their proteolytic cleavage, while in vivo data from mouse models and patient samples shed light on the corresponding processes in cancer. The emerging roles of proteases, e.g., matrix metalloproteinases, cathepsins, and dipeptidyl peptidase 4, in chemokine inactivation define new resistance mechanisms against immunotherapies and identify attractive new targets to enhance immune intervention in cancer.

First description of agonist and antagonist IP-10 in urine of patients with active TB.

OBJECTIVES: Biomarkers for tuberculosis (TB) diagnosis and clinical management are needed to defeat TB. In chronic hepatitis, patients not responding to interferon/ribavirin treatment had high levels of an antagonist form of IP-10. Recently, antagonist IP-10 has been shown to be involved also in TB pathogenesis. Here, we investigated IP-10 agonist/antagonist forms as potential inflammatory biomarkers to support TB diagnosis and monitoring. METHODS: Total IP-10 and its agonist/antagonist forms were measured by SIMOA digital ELISA in urine obtained from patients with active TB at baseline and after treatment. Healthy donors (HD) and patients with pneumonia were enrolled as controls. RESULTS: Patients with active TB had significantly higher levels of total and agonist IP-10 at baseline compared to HD; conversely, no differences were observed between IP-10 levels in active TB vs pneumonia. Moreover, in active TB a decline of total urine IP-10 was observed at therapy completion; agonist/antagonist forms reflected this decline although their differences were not statistically significant. CONCLUSIONS: We showed for the first time that agonist/antagonist IP-10 forms are measurable in urine. IP-10 levels associate with TB and pneumonia disease, suggesting their association with acute inflammation. Further studies are needed to assess their role to monitor TB treatment efficacy.

Designing an improved T-cell mobilising CXCL10 mutant through enhanced GAG binding affinity.

The chemokine CXCL10 is released by a plethora of cells, including immune and metastatic cancer cells, following stimulation with interferon-gamma. It acts via its GPC receptor on T-cells attracting them to various target tissues. Glycosaminoglycans (GAGs) are regarded as co-receptors of chemokines, which enable the establishment of a chemotactic gradient for target cell migration. We have engineered human CXCL10 towards improved T-cell mobilisation by implementing a single site-directed mutation N20K into the protein, which leads to a higher GAG binding affinity compared to the wild type. Interestingly, this mutation not only increased T-cell migration in a transendothelial migration assay, the mutant intensified T-cell chemotaxis also in a Boyden chamber set-up thereby indicating a strong role of T-cell-localised GAGs on leukocyte migration. A CXCL10 mutant with increased GAG-binding affinity could therefore potentially serve as a T-cell mobiliser in pathological conditions where the immune surveillance of the target tissue is impaired, as is the case for most solid tumors.

Integrin α4ß7 switches its ligand specificity via distinct conformer-specific activation.

Chemokine (C-C motif) ligand 25 (CCL25) and C-X-C motif chemokine 10 (CXCL10) induce the ligand-specific activation of integrin α4ß7 to mediate the selective adhesion of lymphocytes to mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) or vascular cell adhesion molecule-1 (VCAM-1). However, the mechanism underlying the selective binding of different ligands by α4ß7 remains obscure. In this study, we demonstrate that CCL25 and CXCL10 induce distinct active conformers of α4ß7 with a high affinity for either MAdCAM-1 or VCAM-1. Single-cell force measurements show that CCL25 increases the affinity of α4ß7 for MAdCAM-1 but decreases its affinity for VCAM-1, whereas CXCL10 has the opposite effect. Structurally, CCL25 induces a more extended active conformation of α4ß7 compared with CXCL10-activated integrin. These two distinct intermediate open α4ß7 conformers selectively bind to MAdCAM-1 or VCAM-1 by distinguishing their immunoglobulin domain 2. Notably, Mn2+ fully opens α4ß7 with a high affinity for both ligands. Thus, integrin α4ß7 adopts different active conformations to switch its ligand-binding specificity.

CXCL10/IP-10 Neutralization Can Ameliorate Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome in Rats.

The role of C-X-C motif chemokine 10 (CXCL10), a pro-inflammatory factor, in the development of acute respiratory distress syndrome (ARDS) remains unclear. In this study, we explored the role of CXCL10 and the effect of CXCL10 neutralization in lipopolysaccharide (LPS)-induced ARDS in rats. The expression of CXCL10 and its receptor chemokine receptor 3(CXCR3) increased after LPS induction. Moreover, neutralization of CXCL10 ameliorated the severity of ARDS by reducing pulmonary edema, inhibiting the release of inflammatory mediators (IFN-γ, IL-6 and ICAM-1) and limiting inflammatory cells (neutrophils, macrophages, CD8+ T cells) influx into the lung, with a reduction in CXCR3 expression in neutrophils and macrophages. Therefore, CXCL10 could be a potential therapeutic target in LPS-induced ARDS.

Identification of a fish specific chemokine CXCL_F2 in large yellow croaker (Larimichthys crocea) reveals its primitive chemotactic function.

Chemokines are a superfamily of cytokines regulating immune cell migration under both inflammatory and normal physiological conditions. Currently, a number of fish specific CXC chemokines, named as CXCL_F1-5, have been identified in several species. However, understanding of their functional characteristics is still limited. In this study, we identified a fish specific chemokine CXCL_F2 (LycCXCL_F2) from large yellow croaker (Larimichthys crocea). The open reading frame (ORF) of LycCXCL_F2 is 348 nucleotides long, encoding a protein of 115 amino acids (aa). The deduced LycCXCL_F2 protein contains a 20-aa signal peptide and a 95-aa mature polypeptide. Phylogenetic analysis showed that LycCXCL_F2 fell into a major clade formed by CXCL_F2 sequences and was separated from CXCL_F1 and CXCL_F3-5 subgroups. LycCXCL_F2 mRNA transcript was constitutively expressed in various tissues, with the highest levels in spleen and head kidney. After stimulation with inactivated trivalent bacterial vaccines, LycCXCL_F2 mRNA transcription was significantly increased in both spleen and head kidney. Moreover, recombinant LycCXCL_F2 protein exhibited obvious chemotaxis to monocytes, lymphocytes and eosnophils of PBLs isolated from large yellow croaker, but could not induce the respiratory burst of macrophages. These results indicate that this fish specific CXC chemokine LycCXCL_F2 possesses primitive chemotactic activity and may play a role in immune response in large yellow croaker.

CXCL10 Acts as a Bifunctional Antimicrobial Molecule against Bacillus anthracis.

UNLABELLED: Bacillus anthracis is killed by the interferon-inducible, ELR(-) CXC chemokine CXCL10. Previous studies showed that disruption of the gene encoding FtsX, a conserved membrane component of the ATP-binding cassette transporter-like complex FtsE/X, resulted in resistance to CXCL10. FtsX exhibits some sequence similarity to the mammalian CXCL10 receptor, CXCR3, suggesting that the CXCL10 N-terminal region that interacts with CXCR3 may also interact with FtsX. A C-terminal truncated CXCL10 was tested to determine if the FtsX-dependent antimicrobial activity is associated with the CXCR3-interacting N terminus. The truncated CXCL10 exhibited antimicrobial activity against the B. anthracis parent strain but not the ΔftsX mutant, which supports a key role for the CXCL10 N terminus. Mutations in FtsE, the conserved ATP-binding protein of the FtsE/X complex, resulted in resistance to both CXCL10 and truncated CXCL10, indicating that both FtsX and FtsE are important. Higher concentrations of CXCL10 overcame the resistance of the ΔftsX mutant to CXCL10, suggesting an FtsX-independent killing mechanism, likely involving its C-terminal α-helix, which resembles a cationic antimicrobial peptide. Membrane depolarization studies revealed that CXCL10 disrupted membranes of the B. anthracis parent strain and the ΔftsX mutant, but only the parent strain underwent depolarization with truncated CXCL10. These findings suggest that CXCL10 is a bifunctional molecule that kills B. anthracis by two mechanisms. FtsE/X-dependent killing is mediated through an N-terminal portion of CXCL10 and is not reliant upon the C-terminal α-helix. The FtsE/X-independent mechanism involves membrane depolarization by CXCL10, likely because of its α-helix. These findings present a new paradigm for understanding mechanisms by which CXCL10 and related chemokines kill bacteria. IMPORTANCE: Chemokines are a class of molecules known for their chemoattractant properties but more recently have been shown to possess antimicrobial activity against a wide range of Gram-positive and Gram-negative bacterial pathogens. The mechanism(s) by which these chemokines kill bacteria is not well understood, but it is generally thought to be due to the conserved amphipathic C-terminal α-helix that resembles cationic antimicrobial peptides in charge and secondary structure. Our present study indicates that the interferon-inducible, ELR(-) chemokine CXCL10 kills the Gram-positive pathogen Bacillus anthracis through multiple molecular mechanisms. One mechanism is mediated by interaction of CXCL10 with the bacterial FtsE/X complex and does not require the presence of the CXCL10 C-terminal α-helix. The second mechanism is FtsE/X receptor independent and kills through membrane disruption due to the C-terminal α-helix. This study represents a new paradigm for understanding how chemokines exert an antimicrobial effect that may prove applicable to other bacterial species.

Serum soluble urokinase-type plasminogen activator receptor and interferon-γ-induced protein 10 levels correlate with significant fibrosis in chronic hepatitis B.

BACKGROUND: Hepatitis B virus (HBV) presents an important public health problem. Liver biopsy is currently the gold standard for assessing the degree of intrahepatic inflammation and for staging liver fibrosis. However, the value of liver biopsies is limited by sampling errors, understaging and interobserver variability in interpretation. There is, therefore, a need to identify novel, non-invasive serologic biomarkers for the development of new predictive models of fibrosis. METHODS: We enrolled patients with chronic hepatitis B infection (CHB) and examined the relationships between serum soluble urokinase plasminogen activator receptor (suPAR) and interferon-induced protein-10 (IP-10), and the results of liver biopsies. Healthy volunteers with normal aminotransferase levels and negative serological results for HBV, hepatitis C virus and human immunodeficiency virus were recruited as controls. RESULTS: Mean platelet volume, serum suPAR and IP-10 were significantly elevated in patients with CHB compared with controls. Median serum suPAR and IP-10 levels were significantly higher in patients with liver fibrosis compared with patients with mild fibrosis. There was no significant difference in mean platelet volume or aspartate aminotransferase-to-platelet ratio index scores between patients with mild and significant fibrosis. CONCLUSION: suPAR and IP-10 were able to distinguish between significant and mild fibrosis with good sensitivity and specificity, and may thus represent useful biomarkers for identifying patients with significant fibrosis.

De novo isolation of antibodies with pH-dependent binding properties.

pH-dependent antibodies are engineered to release their target at a slightly acidic pH, a property making them suitable for clinical as well as biotechnological applications. Such antibodies were previously obtained by histidine scanning of pre-existing antibodies, a labor-intensive strategy resulting in antibodies that displayed residual binding to their target at pH 6.0. We report here the de novo isolation of pH-dependent antibodies selected by phage display from libraries enriched in histidines. Strongly pH-dependent clones with various affinity profiles against CXCL10 were isolated by this method. Our best candidate has nanomolar affinity for CXCL10 at pH 7.2, but no residual binding was detected at pH 6.0. We therefore propose that this new process is an efficient strategy to generate pH-dependent antibodies.

Development of a highly sensitive liquid chromatography/tandem mass spectrometry method to quantify total and free levels of a target protein, interferon-gamma-inducible protein-10, at picomolar levels in human serum.

RATIONALE: Liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays are increasingly being used for absolute quantitation of proteins due to high specificity and low cost. However, the major challenge for the LC/MS method is insufficient sensitivity. This paper details the strategies developed to maximize the sensitivity from aspects of chromatography, mass spectrometry, and sample preparation to achieve a highly sensitive LC/MS method. METHODS: The method is based on the LC/MS/MS measurement of a surrogate peptide generated from trypsin digestion of interferon-gamma-inducible protein-10 (IP-10). The sample preparation strategy involved selectively extracting IP-10 and removing high-abundance serum proteins through acidified protein precipitation (PPT). It was revealed in this work that these high-abundance serum proteins, if not separated from the protein of interest, could cause significant ionization saturation and high background noise in selected reaction monitoring (SRM), leading to a 100-fold higher lower limit of quantification (LLOQ). RESULTS: Our method demonstrated that the acidified PPT could be optimized to selectively extract the protein of interest with full recovery of 97% to 103%, while the high-abundance serum proteins could be effectively removed with minimal matrix effect of 90% to 93%. For the first time, a highly sensitive LC/MS method with a LLOQ of 31.62 pM for the quantitation of IP-10 has been achieved, which is a 100-fold improvement over the generic method. CONCLUSIONS: The described method offers excellent sensitivity with advantages of being antibody reagent independent and leads to significant cost and time savings. It has been successfully employed to determine both total and free IP-10 levels in human serum samples. This method development strategy may also be applied to other small proteins.

Evidence for the antagonistic form of CXC-motif chemokine CXCL10 in serous epithelial ovarian tumours.

Patients with high-grade, serous epithelial ovarian carcinoma (HGSOC) are generally diagnosed with extensive peritoneal metastases, and exhibit 5-year survival rates <30%. A subset of these tumours, defined as "immunoreactive," overexpress mRNA encoding the T-cell-recruiting chemokine CXCL10 (10-kDa interferon gamma-induced protein; C-X-C motif chemokine 10). Tumour-infiltrating CD4(+) CD8(+) T-cells are a well-documented, positive prognostic indicator for HGSOC patients; paradoxically, however, patients diagnosed with HGSOC (overexpressing CXCL10 and therefore theorised to recruit T-cells) typically exhibit poor survival. Recently, an "antagonistic" CXCL10 variant was identified that inhibited leucocyte recruitment to inflamed liver in vivo (Casrouge et al., J Clin Invest 2011;121:308-17). We hypothesised that "immunoreactive" HGSOC might also express antagonistic CXCL10, interfering with leucocyte recruitment and contributing to poor patient prognosis. CXCL10 expression was analysed in HGSOC tissues grouped according to pathology, grade and FIGO stage at diagnosis, and its localisation and association with T-cells established by immunohistochemical staining in tissue microarrays. CXCL10 expression was increased in a subset of serous epithelial tumour samples; however, it did not correlate well with CD45-positive tumour infiltrate. Immunoprecipitation and de novo sequence analysis of CXCL10 identified the N-terminally cleaved, "antagonistic" variant of CXCL10 specifically in malignant tumours, and not in benign ovarian disease. The data demonstrate the presence of the antagonistic form of CXCL10 in HGSOC for the first time, and provide a partial explanation for reduced leucocyte infiltration observed in these tumours. We suggest that CXCL10 cleavage and subsequent antagonism of immune cell recruitment may be a feature of the "immunoreactive" HGSOC subtype, leading to early impairment of the immune response and subsequently worsening patient prognosis.

CXCL10 decreases GP73 expression in hepatoma cells at the early stage of hepatitis C virus (HCV) infection.

Golgi protein 73 (GP73), which is up-regulated in hepatocellular carcinoma (HCC), has recently been identified as a novel serum marker for HCC diagnosis. Several reports also noted the increased levels of GP73 expression in chronic liver disease in patients with acute hepatitis of various etiologies, chronic Hepatitis C virus (HCV) infection and alcoholic liver disease. The molecular mechanisms of GP73 expression in HCV related liver disease still need to be determined. In this study, we aimed to evaluate the effect of HCV infection on GP73 expression. GP73 was highly expressed in Huh7, Hep3B, 293T and HUVEC cells, and was low-expressed in HepG2 cells. HCV infection led to down-regulation of GP73 in Huh7 and HepG2/CD81 cells at the early stage of infection. CXCL10 decreased GP73 expression in Huh7 and HepG2 cells. Up-regulation of GP73 was noted in hepatocytes with cytopathic effect at advanced stage of HCV infection, and further research is needed to determine the unknown factors affecting GP73 expression. In conclusion, our study provided additional evidence for the roles of GP73 in liver disease.

Study on characteristics of chemokine CXCL10 gene cloned from cDNA expression library of Ujumqin sheep.

Chemokines were a major regulator of body's inflammatory and immune responses. In this study, the cDNA fragment of chemokine CXC ligand 10 (CXCL10) was cloned from the Ujumqin sheep ear marginal tissue cDNA expression library; the CXCL10 gene had 103 amino acids and a molecular weight of 11.47 kDa, and it shared a high homology among cattle, sheep, and goat, while a low homology compared with mouse. The CXCL10 protein had 4 conservative cysteine residues, located in 28, 30, 55, and 72 sites. The expression pattern and intracellular distribution of recombinant CXCL10 proteins in Ujumqin sheep fibroblast cells showed that there were green fluorescence signals both in cytoplasm and nucleolus after 24 h of transfection, the number of positive cells was increased with time, the peak level of fluorescence signal was reached after 48 h of transfection and the transfection efficiency was 33.3%; there was a significant decrease in fluorescence intensity after 72 h of transfection. Expression of recombinant CXCL10 gene in Escherichia coli had a time- and temperature-dependency on the amount of protein expression, and a small quantity of inducer was needed.

An IP-10 (CXCL10)-derived peptide inhibits angiogenesis.

Angiogenesis plays a critical role in processes such as organ development, wound healing, and tumor growth. It requires well-orchestrated integration of soluble and matrix factors and timely recognition of such signals to regulate this process. Previous work has shown that newly forming vessels express the chemokine receptor CXC receptor 3 (CXCR3) and, activation by its ligand IP-10 (CXCL10), both inhibits development of new vasculature and causes regression of newly formed vessels. To identify and develop new therapeutic agents to limit or reverse pathological angiogenesis, we identified a 21 amino acid fragment of IP-10, spanning the α-helical domain residues 77-98, that mimic the actions of the whole IP-10 molecule on endothelial cells. Treatment of the endothelial cells with the 22 amino acid fragment referred to as IP-10p significantly inhibited VEGF-induced endothelial motility and tube formation in vitro, properties critical for angiogenesis. Using a Matrigel plug assay in vivo, we demonstrate that IP-10p both prevented vessel formation and induced involution of nascent vessels. CXCR3 neutralizing antibody was able to block the inhibitory effects of the IP-10p, demonstrating specificity of the peptide. Inhibition of endothelial function by IP-10p was similar to that described for IP-10, secondary to CXCR3-mediated increase in cAMP production, activation of PKA inhibiting cell migration, and inhibition of VEGF-mediated m-calpain activation. IP-10p provides a novel therapeutic agent that inhibits endothelial cell function thus, allowing for the modulation of angiogenesis.