The complex pathophysiology of lung allergic inflammation and bronchial hyperresponsiveness (BHR) that characterize asthma is achieved by the regulated accumulation and activation of different leukocyte subsets in the lung. The development and maintenance of these processes correlate with the coordinated production of chemokines. Here, we have assessed the role that different chemokines play in lung allergic inflammation and BHR by blocking their activities in vivo. Our results show that blockage of each one of these chemokines reduces both lung leukocyte infiltration and BHR in a substantially different way. Thus, eotaxin neutralization reduces specifically BHR and lung eosinophilia transiently after each antigen exposure. Monocyte chemoattractant protein (MCP)-5 neutralization abolishes BHR not by affecting the accumulation of inflammatory leukocytes in the airways, but rather by altering the trafficking of the eosinophils and other leukocytes through the lung interstitium. Neutralization of RANTES (regulated upon activation, normal T cell expressed and secreted) receptor(s) with a receptor antagonist decreases significantly lymphocyte and eosinophil infiltration as well as mRNA expression of eotaxin and RANTES. In contrast, neutralization of one of the ligands for RANTES receptors, macrophage-inflammatory protein 1α, reduces only slightly lung eosinophilia and BHR. Finally, MCP-1 neutralization diminishes drastically BHR and inflammation, and this correlates with a pronounced decrease in monocyte- and lymphocyte-derived inflammatory mediators. These results suggest that different chemokines activate different cellular and molecular pathways that in a coordinated fashion contribute to the complex pathophysiology of asthma, and that their individual blockage results in intervention at different levels of these processes.

IB kinase (IKK) ␤ is essential for inflammatory cytokine-induced activation of nuclear factor B (NF-B). NF-B plays a pivotal role in the function of major cell types that contribute to the pathophysiological process of rheumatoid arthritis (RA). Here, we report the mechanism and the effect of the IKK␤ inhibitor N- (6-chloro-7-methoxy-9H-␤-carbolin-8-yl)-2-methylnicotinamide (ML120B), a ␤-carboline derivative, on NF-B signaling and gene activation in RA-relevant cell systems. ML120B is a potent, selective, reversible, and ATP-competitive inhibitor of IKK␤ with an IC 50 of 60 nM when evaluated in an IB␣ kinase complex assay. ML120B does not inhibit other IKK isoforms or a panel of other kinases. ML120B concentrationdependently inhibits tumor necrosis factor ␣ (TNF␣)-stimulated NF-B signaling via inhibition of IB␣ phosphorylation, degradation, and NF-B translocation into the nucleus. For the first time, we have demonstrated that in human fibroblast-like synoviocytes, TNF␣-or interleukin (IL)-1␤-induced monocyte chemoattractant protein-1 regulated on activation, normal T cell expressed and secreted and production is IKK␤-dependent. In addition, for the first time, we have demonstrated that lipopolysaccharide-or peptidoglycan-induced cytokine production in human cord blood-derived mast cells is IKK␤-dependent. In addition, in human chondrocytes, ML120B inhibited IL-1␤-induced matrix metalloproteinase production with an IC 50 of approximately 1 M. ML120B also blocked IL-1␤-induced prostaglandin E 2 production. In summary, ML120B blocked numerous NF-B-regulated cell responses that are involved in inflammation and destructive processes in the RA joint. Our findings support the evaluation of IKK␤ inhibitors as anti-inflammatory agents for the treatment of RA.

The transcription factor NF-B plays a central role in regulating inflammation and apoptosis, making it a compelling target for drug development. We identified a small molecule inhibitor (ML120B) that specifically inhibits IKK␤, an Ikappa IntroductionNF-B plays a central role in inflammation and apoptosis, making it a primary target for inhibition for treatment of inflammatory disease and cancer. 1,2 NF-B consists of subunits that associate in dimers. 3,4 The commonly described dimer forms are p50 (NF-B1) with RelA (p65) and p52 (NF-B2) with RelB. IB␣ holds the p50/RelA complex in the cytoplasm in an inactive form. NF-B nuclear translocation is regulated by upstream signaling events initiated primarily by proinflammatory stimuli, resulting in activation of IKK. 5 IKK contains 3 related subunits: ␣, ␤, and ␥. IKK␤ is the subunit responsible for phosphorylating IB␣, resulting in its ubiquitination and subsequent proteasomal degradation. [6][7][8] Released NF-B translocates to the nucleus to initiate transcription of response genes, which include proinflammatory and antiapoptotic genes. 9 In addition to phosphorylating IB␣, IKK␤ also phosphorylates the transcription factor FOXO3a, which is also degraded by the proteasome. 10 FOXO3a that is not phosphorylated by IKK␤ can translocate to the nucleus where it upregulates expression of genes that control cell cycle progression [11][12][13] and induce apoptosis. 14,15 NF-B activation has been associated with several human immune modulated diseases including arthritis, asthma, diabetes, stroke, inflammatory bowel disease, and atherosclerosis. 16 In addition, IKK␤ and NF-B have been shown to play a direct role in regulating the development of insulin resistance 17,18 and in severe muscle wasting. 19 Recently, it has been demonstrated that IKK␤ and NF-B play a key role in inflammation-associated cancer and tumor progression. [20][21][22][23][24] These data suggest that inhibition of IKK␤ in vivo with a specific small molecule may be a potential therapeutic approach for several human diseases.In addition to controlling inflammatory response and apoptosis, IKK␤ and NF-B are required for normal lymphocyte development. Mice deficient in the p65 (RelA) subunit of NF-B or IKK␤ die during fetal development through a TNF-dependent mechanism. 6,25,26 Generation of p65-or IKK␤-deficient mice on a TNF␣-or TNFR-deficient background rescues mice from embryonic lethality. [27][28][29] Studies evaluating the requirement for NF-B in hematopoiesis using transplantation of fetal liver stem cells from p65-or IKK␤-deficient mice into lethally irradiated hosts have revealed a specific requirement for these molecules in development of T cells, B cells, and common lymphoid progenitors but not myeloid cells or stem cells and a role in regulating the production of granulocytes. [30][31][32] We identified ML120B, a specific small chemical inhibitor of IKK␤ that protects against cartilage and bone destruction in adjuvant and collagen-induced models of arthritis. 33 Here we demonstrate that IKK␤ inhib...

Objective. The IKK complex regulates NF-B activation, an important pathway implicated in the rheumatoid arthritis (RA) disease process. This study was undertaken to assess the efficacy of N-(6-chloro-7-methoxy-9H-␤-carbolin-8-yl)-2-methylnicotinamide (ML120B), a potent and selective small molecule inhibitor of IKK␤.Methods. Polyarthritis was induced in rats by injection of Freund's complete adjuvant into the hind footpad. ML120B was administered orally twice daily, either prophylactically or therapeutically. Paw volumes and body weights were measured every 2-3 days throughout the study. We assessed bone erosions by several methods: histologic evaluation, quantitative micro-computed tomography (micro-CT) imaging analysis, and measurement of type I collagen fragments in the serum. Quantitative polymerase chain reaction was used to evaluate expression of messenger RNA for genes related to inflammation and to bone and cartilage integrity.Results. Oral administration of ML120B inhibited paw swelling in a dose-dependent manner (median effective dosage 12 mg/kg twice daily) and offered significant protection against arthritis-induced weight loss as well as cartilage and bone erosion. We were able to directly demonstrate that NF-B activity in arthritic joints was reduced after ML120B administration. Also, we observed that down-regulation of the NF-B pathway via IKK␤ inhibition dampened the chronic inflammatory process associated with rat adjuvant-induced arthritis.Conclusion. The results of the present study suggest that IKK␤ inhibition is an effective therapeutic approach to treat both the inflammation and the bone/ cartilage destruction observed in RA. Methods for the determination of serum markers for bone and cartilage destruction, as well as micro-CT analysis, may aid in predicting and evaluating the therapeutic efficacy of IKK␤ inhibition therapy in humans.

BackgroundPatient-derived organoids and xenografts (PDXs) have emerged as powerful models in functional diagnostics with high predictive power for anticancer drug response. However, limitations such as engraftment failure and time-consuming for establishing and expanding PDX models followed by testing drug efficacy, and inability to subject to systemic drug administration for ex vivo organoid culture hinder realistic and fast decision-making in selecting the right therapeutics in the clinic. The present study aimed to develop an advanced PDX model, namely MiniPDX, for rapidly testing drug efficacy to strengthen its value in personalized cancer treatment.MethodsWe developed a rapid in vivo drug sensitivity assay, OncoVee® MiniPDX, for screening clinically relevant regimens for cancer. In this model, patient-derived tumor cells were arrayed within hollow fiber capsules, implanted subcutaneously into mice and cultured for 7 days. The cellular activity morphology and pharmacokinetics were systematically evaluated. MiniPDX performance (sensitivity, specificity, positive and negative predictive values) was examined using PDX as the reference. Drug responses were examined by tumor cell growth inhibition rate and tumor growth inhibition rate in PDX models and MiniPDX assays respectively. The results from MiniPDX were also used to evaluate its predictive power for clinical outcomes.ResultsMorphological and histopathological features of tumor cells within the MiniPDX capsules matched those both in PDX models and in original tumors. Drug responses in the PDX tumor graft assays correlated well with those in the corresponding MiniPDX assays using 26 PDX models generated from patients, including 14 gastric cancer, 10 lung cancer and 2 pancreatic cancer. The positive predictive value of MiniPDX was 92%, and the negative predictive value was 81% with a sensitivity of 80% and a specificity of 93%. Through expanding to clinical tumor samples, MiniPDX assay showed potential of wide clinical application.ConclusionsFast in vivo MiniPDX assay based on capsule implantation was developed-to assess drug responses of both PDX tumor grafts and clinical cancer specimens. The high correlation between drug responses of paired MiniPDX and PDX tumor graft assay, as well as translational data suggest that MiniPDX assay is an advanced tool for personalized cancer treatment.

Hepatocellular carcinoma (HCC) is a common cancer with poor prognosis worldwide and the molecular mechanism is not well understood. This study aimed to establish a collection of human HCC cell lines from patient-derived xenograft (PDX) models. From the 20 surgical HCC sample collections, 7 tumors were successfully developed in immunodeficient mice and further established 7 novel HCC cell lines (LIXC002, LIXC003, LIXC004, LIXC006, LIXC011, LIXC012 and CPL0903) by primary culture. The characterization of cell lines was defined by morphology, growth kinetics, cell cycle, chromosome analysis, short tandem repeat (STR) analysis, molecular profile, and tumorigenicity. Additionally, response to clinical chemotherapeutics was validated both in vitro and in vivo. STR analysis indicated that all cell lines were unique cells different from known cell lines and free of contamination by bacteria or mycoplasma. The other findings were quite heterogeneous between individual lines. Chromosome aberration could be found in all cell lines. Alpha-fetoprotein was overexpressed only in 3 out of 7 cell lines. 4 cell lines expressed high level of vimentin. Ki67 was strongly stained in all cell lines. mRNA level of retinoic acid induced protein 3 (RAI3) was decreased in all cell lines. The 7 novel cell lines showed variable sensitivity to 8 tested compounds. LIXC011 and CPL0903 possessed multiple drug resistance property. Sorafenib inhibited xenograft tumor growth of LIXC006, but not of LIXC012. Our results indicated that the 7 novel cell lines with low passage maintaining their clinical and pathological characters could be good tools for further exploring the molecular mechanism of HCC and anti-cancer drug screening.

To investigate structure-function relationships of erythropoietin (Epo), we have obtained cDNA sequences that encode the mature Epo protein of a variety of mammals. A first set of primers, corresponding to conserved nucleotide sequences between mouse and human DNAs, allowed us to amplify by polymerase chain reaction (PCR) intron 1/exon 2 fragments from genomic DNA of the hamster, cat, lion, dog, horse, sheep, dolphin, and pig. Sequencing of these fragments permitted the design of a second generation of species-specific primers. RNA was prepared from anemic kidneys and reverse-transcribed. Using our battery of species-specific 5′ primers, we were able to successfully PCR- amplify Epo cDNA from Rhesus monkey, rat, sheep, dog, cat, and pig. Deduced amino acid sequences of mature Epo proteins from these animals, in combination with known sequences for human, Cynomolgus monkey, and mouse, showed a high degree of homology, which explains the biologic and immunological cross-reactivity that has been observed in a number of species. Human Epo is 91% identical to monkey Epo, 85% to cat and dog Epo, and 80% to 82% to pig, sheep, mouse, and rat Epos. There was full conservation of (1) the disulfide bridge linking the NH2 and COOH termini; (2) N-glycosylation sites; and (3) predicted amphipathic alpha- helices. In contrast, the short disulfide bridge (C29/C33 in humans) is not invariant. Cys33 was replaced by a Pro in rodents. Most of the amino acid replacements were conservative. The C-terminal part of the loop between the C and D helices showed the most variation, with several amino acid substitutions, deletions, and/or insertions. Calculations of maximum parsimony for intron 1/exon 2 sequences as well as coding sequences enabled the construction of cladograms that are in good agreement with known phylogenetic relationships.

BackgroundTreatment guidelines for a variety of cancers have been increasingly used in clinical practice, and have resulted in major improvement in patient outcomes. However, recommended regimens (even first-line treatments) are clearly not ideal for every patients. In the present study, we used mini patient-derived xenograft (mini-PDX) and next-generation sequencing to develop personalized treatment in a patient with metastatic duodenal adenocarcinoma.MethodsResected metachronous metastatic tumor tissues were implanted into SCID mice to determine the sensitivity to a variety of drug regimens. Mutation profiles were assessed using both DNA whole-exome sequencing (DNA–WES) and RNA sequencing. The results of the analyses were used to select optimal treatment for the patient with metastatic duodenal adenocarcinoma.ResultsAssessment with mini-PDX models took only 7 days. The results showed high sensitivity to S-1 plus cisplatin, gemcitabine plus cisplatin and everolimus alone. The patient received gemcitabine plus cisplatin initially, but the treatment was terminated due to toxicity. The patient was then switched to treatment with S-1 alone. The overall disease-free survival was 34 months. DNA–WES and RNA sequencing identified KRAS mutation (A146T), TP53 (C229Yfs*10) and RICTOR amplification in the metastatic duodenal adenocarcinoma. These findings provided further support to the results of the mini-PDX, and suggest mTOR inhibitors should be used if and when relapse eventually occurs in this patient.ConclusionsMini-PDX model combined with WES/RNA sequencing can rapidly assess drug sensitivity in cancer patients and reveal key genetic alterations. Further research on this technology for personalized therapy in patients with refractory malignant tumors is warranted.Electronic supplementary materialThe online version of this article (10.1186/s40880-018-0323-y) contains supplementary material, which is available to authorized users.