G-protein Coupled Chemokine Receptor Research Articles

Genetic Characterization of Lumpy Skin Disease Viruses Circulating in Lesotho Cattle

Lumpy skin disease is one of the fast-spreading viral diseases of cattle and buffalo that can potentially cause severe economic impact. Lesotho experienced LSD for the first time in 1947 and episodes of outbreaks occurred throughout the decades. In this study, eighteen specimens were collected from LSD-clinically diseased cattle between 2020 and 2022 from Mafeteng, Leribe, Maseru, Berea, and Mohales’ Hoek districts of Lesotho. A total of 11 DNA samples were analyzed by PCR and sequencing of the extracellular enveloped virus (EEV) glycoprotein, G-protein-coupled chemokine receptor (GPCR), 30 kDa RNA polymerase subunit (RPO30), and B22R genes. All nucleotide sequences of the above-mentioned genes confirmed that the PCR amplicons of clinical samples are truly LSDV, as they were identical to respective LSDV isolates on the NCBI GenBank. Two of the elevem samples were further characterized by whole-genome sequencing. The analysis, based on both CaPV marker genes and complete genome sequences, revealed that the LSDV isolates from Lesotho cluster with the NW-like LSDVs, which includes the commonly circulating LSDV field isolates from Africa, the Middle East, the Balkans, Turkey, and Eastern Europe.

Abstract 4119: CXCR4/CB2 heterodimer antagonizes CXCR4-mediated metastasis and tumor growth in vivo

Abstract The G-protein-coupled chemokine receptor (GPCR), CXCR4, is ubiquitously expressed in malignant tumor tissues, and controls migration and other tumor survival mechanisms upon interaction with its chemokine agonist, stromal cell-derived 1α (SDF1α). The CXCR4/SDF1α axis is relevant to shaping the tumor microenvironment mainly the metastatic spread of primary tumors to distal organs and immune response; thus, expression of CXCR4 in tumors is vital to tumor aggressiveness, survival probability, and metastasis-associated mortality. CXCR4 signals through a homodimer, but also forms heterodimers with other chemokine and non-chemokine GPCRs. We’ve demonstrated that CXCR4 forms a heterodimer with the GPCR, cannabinoid receptor 2 (CB2), in human breast and prostate cell lines in vitro. The cannabinoid system has earned interest as anticancer therapeutic targets by modulating growth, migration, angiogenesis, etc. In our model, CXCR4-mediated, pro-tumor signaling was reduced by the physical heterodimer, and the dimer was induced by simultaneous treatment with agonists (SDF1α and AM1241) for respective receptors. The resultant heterodimer negatively interfered with migration, cytoskeletal rearrangement, motility associated proteins, and signaling that coordinates movement. Given that cannabinoids have been shown to reduce metastasis and profession of clinical tumors, we hypothesize that a heterodimer of CXCR4 and CB2 disrupts CXCR4-mediated metastasis, and ultimately, decrease tumor progression in vivo.Herein, we tested our hypothesis in an in vivo model of spontaneous metastasis NSG male mice. Luciferase-containing human prostate cancer cells (DU145 RFP/Luc) were implanted subcutaneously. Once tumors reached 40mm3, mice were randomized by volume, weight and age, and divided into 4 treatments: vehicle, SDF1α, AM124, and SDF1α/AM124(combo) for daily intraperitoneal injections. Tumors reaching 400mm3 were excised, and treatments continued for 3 weeks. Spontaneous metastasis was monitored via IVIS imaging at 2- and 3-weeks post-tumor removal. We observed reduced tumor growth, and decreased incidence rates of metastasis to the lung in the combination treatment group, which, based on our in vitro data, is representative of a CXCR4/CB2 heterodimer. This novel definition of heterodimerization could reveal and exploit new mechanisms for CXCR4 regulation in tumor metastasis. The CXCR4/CB2 heterodimer may attenuate responses triggered individually by CXCR4 without toxicity and side effects experienced with current CXCR4 antagonists. Citation Format: Nakea Michelle Pennant, Alifiani B. Hartono, Shiqin Liu, Christopher Massey, Chung Lee, Tanya Stoyanova, Cimona V. Hinton. CXCR4/CB2 heterodimer antagonizes CXCR4-mediated metastasis and tumor growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4119.

Genetic Diversity of LSDV Isolates from Menofeia Governorate, Egypt, Summer 2019 based on the G-Protein Coupled Chemokine Receptor (GPCR) Gene

Genetic Diversity of LSDV Isolates from Menofeia Governorate, Egypt, Summer 2019 based on the G-Protein Coupled Chemokine Receptor (GPCR) Gene

Abstract A12: Pervasive hypermutation of super-enhancer regions dysregulates oncogene expression in diffuse large B-cell lymphoma

Abstract Coding-genome sequencing efforts have identified several genes/pathways altered in Diffuse Large B-cell Lymphoma (DLBCL), including new potential therapeutic targets. However, the mutational contribution of the non-coding genome of DLBCL remains largely unexplored. To identify functionally relevant non-coding mutations targeting regulatory domains, we integrated enhancer (E)/super-enhancer (SE) identification, by ChIP-seq analysis of the hallmark H3K27 histone mark, with whole genome sequencing (WGS) and RNA-seq analysis in 29 DLBCL cell lines representative of the major DLBCL subtypes, along with germinal center (GC) B cells representing the normal counterpart of DLBCL. Following validation in 93 normal/tumor DLBCL biopsies, we found that active SEs are specifically hypermutated (≥3 somatic mutations/Kb) in 99% of DLBCLs, as compared to the same loci when not active as SE (in total, 159 hypermutated SEs, with at least 2/case). As evidence of oncogenic relevance, we have shown that the hypermutated SEs linked to the BCL6 and BCL2 proto-oncogenes prevent the binding and transcriptional downregulation by transcriptional repressors. Genetic correction of selected mutations using the CRISPR/Cas9 technology restored repressor DNA-binding, downregulated target gene expression, and led to the counter-selection of cells harboring corrected alleles, indicating oncogenic dependency from the SE mutations. A third recurrently hypermutated SE was the CXCR4-SE, affected in 19% of cases. CXCR4 encodes for a G-protein coupled chemokine receptor involved in cell migration within the GC, and is mutationally activated in 40% of Waldenström macrobulinemia (WM), a post-GC lymphoproliferative disease. We identified a mutational hotspot preventing the binding of the NR3C1 glucocorticoid receptor in 7.5% of DLBCL cases. Correction of the mutations in cell lines led to counter selection and reduced expression of CXCR4. Conversely, the introduction of a mutation disrupting NR3C1 binding in WT DLBCL cells increased CXCR4 expression. Together, these results are consistent with dependency of the mutant cells on the CXCR4-SE mutation, and confirm a direct link between SE mutations in this region and deregulated gene expression through escape from NR3C1-mediated suppression. In primary cases, mutations in the NR3C1 binding site of the CXCR4-SE were observed in the absence of NR3C1 coding mutations and correlated with increased CXCR4 transcript levels, as documented by RNA-seq. Together with the oncogenic role of CXCR4 in WM, these findings reveal this chemokine receptor as a potential oncogenic target in DLBCL. Collectively, these findings reveal a new layer of genetic alterations, which identifies novel mechanisms of dysregulation for known oncogenes, as well as new dysregulated genes and pathways, with implications for precision classification and therapeutic targeting of DLBCL. * Co-senior authors. Citation Format: Elodie Bal, Rahul Kumar, Mohammad Hadigol, Antony B. Holmes, Laura K. Hilton, Jui Wan Loh, Kostiantyn Dreval, Jasper Wong, Sofija Vlasevska, Clarissa Corinaldesi, Rajesh Kumar Soni Soni, Katia Basso, Ryan D. Morin, Hossein Khiabanian, Laura Pasqualucci, Riccardo Dalla-Favera. Pervasive hypermutation of super-enhancer regions dysregulates oncogene expression in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A12.

Isolation and molecular characterization of lumpy skin disease virus from hard ticks, Rhipicephalus (Boophilus) annulatus in Egypt

BackgroundLumpy skin disease (LSD), a disease of cattle and buffaloes, has recently become widely prevalent in Egypt. The aim of this study was to ascertain the potential role of Rhipicephalus (Boophilus) annulatus ticks in the transmission of this disease. Samples collected from suspected lumpy skin disease virus (LSDV) infected cows that had previously been vaccinated with the Romanian sheep pox virus (SPPV) in various Egyptian governorates were obtained between May to November over two consecutive years, namely 2018 and 2019. Ticks were morphologically identified and the partial cytochrome oxidase subunit I gene (COI) were sequenced, revealing that they were closely related to R. (Boophilus) annulatus. The G-protein-coupled chemokine receptor (GPCR) gene of the LSDV was used to test hard ticks.ResultsTwo positive samples from Kafr El-Sheikh province and one positive sample from Al-Behera province were reported. BLAST analysis revealed that the positive samples were closely related to the Kazakhstani Kubash/KAZ/16 strain (accession number MN642592). Phylogenetic analysis revealed that the GPCR gene of the LSDV recently circulating in Egypt belongs to a global cluster of field LSDV with a nucleotide identity of 98–100%. LSDV isolation was successfully performed four days after inoculation using 9 to 11-day-old embryonated chicken eggs showing characteristic focal white pock lesions dispersed on the choriallantoic membrane after three blind passages. Intracytoplasmic inclusion bodies, cell rupture, vacuoles in cells, and virus particles ovoid in shape were demonstrated by electron microscopy.ConclusionIn this study the role of hard ticks in the transmission of the LSDV to susceptible animals in Egypt was revealed and confirmed by various methods.

Molecular Characterization and Phylogenetic Analysis of Lumpy Skin Disease Virus Collected from Outbreaks in Northern Thailand in 2021.

Understanding molecular epidemiology is essential for the improvement of lumpy skin disease (LSD) eradication and control strategies. The objective of this study was to perform a molecular characterization and phylogenetic analysis of lumpy skin disease virus (LSDV) isolated from dairy cows presenting LSD-like clinical signs in northern Thailand. The skin nodules were collected from 26 LSD-suspected cows involved in six outbreaks during the period from July to September of 2021. LSDVs were confirmed from clinical samples using the polymerase chain reaction (PCR). The PCR-positive samples were subsequently amplified and sequenced using a G-protein-coupled chemokine receptor (GPCR) gene for molecular characterization and phylogenetic analyses. All 26 samples were positive for LSDV by PCR. A phylogenetic analysis indicated that the 24 LSDV isolates obtained from cattle in northern Thailand were closely related to other LSDV sequences acquired from Asia (China, Hong Kong, and Vietnam). On the other hand, two LSDV isolates of the cows presenting LSD-like clinical signs after vaccination were clustered along with LSDV Neethling-derived vaccines. The outcomes of this research will be beneficial in developing effective control strategies for LSDV.

CXCL12-CXCR4/CXCR7 Axis in Colorectal Cancer: Therapeutic Target in Preclinical and Clinical Studies.

Chemokines are chemotactic cytokines that promote cancer growth, metastasis, and regulate resistance to chemotherapy. Stromal cell-derived factor 1 (SDF1) also known as C-X-C motif chemokine 12 (CXCL12), a prognostic factor, is an extracellular homeostatic chemokine that is the natural ligand for chemokine receptors C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or cluster of differentiation 184 (CD184) and chemokine receptor type 7 (CXCR7). CXCR4 is the most widely expressed rhodopsin-like G protein coupled chemokine receptor (GPCR). The CXCL12–CXCR4 axis is involved in tumor growth, invasion, angiogenesis, and metastasis in colorectal cancer (CRC). CXCR7, recently termed as atypical chemokine receptor 3 (ACKR3), is amongst the G protein coupled cell surface receptor family that is also commonly expressed in a large variety of cancer cells. CXCR7, like CXCR4, regulates immunity, angiogenesis, stem cell trafficking, cell growth and organ-specific metastases. CXCR4 and CXCR7 are expressed individually or together, depending on the tumor type. When expressed together, CXCR4 and CXCR7 can form homo- or hetero-dimers. Homo- and hetero-dimerization of CXCL12 and its receptors CXCR4 and CXCR7 alter their signaling activity. Only few drugs have been approved for clinical use targeting CXCL12-CXCR4/CXCR7 axis. Several CXCR4 inhibitors are in clinical trials for solid tumor treatment with limited success whereas CXCR7-specific inhibitors are still in preclinical studies for CRC. This review focuses on current knowledge of chemokine CXCL12 and its receptors CXCR4 and CXCR7, with emphasis on targeting the CXCL12–CXCR4/CXCR7 axis as a treatment strategy for CRC.

Genetic Evidence of Multiple Introductions of Lumpy Skin Disease Virus into Saratov Region, Russia.

Lumpy skin disease virus (LSDV) is the causative agent of lumpy skin disease (LSD) that has been recently reported in the South-East and North Asian parts of the Russian Federation. During 2017–2019, there were more than 30 LSD outbreaks in Saratov Region despite active inoculation of cattle with heterologous vaccine. Importantly, the first case of the novel recombinant LSDV strain was reported here in 2017. This study aimed to determine the main clonal lineage(s) of LSDV strains circulated within Saratov Region and other regions of Russia since the first introduction of LSDV. The molecular typing and subtyping based on the coding regions of the G-protein-coupled chemokine receptor (GPCR) gene resulted in a discrimination of all outbreak-related LSDV strains into two main types, such as Type I and Type II, and subtypes Ia-d and IIa-g. Phylogenetically, eleven LSDV lineages were revealed in Russia including the five ones in Saratov Region. They were the following: (i) the Neethling wild Type Ia/2017; (ii) the recombinant Saratov IIc/2017/2019; (iii) the specific Dergachevskyi IId/2017; (iv) the Khvalynsky IIg/2018, and (v) the Haden-Type IIa lineage for the six LSDV strains detected in cattle immunized with heterologous vaccine during the last LSD outbreak in the Saratov Region, Nesterovo Village, in 2019 (Nesterovo-2019 strains). A single LSDV strain detected in Saratov Region in 2017 had the same Type Ia that was identified in 2016 in the bordered Republic of Kazakhstan. Phylogeographic analysis demonstrated three nominal clusters of LSDV types in the following Russian Federation territories: (I) the Central European part; (II) the South-East of the European part; (III) the North Asian part. Cluster I was represented by mainly Type I strains, while both Clusters 2 and 3 contained predominantly Type II strains. The Clusters I and II partially overlapped, while Cluster 3 was separate. Multiple introductions of LSDV into Saratov Region in 2017–2019 using GPCR-based molecular typing and subtyping were revealed. This scheme is a promising tool for molecular discrimination of LSDV strains derived from both vaccinated and unvaccinated against LSD cattle as well as for molecular epidemiology.

Use of an Alignment-Free Method for the Geographical Discrimination of GTPVs Based on the GPCR Sequences.

Goatpox virus (GTPV) belongs to the genus Capripoxvirus, together with sheeppox virus (SPPV) and lumpy skin disease virus (LSDV). GTPV primarily affects sheep, goats and some wild ruminants. Although GTPV is only present in Africa and Asia, the recent spread of LSDV in Europe and Asia shows capripoxviruses could escape their traditional geographical regions to cause severe outbreaks in new areas. Therefore, it is crucial to develop effective source tracing of capripoxvirus infections. Earlier, conventional phylogenetic methods, based on limited samples, identified three different nucleotide sequence profiles in the G-protein-coupled chemokine receptor (GPCR) gene of GTPVs. However, this method did not differentiate GTPV strains by their geographical origins. We have sequenced the GPCR gene of additional GTPVs and analyzed them with publicly available sequences, using conventional alignment-based methods and an alignment-free approach exploiting k-mer frequencies. Using the alignment-free method, we can now classify GTPVs based on their geographical origin: African GTPVs and Asian GTPVs, which further split into Western and Central Asian (WCA) GTPVs and Eastern and Southern Asian (ESA) GTPVs. This approach will help determine the source of introduction in GTPV emergence in disease-free regions and detect the importation of additional strains in disease-endemic areas.

Targeting the MIF/CXCR7/AKT Signaling Pathway in Castration-Resistant Prostate Cancer.

Although androgen deprivation therapy (ADT) is an effective treatment for metastatic prostate cancer, incurable castration-resistant prostate cancer (CRPC) inevitably develops. Importantly, androgen receptor (AR) continues to be critical for prostate cancer growth and progression after ADT. One of the underlying molecular mechanisms is derepression of AR-repressed genes involved in cell cycle and proliferation after ADT. Here, the data demonstrate that C-X-C chemokine receptor type 7 (CXCR7), a seven-transmembrane G-protein-coupled chemokine receptor, is an AR-repressed gene and is upregulated after ADT. AR directly regulates CXCR7 using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing. Macrophage migration inhibitory factor (MIF) was identified as a ligand for CXCR7, which induces expression of cell-cycle genes through activating AKT signaling pathway. Previous studies have been focused on chemokine CXCL12 and its receptor CXCR4 in mediating metastasis of various cancer types, including prostate cancer. The critical roles of CXCL12/CXCR4 axis in the interaction between cancer cells and their microenvironment render it a promising therapeutic target in cancer treatment. The data suggest that the MIF/CXCR7/AKT pathway drives CRPC growth and metastasis independent of the CXCL12/CXCR4 axis. Furthermore, CXCR7 blockade in combination with anti-androgen enzalutamide inhibits CRPC tumor growth and potentially prevents metastasis. Notably, both MIF and CXCR7 are overexpressed in CRPC patient specimens and therefore are attractive therapeutic targets for these patients. IMPLICATIONS: This work suggests that CXCR7 plays more important roles than CXCR4 in CRPC progression; thus, targeting CXCR7 in combination with anti-androgen is a promising therapeutic approach for metastatic CRPC.

Outbreak of lumpy skin disease in a dairy farm in Keffi, Nasarawa State, Nigeria

A dairy farm in Keffi, Nasarawa State, North Central Nigeria reported cases of skin infections in cattle suggestive of lumpy skin disease (LSD) in the years 2014 and 2016. During the 2016 outbreak, skin biopsies were collected for laboratory investigation using histopathology and polymerase chain reaction (PCR). The samples were subjected to PCR using two sets of primers which amplified the G-protein-coupled chemokine receptor (GPCR) gene and RNA polymerase subunit gene (RPO30) of the LSD virus (LSDV). Clinical signs observed in the two outbreaks were similar and included ocular and nasal discharges, nodular skin lesion, and pyrexia and reduced milk production. Morbidity rate was 6.25% (25/400) in 2014 and the mortality rate was 2.5% (10/400). Furthermore, the second outbreak in 2016 had morbidity and mortality rates of 5% (20/400) and 0.5 % (2/400) respectively. From animals that died in the 2014 outbreak, necropsy findings included nodular lesions on the skin, muzzle, in the buccal cavity and nasal mucosa. Also, congested and oedematous lungs and enlarged lymph nodes were observed. The nodular skin lesions observed during the 2016 outbreak were larger and more numerous in the Sokoto Gudali, while the Holstein Friesian cross had smaller gross skin nodules with acute to sub-acute histopathologic changes. The histopathological examination of the skin biopsy of the HF was consistent with LSD viral lesions, while PCR detected the RPO30 and GPCR gene fragments of LSDV. Keywords: Dairy, histopathology, Lumpy skin disease, polymerase chain reaction, Nigeria

Sporadic cases of lumpy skin disease among cattle in Sharkia province, Egypt: Genetic characterization of lumpy skin disease virus isolates and pathological findings.

Background and Aim:Lumpy skin disease (LSD) is a highly infectious viral disease upsetting cattle, caused by LSD virus (LSDV) within the family Poxviridae. Sporadic cases of LSD have been observed in cattle previously vaccinated with the Romanian sheep poxvirus (SPPV) vaccine during the summer of 2016 in Sharkia province, Egypt. The present study was undertaken to perform molecular characterization of LSDV strains which circulated in this period as well as investigate their phylogenetic relatedness with published reference capripoxvirus genome sequences.Materials and Methods:A total of 82 skin nodules, as well as 5 lymph nodes, were collected from suspect LSD cases, and the virus was isolated in embryonated chicken eggs (ECEs). LSD was confirmed by polymerase chain reactions amplification of the partial and full-length sequences of the attachment and G-protein-coupled chemokine receptor (GPCR) genes, respectively, as well as a histopathological examination of the lesions. Molecular characterization of the LSDV isolates was conducted by sequencing the GPCR gene.Results:Characteristic skin nodules that covered the whole intact skin, as well as lymphadenopathy, were significant clinical signs in all suspected cases. LSDV isolation in ECEs revealed the characteristic focal white pock lesions dispersed on the chorioallantoic membranes. Histopathologic examination showed characteristic eosinophilic intracytoplasmic inclusion bodies within inflammatory cell infiltration. Phylogenetic analysis revealed that the LSDV isolates were clustered together with other African and European LSDV strains. In addition, the LSDV isolates have a unique signature of LSDVs (A11, T12, T34, S99, and P199).Conclusion:LSDV infections have been detected in cattle previously vaccinated with Romanian SPPV vaccine during the summer of 2016 and making the evaluation of vaccine efficacy under field conditions necessary.

Chemerin induces endothelial cell inflammation: activation of nuclear factor-kappa beta and monocyte-endothelial adhesion.

Chemerin, a chemoattractant protein, acts via a G-protein coupled chemokine receptor, i.e. Chemokine like Receptor 1/ChemR23; levels of which are elevated in pro-inflammatory states such as obesity and type 2 diabetes mellitus (T2DM). Obesity and T2DM patients are at high risk of developing cardiovascular disorders such as atherosclerosis. We have reported that chemerin induces human endothelial cell angiogenesis and since dysregulated angiogenesis and endothelial dysfunction are hallmarks of vascular disease; we sought to determine the effects of chemerin on monocyte-endothelial adhesion, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), a critical pro-inflammatory transcription factor. Human endothelial cells were transfected with pNF-kappaB-Luc plasmid. Chemerin induced NF-κB activation via the MAPK and PI3K/Akt pathways. Western blot analyses and monocyte-endothelial adhesion assay showed that chemerin increased endothelial cell adhesion molecule expression and secretion, namely E-selectin (Endothelial Selectin), VCAM-1 (Vascular Cell Adhesion Molecule-1) and ICAM-1 (Intracellular Adhesion Molecule-1), leading to enhancement of monocyte-endothelial adhesion. Additionally, we showed a synergistic response of the pro-inflammatory mediator, Interleukin-1β with chemerin induced effects. Chemerin plays an important role in endothelial inflammation, as it induces monocyte-endothelial adhesion, a critical step in the development of atherosclerosis.

Development of real-time and lateral flow dipstick recombinase polymerase amplification assays for rapid detection of goatpox virus and sheeppox virus

BackgroundGoatpox virus (GTPV) and sheeppox virus (SPPV), which belong to the Capripoxvirus (CaPV), are economically important pathogens of small ruminants. Therefore, a sensitive, specific and rapid diagnostic assay for detection of GTPV and SPPV is necessary to accurately and promptly control these diseases.MethodsRecombinase polymerase amplification (RPA) assays combined with a real-time fluorescent detection (real-time RPA assay) and lateral flow dipstick (RPA LFD assay) were developed targeting the CaPV G-protein-coupled chemokine receptor (GPCR) gene, respectively.ResultsThe sensitivity of both CaPV real-time RPA assay and CaPV RPA LFD assay were 3 × 102 copies per reaction within 20 min at 38 °C. Both assays were highly specific for CaPV, with no cross-reactions with peste des petits ruminants virus, foot-and-mouth disease virus and Orf virus. The evaluation of the performance of these two assays with clinical sample (n = 107) showed that the CaPV real-time RPA assay and CaPV RPA LFD assay were able to specially detect SPPV or GTPV present in samples of ovine in liver, lung, kidney, spleen, skin and blood.ConclusionsThis study provided a highly time-efficient and simple alternative for rapid detection of GTPV and SPPV.

CXCL10/CXCR3 axis promotes the invasion of gastric cancer via PI3K/AKT pathway-dependent MMPs production.

CXCR3, a G-protein coupled chemokine receptor, has been found to be overexpressed in many tumors and act as an independent prognostic marker. However, it is still unclear whether CXCR3 is involved in gastric cancer progression. In this study, we found that CXCR3 was markedly expressed in gastric cancer cells and tissues. High CXCR3 expression correlated with advanced tumor stage, vascular invasion, lymph node metastasis and poor survival of gastric cancer patients. Activation of CXCR3 by one of its ligands CXCL10 promoted the invasion and migration of gastric cancer BGC-823 and MGC-803 cells, and increased the secretion and activities of MMP-2 and MMP-9. However, the effects of CXCL10 on gastric cancer cells were attenuated by CXCR3 siRNA transfection. Furthermore, overexpression of CXCR3 enhanced CXCL10-mediated cell invasion and migration of gastric cancer MKN28 cells. In addition, CXCR3 time-dependently induced activation of AKT. PI3K/AKT pathway was required for CXCR3-mediated gastric cancer cell invasion, migration and MMP-2/9 production. Together, our findings suggest that CXCL10/CXCR3 axis promotes gastric cancer cell invasion and migration by upregulating MMP-2 and MMP-9 production via PI3K/AKT pathway. Thus, CXCR3 could be a potential target for the gastric cancer treatment.

Blockade of CXC chemokine receptor 3 on endothelial cells protects against sepsis-induced acute lung injury

BackgroundCXCR3, a G-protein coupled chemokine receptor, has been shown to play a critical role in recruiting inflammatory cells into lungs in several studies. However, its roles in polymicrobial septic acute lung injury (ALI) is yet unknown. Therefore, the purpose of this study was to elucidate the protective effects of CXCR3 blockade on pulmonary microvascular endothelial cells (PMVECs) in septic ALI and explore potential mechanisms. Materials and methodsALI was induced by polymicrobial sepsis through cecal ligation and puncture surgery. The expression of CXCR3 on pulmonary microvascular endothelial cells was measured 24 h after cecal ligation and puncture surgery. In addition, the protective effects of neutralizing antibody were detected, including protein concentration, inflammation cell counts, lung ​wet-to-dry ratio, and lung damages. In human umbilical vein endothelial cells (HUVECs) culture condition, CXCR3 expression was measured after exposure to tumor necrosis factor-α. The permeability and apoptosis ratio were detected through CXCR3 gene silencing on HUVECs. The p38 mitogen-activated protein kinase (MAPK) was analyzed with Western blot. ResultsCXCR3 expression was upregulated both in vivo and in vitro. After CXCR3 neutralizing antibody administrated intraperitoneally, the protein concentration, inflammatory cell counts in BALF and lung ​wet-to-dry ratio were decreased significantly, as well as the lung tissue damages. In vitro, CXCR3 gene silencing inhibited tumor necrosis factor-α and CXCL10-induced hyperpermeability and apoptosis in HUVECs. In addition, p38 mitogen-activated protein kinase activation was essential for CXCR3-mediated apoptosis. ConclusionsCXCR3 blockade exerts protective effects on ALI at least partly by inhibiting endothelial cells apoptosis and decreasing the leakage of protein-rich fluid and inflammatory cells. Blockade of CXCR3 may be a promising therapeutic strategy for severe sepsis-induced ALI.

Identification of CXCR4 Expression By Targeted PET/MR Imaging in Patients with Acute Myeloid Leukemia (AML)

IntroductionAML is an aggressive hematological neoplasm originating from hematopoietic precursor or stem cells and is rapidly fatal if untreated. Although rates of complete remission (CR) approach 80% in patients with favorable risk profile according to European Leukemia Net classification, CR rates drop dramatically within the intermediate or adverse prognostic groups, and most patients relapse in spite of CR. The origin of refractory or relapsed disease is - according to several studies - the bone marrow (BM) niche that protects the leukemia initiating cells (LIC) from cytotoxic drugs.The chemokine receptor CXCR4 is a key factor mediating the crosstalk between the BM niche and LICs. CXCR4 is a G-protein coupled chemokine receptor with Stromal cell-derived factor-1α (SDF-1α) as its single known chemokine-ligand, and high expression is associated with poor prognosis in AML. Targeting the CXCR4/SDF-1αaxis with small peptide inhibitors or monoclonal antibodies has shown promising results in preclinical and clinical studies, thus rendering it an attractive target for future therapeutic strategies (Peled & Tavor, Theranostics, 2014). In this observational study we analyzed whether the new CXCR4-targeted positron emission tomography (PET)-probe [68Ga]-Pentixafor (Philipp-Abbrederis et al, EMBO Mol Med, 2015; Wester et al, Theranostics, 2015) is applicable for molecular imaging of patients with AML with the goal to develop a CXCR4- targeted peptide-receptor-radiotherapy (PRRT).MethodsUsing myeloid malignancies (MDS, active AML) as an exemplary CXCR4-expressing cancer entity we imaged 10 patients with the CXCR4 specific PET-probe [68Ga]-Pentixafor by whole body PET/magnetic resonance imaging (PET/MR) after they signed informed consent for this observational assessment. Maximum standardized uptake values (SUVmax) of the involved BM areas were compared to BM from patients without BM malignancy assessed in a different study. CXCR4 surface expression of leukemic BM cells of an unselected cohort of 67 patients with myeloid malignancies was analyzed by flow cytometry. CXCR4 expression in 14 established AML cell lines was measured by flow cytometry and quantitative PCR.Results Out of 10 patients who were imaged with [68Ga]-Pentixafor-PET/MR, 4 - all of them with the diagnosis of an AML - showed visually positive PET signal of the BM. Those areas correlated well with disease infiltration as determined by MR imaging and the meanSUVmax was significantly higher as compared to visually CXCR4 negative patients (meanSUVmax 8.23 ± 5.23 vs. 2.26 ± 0.47; p=0.036). One of the 10 patients presenting with proven extramedullary relapse showed a CXCR4 positive PET signal within the lesion (SUVmax 5.12), whereas the cytological non-involved BM was PET negative (meanSUVmax 1.82). CXCR4 surface expression in established AML cell lines was variable and ranged from low to high, and correlated with mRNA levels. These data reflect the different expression values we found in the cohort of patients with myeloid malignancies. There was a trend to higher CXCR4 surface expression on blasts when AML patients were compared to MDS patients (median fluorescent intensity CXCR4/isotype 1.73 ± 0.25 vs. 0.96 ± 0.06; p=0.063) that became significant when blast frequency exceeds 30% (≤30% vs. >30% blasts: 1.07 ± 0.11 vs. 2.08 ± 0.38; p=0.006). In this patient cohort we did not observe any correlation between CXCR4 expression and factors such as diagnosis (secondary vs. de novo AML), disease status (refractory or relapse vs. no pretreatment), molecular subtypes or cytogenetic aberrations.DiscussionImaging of CXCR4 by PET/MR with [68Ga]-Pentixafor is feasible in patients with AML. Due to the higher uptake in leukemic BM compared to control BM and its low uptake in other organs (e.g. liver, brain and gut), CXCR4 could serve as an attractive new PRRT-target in selected patients with AML. Such PRRT approaches are of special interest in the setting of allogeneic BM transplantation as conventional myeloablative conditioning regimens - although they are associated with a better relapse free survival - show substantial rates of transplant-related mortality and are therefore not applicable in many AML patients due to their age. In particular, therapeutic targeting of CXCR4 by PRRT provides a unique opportunity of integrating a LIC/niche-directed treatment into the conditioning regimen. Such clinical studies are in development. DisclosuresWester:Scintomics GmbH: Other: General Manager. Keller:Roche: Consultancy, Honoraria; Pfizer: Consultancy.

CXCR4 and Glioblastoma.

This article focuses on the possible application of antagonists of the G-protein coupled chemokine receptor, CXCR4, for the treatment of glioblastoma and summarises the evidence for CXCR4 antagonism being a viable therapeutic approach. Particular attention is paid to the role of this receptor in cancer stem cell biology, and the maintenance of CXCR4 expression by the glioblastoma key driver mutations. The expression of the CXCR4 receptor, and of its ligand stromal derived factor 1 (SDF-1, CXCL12), is maintained by intracellular pathways via positive feedback loops, and is associated with the epithelial mesenchymal transition (EMT) and the generation and self-renewal of cancer stem cells. SDF-1 and CXCR4 also play a role in the generation and maintenance of the perivascular stem cell niche which contains these cancer stem cells. The available data suggest that most, if not all, glioblastoma cancer stem cells rely on CXCR4 mediated signalling to maintain their phenotype. SDF-1 and CXCR4 are alsoinvolved in many other aspects of brain tumour biology including resistance to radio- and chemotherapy, the migration of cancer cells through the brain, the generation of the tumour blood supply and the recruitment of vascular progenitor cells. These properties suggest that a CXCR4 antagonist would help in the control of this disease.

Homology Modeling of Chemokine Receptor CXCR3: A Novel Therapeutic Target against Inflammatory Diseases

CXCR3 is a C-X-C chemokine receptor type 3 also known as GPR9 and CD183. CXCR3 is a G-Protein coupled chemokine receptor which interacts with three endogenous interferon inducible chemokine's (CXCL9, CXCL10 and CXCL11) and is proved to play a vital role in the Th1 inflammatory responses. CXCR3 has been implicated to be associated with various disease conditions like inflammatory diseases, autoimmune diseases, type I diabetes and acute cardiac allograft rejection. Therefore CXCR3 receptor is found to be an attractive therapeutic target for the treatment of inflammatory diseases. Inorder to decipher the biological function of a CXCR3, 3D structure is of much important but the crystal structure for CXCR3 has not yet been resolved. Hence, in the current study Homology modeling of CXCR3 was performed against various templates and validated using different parameters to suggest the best model for CXCR3. The reported best model can be used for further studies such as docking to identify the important binding site residues.

G-Protein-Coupled Chemokine Receptor Gene in Lumpy Skin Disease Virus Isolates from Cattle and Water Buffalo (Bubalus bubalis) in Egypt.

Lumpy skin disease virus (LSDV), sheep poxvirus (SPV) and goat poxvirus (GPV) are the most serious poxviruses of ruminants. In this study, we analysed the G-protein-coupled chemokine receptor (GPCR) genes of LSDV isolates from cattle and water buffalo (Bubalus bubalis) in Egypt during the summer of 2011. Multiple alignments of the nucleotide sequences revealed that the water buffalo LSDV isolate differed from the cattle isolate at four nucleotide positions, and both isolates had nine nucleotide mutations from the reference strain, Egyptian tissue culture-adapted cattle LSDV/Ismailyia88 strain. Compared with the GPCR sequences of SPV and GPV strains, a 21 nucleotide insertion and a 12 nucleotide deletion were identified in the GPCR genes of our used isolates and other LSDVs. The amino acid sequences of GPCR genes of our isolates contained the unique signature of LSDV (A11 , T12 , T34 , S99 and P199 ). Phylogenetic analyses showed that the GPCR genes of cattle and water buffalo LSDVs were closest genetically, indicating a potential transmission of cattle LSDV to water buffalo.