A phase 1 trial of xentuzumab, an IGF-neutralizing antibody, in Japanese patients with advanced solid tumors.

Xentuzumab is an insulin-like growth factor (IGF) ligand-neutralizing antibody. This phase 1 trial assessed xentuzumab in Japanese patients with solid tumors. Patients aged ≥20 y old with solid tumors that were refractory or not amenable to standard therapy were enrolled. Patients received xentuzumab intravenously at a starting dose of 750 mg/wk. Dose escalation used a 3 + 3 design with dose de-escalation. The primary endpoint was to determine the maximum tolerated dose (MTD) of xentuzumab. Safety, pharmacokinetics, pharmacodynamics, and anti-tumor activity were also assessed. Fifteen patients received xentuzumab in the dose escalation part (750 mg/wk [n = 6]; 1000 mg/wk [n = 3]; 1400 mg/wk [n = 6]). There were no dose-limiting toxicities at any dose; the MTD of xentuzumab was not reached. Xentuzumab 1000 mg/wk was recommended as the relevant biological dose. Six further patients received xentuzumab 1000 mg/wk in an expansion cohort. Of 21 patients, 13 (61.9%) experienced a drug-related adverse event, most commonly fatigue (23.8%), neutropenia (19.0%), diarrhea, nausea, white blood cell count decrease, and muscle spasms (14.3% each). No relevant deviations from dose linearity of xentuzumab exposure were observed during dose escalation. Total IGF-1 and IGF-2 levels increased and bioactive IGF levels decreased from baseline to 24 h after the first infusion in cycle 1. Partial response was observed in 2 (9.5%) patients with desmoid-type fibromatosis. Disease control was achieved in 6 (28.6%) patients (median duration 42.4 mo). Xentuzumab monotherapy was well tolerated in Japanese patients and showed evidence of anti-tumor activity. This study was registered with www.clinicaltrials.gov (NCT02145741).

A novel epitope-presenting thermostable scaffold for the development of highly specific insulin-like growth factor-1/2 antibodies.

High sequence and structural homology between mature human insulin-like growth factors IGF-1 and IGF-2 makes serological discrimination by immunodiagnostic IGF tests a challenging task. There is an urgent need for highly specific IGF-1 and IGF-2 antibodies, yet only a short sequence element, i.e. the IGF loop, provides enough difference in sequence to discriminate between the two molecules. We sought to address this unmet demand by investigating novel chimeric immunogens as carriers for recombinant peptide motif grafting. We found Thermus thermophilus sensitive to lysis D (SlyD) and Thermococcus gammatolerans SlyD FK-506-binding protein (FKBP) domains suitable for presentation of the predefined epitopes, namely the IGF-1 and IGF-2 loops. Chimeric SlyD-IGF proteins allowed for the development of exceptionally specific IGF-1 and IGF-2 monoclonal antibodies. The selected antibodies bound with high affinity to the distinct IGF epitopes displayed on the protein scaffolds, as well as on the mature human IGF isoforms. The respective SlyD scaffolds display favorable engineering properties in that they are small, monomeric, and cysteine-free and can be produced in high yields in a prokaryotic host, such as Escherichia coli In conclusion, FKBP domains from thermostable SlyD proteins are highly suitable as a generic scaffold platform for epitope grafting.

Two first-in-human studies of xentuzumab, a humanised insulin-like growth factor (IGF)-neutralising antibody, in patients with advanced solid tumours.

BACKGROUND: Xentuzumab, an insulin-like growth factor (IGF)-1/IGF-2-neutralising antibody, binds IGF-1 and IGF-2, inhibiting their growth-promoting signalling. Two first-in-human trials assessed the maximum-tolerated/relevant biological dose (MTD/RBD), safety, pharmacokinetics, pharmacodynamics, and activity of xentuzumab in advanced/metastatic solid cancers. METHODS: These phase 1, open-label trials comprised dose-finding (part I; 3 + 3 design) and expansion cohorts (part II; selected tumours; RBD [weekly dosing]). Primary endpoints were MTD/RBD. RESULTS: Study 1280.1 involved 61 patients (part I: xentuzumab 10-1800 mg weekly, n = 48; part II: 1000 mg weekly, n = 13); study 1280.2, 64 patients (part I: 10-3600 mg three-weekly, n = 33; part II: 1000 mg weekly, n = 31). One dose-limiting toxicity occurred; the MTD was not reached for either schedule. Adverse events were generally grade 1/2, mostly gastrointestinal. Xentuzumab showed dose-proportional pharmacokinetics. Total plasma IGF-1 increased dose dependently, plateauing at ~1000 mg/week; at ≥450 mg/week, IGF bioactivity was almost undetectable. Two partial responses occurred (poorly differentiated nasopharyngeal carcinoma and peripheral primitive neuroectodermal tumour). Integration of biomarker and response data by Bayesian Logistic Regression Modeling (BLRM) confirmed the RBD. CONCLUSIONS: Xentuzumab was well tolerated; MTD was not reached. RBD was 1000 mg weekly, confirmed by BLRM. Xentuzumab showed preliminary anti-tumour activity. CLINICAL TRIAL REGISTRATION: NCT01403974; NCT01317420.

Identification of a Novel Autoimmune Peptide Epitope of Prostein in Prostate Cancer.

There is a demand for novel targets and approaches to diagnose and treat prostate cancer (PCA). In this context, serum and plasma samples from a total of 609 individuals from two independent patient cohorts were screened for IgG reactivity against a sum of 3833 human protein fragments. Starting from planar protein arrays with 3786 protein fragments to screen 80 patients with and without PCA diagnosis, 161 fragments (4%) were chosen for further analysis based on their reactivity profiles. Adding 71 antigens from literature, the selection of antigens was corroborated for their reactivity in a set of 550 samples using suspension bead arrays. The antigens prostein (SLC45A3), TATA-box binding protein (TBP), and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) showed higher reactivity in PCA patients with late disease compared with early disease. Because of its prostate tissue specificity, we focused on prostein and continued with mapping epitopes of the 66-mer protein fragment using patient samples. Using bead-based assays and 15-mer peptides, a minimal peptide epitope was identified and refined by alanine scanning to the KPxAPFP. Further sequence alignment of this motif revealed homology to transmembrane protein 79 (TMEM79) and TGF-beta-induced factor 2 (TGIF2), thus providing a reasoning for cross-reactivity found in females. A comprehensive workflow to discover and validate IgG reactivity against prostein and homologous targets in human serum and plasma was applied. This study provides useful information when searching for novel biomarkers or drug targets that are guided by the reactivity of the immune system against autoantigens.

Insulin-like growth factor-2 enhances functions of antigen (Ag)-specific regulatory B cells.

Regulatory B cells (Bregs) are important in immune regulation. The factors that regulate Breg functions are less clear. Insulin-like growth factor 2 (IGF2) is capable of inducing hematopoietic stem cell differentiation. This study aimed to investigate the role of IGF2 in the development of Bregs and the enhancement of their function. In this study, the expression of IGF1 receptor (IGF1R) and IGF2R in ovalbumin (OVA)-specific B cells (OVAsBCs) was assessed by real time RT-PCR and Western blotting. The release of interleukin (IL)-10 from OVAsBCs and OVAsBC proliferation were assessed by enzyme-linked immunoassay and proliferation assay. The role of IGF2 in enhancing the function of OVAsBCs was tested with an intestinal allergic inflammation mouse model. The results showed that OVAsBCs expressed high levels of IGF2R. Exposure to both IGF2 and a specific antigen (Ag), OVA, markedly enhanced the expression of IL-10 in OVAsBCs as well as enhanced the IL-10(+) OVAsBC proliferation. The concurrent exposure to IGF2 and specific Ag markedly induced the IL-10 promoter DNA demethylation via activating the STAT5 pathway. IGF2 also enhanced both the OVAsBC proliferation in vivo and the effect of Ag-specific immunotherapy on inhibiting allergic inflammation in the intestine. We conclude that OVAsBCs express high levels of IGF2R and that IGF2 increases the expression of IL-10 in OVAsBCs and enhances OVAsBC proliferation and the inhibitory effect on allergic inflammation.

A dual-specific anti-IGF-1/IGF-2 human monoclonal antibody alone and in combination with temsirolimus for therapy of neuroblastoma.

The insulin-like growth factors (IGFs), IGF-1 and IGF-2, have been implicated in the growth, survival and metastasis of a broad range of malignancies including pediatric tumors. They bind to the IGF receptor type 1 (IGF-1R) and the insulin receptor (IR) which are overexpressed in many types of solid malignancies. Activation of the IR by IGF-2 results in increased survival of tumor cells. We have previously identified a novel human monoclonal antibody, m708.5, which binds with high (pM) affinity to both human IGF-1 and IGF-2, and potently inhibits phosphorylation of the IGF-1R and the IR in tumor cells. m708.5 exhibited strong antitumor activity as a single agent against most cell lines derived from neuroblastoma, Ewing family of tumor, rhabdomyosarcoma and osteosarcoma. When tested in neuroblastoma cell lines, it showed strong synergy with temsirolimus and synergy with chemotherapeutic agents in vitro. In xenograft models, the combination of m708.5 and temsirolimus significantly inhibited neuroblastoma growth and prolonged mouse survival. Taken together, these results support the clinical development of m708.5 for pediatric solid tumors with potential for synergy with chemotherapy and mTOR inhibitors.

Phase I dose-escalation study evaluating safety, tolerability and pharmacokinetics of MEDI-573, a dual IGF-I/II neutralizing antibody, in Japanese patients with advanced solid tumours.

PURPOSE: This Phase I, open-label, single-arm, dose-escalation study aimed to evaluate the safety and tolerability of the insulin-like growth factor (IGF-I/II) neutralizing antibody, MEDI-573, in Japanese patients with advanced solid tumours refractory to standard therapy or for which no standard therapy exists. The pharmacokinetics, pharmacodynamics and antitumour activity of MEDI-573 were also evaluated. METHODS: Three cohorts of patients received MEDI-573 in escalating order: cohort 1, 5 mg/kg on Day 1, 8 and 15; cohort 2, 15 mg/kg on Day 1, 8 and 15; cohort 3, 45 mg/kg on Day 1, of 21-day cycles. RESULTS: Ten patients who received at least one dose of MEDI-573 were evaluated. The median number of treatment cycles was 2.0 (range 1-6) and the median number of MEDI-573 doses received was 4.0 (range 1-17). The most commonly reported drug-related adverse events were fatigue (n = 2 patients), pyrexia (n = 2), diarrhoea (n = 2) and electrocardiogram QT prolongation (n = 2). No patients experienced a dose-limiting toxicity. Pharmacokinetics of MEDI-573 were linear with a dose-dependent increase. There were no complete or partial responses; four patients had an overall best response of stable disease. CONCLUSIONS: MEDI-573 is well tolerated at the doses investigated.

Co-targeting the IGF system and HIF-1 inhibits migration and invasion by (triple-negative) breast cancer cells.

BACKGROUND: Metastatic triple-negative breast cancer is mostly incurable, due to lack of suitable drug targets. The insulin-like growth factor (IGF) system could provide such a target, and IGF-1 receptor (IGF-1R)-directed agents are already available, but seem unable to control all the complexities of the system, including crosstalk with hypoxia-inducible pathways. METHODS: Migration of triple-negative MDA-231 breast cancer cells and its modulation by IGFs, the IGF-1R inhibitor NVP-AEW541 and the IGF-2-sequestering monoclonal antibody MAB292 were assessed by the scratch wound healing and Boyden chamber assays; the effect of topotecan (inhibiting hypoxia-inducible factor-1 (HIF-1)) under hypoxia was also evaluated. Constitutive as well as drug-modulated levels of components of the IGF and HIF-1 pathways were evaluated by western blotting and qPCR. RESULTS: IGF-induced migration of MDA-231 cells was not abrogated by the IGF-1R inhibitor NVP-AEW541, whereas IGF-2 sequestration by MAB292 significantly reduced cell migration. Under hypoxia, topotecan was also effective, likely by reducing HIF-1-induced IGF-2 release. Simultaneous targeting of IGF-1R and IGF-2 or HIF-1 completely abolished cell migration. CONCLUSIONS: IR activation may account for the failure of NVP-AEW541 to suppress MDA-231 cell migration. Ligand-targeting compounds, or co-inhibition of the IGF and HIF-1 systems, may prevent activation of compensatory signalling, thereby providing a valuable addition to IGF-1R inhibitor-based therapies.

A new bispecific antibody targeting non-overlapping epitopes on IGF2: design, in vitro characterization and pharmacokinetics in macaques.

The insulin-like growth factor 2 (IGF2) is an important target for cancer therapy. We have previously proposed an approach for fast and irreversible removal of IGF2 from the circulation by using monoclonal antibodies (mAbs) that bind to two or more non-overlapping epitopes on the same molecule. We provided initial evidence for the formation of oligomeric antibody-ligand complexes that can bind to cells expressing Fc gamma receptors (FcγRs) with high avidity using an antibody domain with relatively low affinity as one of the anti-IGF2 mAbs. Recently, we identified a mAb, m708.5, in a scFv format which binds to both IGF2 and IGF1 with very high (pM) affinity. Interestingly, and rather surprisingly, this mAb did not compete with our other high affinity mAb, m610.27, for binding to IGF2. Therefore, we generated a new bispecific mAb, m67, by combining m708.5 and m610.27. As expected m67 potently inhibited binding of IGF2 to cells expressing the IGF1R and its phosphorylation, and resulted in formation of multimolecular complexes when incubated with IGF2 and bound with high avidity to cells expressing FcγRII; the complexes were internalized in a macrophage-like cell line. However, although m67 exhibited a reasonably long half-life (6.4 ± 0.6 days) in cynomolgus macaques and high stability in serum, its administration to three animals did not result in any measurable decrease in the IGF2 concentration likely due to the complexity of the IGF2 interactions in the blood and the relatively low (2mg/kg) dose of the mAb leading to a relatively low maximal blood concentration of 120nM. In spite of the lack of effect on the IGF2 concentration in this particular experimental setup, m67 exhibited good drugability properties and could be highly effective in other animal models and in humans. Studies with animal models of cancer are ongoing to evaluate the potential of m67 as a new candidate mAb-based therapeutic.

The IGF-I/IGF-R1 pathway regulates postnatal lung growth and is a nonspecific regulator of alveologenesis in the neonatal rat.

IGF-I, IGF-II, and the IGF-I receptor are widely distributed throughout the neonatal rat lung on days 4, 7, 10, and 14 of life, with a similar abundance at each of these time points. Injection of 20 µg/g of a truncated soluble IGF-I receptor on days 2 and 5 of life, to decoy ligand away from the endogenous IGF-I receptor, reduced lung weight and lung-to-body weight ratio, reduced lung tissue fraction, and impaired alveolar formation, as assessed by secondary crest formation and mean linear intercepts on day 7 of life. Lung procollagen I content and elastin fiber density were also reduced. Injection of 100 µg/day of neutralizing anti-IGF-I, to prevent IGF-I from binding to the IGF-I receptor, on days 3, 4, and 5 of life reduced tissue fraction and elastin fiber density and impaired alveolar formation on day 6 of life. Both interventions reduced total lung cell and secondary crest cell DNA synthesis and small vessel counts per unit area, but these effects were lost after normalization to the reduced tissue fraction. These findings are consistent with a role for IGF-I binding to the IGF-I receptor in postnatal lung growth and on alveologenesis through a nonspecific positive effect on DNA synthesis. Injection of 100 µg/day of neutralizing anti-IGF-II, to prevent IGF-II from binding to the IGF-I receptor, on days 3, 4, and 5 of life had no effect on total lung cell DNA synthesis per unit area on day 6 of life, and a role for IGF-II in postnatal alveologenesis was not further pursued.

Expression of the growth hormone/insulin-like growth factor axis during Balb/c thymus ontogeny and effects of growth hormone upon ex vivo T cell differentiation.

AIMS: We address the question of the expression and the role of the growth hormone/insulin-like growth factor (GH/IGF) axis in the thymus. METHODS: Using RT-qPCR, the expression profile of various components of the somatotrope GH/IGF axis was measured in different thymic cell types and during thymus embryogenesis in Balb/c mice. The effect of GH on T cell differentiation was explored via thymic organotypic culture. RESULTS: Transcription of Gh, Igf1, Igf2 and their related receptors predominantly occurred in thymic epithelial cells (TEC), while a low level of Gh and Igf1r transcription was also evidenced in thymic T cells (thymocytes). Gh, Ghr, Ins2, Igf1, Igf2, and Igfr1 displayed distinct expression profiles depending on the developmental stage. The protein concentrations of IGF-1 and IGF-2 were in accordance with the profile of their gene expression. In fetal thymus organ cultures (FTOC) derived from Balb/c mice, treatment with exogenous GH resulted in a significant increase of double negative CD4-CD8- T cells and CD4+ T cells, together with a decrease in double positive CD4+CD8+ T cells. These changes were inhibited by concomitant treatment with GH and the GH receptor (GHR) antagonist pegvisomant. However, GH treatment also induced a significant decrease in FTOC Gh, Ghr and Igf1 expression. CONCLUSION: These data show that the thymotropic properties of the somatotrope GH/IGF-1 axis involve an interaction between exogenous GH and GHR expressed by TEC. Since thymic IGF-1 is not increased by GH treatment, the effects of GH upon T cell differentiation could implicate a different local growth factor or cytokine.

Thymic self-antigen expression for the design of a negative/tolerogenic self-vaccine against type 1 diabetes.

Before being able to react against infectious non-self-antigens, the immune system has to be educated in the recognition and tolerance of neuroendocrine proteins, and this critical process essentially takes place in the thymus. The development of the autoimmune diabetogenic response results from a thymus dysfunction in programming central self-tolerance to pancreatic insulin-secreting islet ß cells, leading to the breakdown of immune homeostasis with an enrichment of islet ß cell reactive effector T cells and a deficiency of ß cell-specific natural regulatory T cells (nTreg) in the peripheral T-lymphocyte repertoire. Insulin-like growth factor 2 (IGF-2) is the dominant member of the insulin family expressed during fetal life by the thymic epithelium under the control of the autoimmune regulator (AIRE) gene/protein. Based on the close homology and cross-tolerance between insulin, the primary T1D autoantigen, and IGF-2, the dominant self-antigen of the insulin family, a novel type of vaccination, so-called "negative/tolerogenic self-vaccination", is currently developed for prevention and cure of T1D. If this approach were found to be effective for reprogramming immunological tolerance in T1D, it could pave the way for the design of negative self-vaccines against autoimmune endocrine diseases, as well as other organ-specific autoimmune diseases.

Insulin-like growth factors are more effective than progastrin in reversing proapoptotic effects of curcumin: critical role of p38MAPK.

Progastrin and insulin-like growth factors (IGFs) stimulate hyperproliferation of intestinal epithelial cells (IECs) via endocrine/paracrine routes; hyperproliferation is a known risk factor for colon carcinogenesis. In the present study, inhibitory potency of curcumin in the presence or absence of progastrin and/or IGF-II was examined. Progastrin and IGF-II significantly increased proliferation of an immortalized IEC cell line, IEC-18, whereas curcumin decreased the proliferation in a dose-dependent manner. IGF-II was significantly more effective than progastrin in reversing antiproliferative effects of curcumin and reversed proapoptotic effects of curcumin by >80%; progastrin was relatively ineffective toward reversing proapoptotic effects of curcumin. IEC-18 clones were generated to overexpress either progastrin (IEC-PG) or hIGF-II (IEC-IGF). Proliferation of IEC-PG and IEC-IGF clones was increased, compared with that of control clones. Curcumin significantly reduced proliferation of IEC-PG, but not IEC-IGF, clones. Similarly, a human colon cancer cell line, Caco-2 (which expresses autocrine IGF-II), was relatively resistant to inhibitory effects of curcumin. However, Caco-2 cells treated with anti-IGF-II-antibodies were rendered sensitive to inhibitory effects of curcumin. Significant differences in inhibitory potency of curcumin against PG- vs. IGF-II-stimulated growth of IEC-18 cells were not reflected by differences in curcumin-mediated inhibition of activated (phosphorylated) ERKs/IKK(alpha/beta)/p65NF-kappaB and c-Src in wild-type (wt)IEC-18 cells, in response to the two growth factors. Surprisingly, curcumin was almost ineffective in reducing IGF-II-stimulated activation of p38MAPK but significantly reduced progastrin-stimulated phosphorylation of p38. Treatment with a p38MAPK inhibitor resulted in loss of protective effects of IGF-II against inhibitory effects of curcumin. These novel findings suggest that growth factor profile of patients and tumors may dictate inhibitory potency of curcumin and that combination of curcumin + p38MAPK inhibitor may be required for reducing hyperproliferative or tumorigenic response of IECs to endocrine and autocrine IGFs.

Effects of stocking density and feed ration on growth and gene expression in the Senegalese sole (Solea senegalensis): potential effects on the immune response.

Stocking density and ration size are two major factors influencing aquaculture production. To evaluate their effects on growth and immune system in Senegalese sole (Solea senegalensis) juveniles, a 2 x 2 experimental design using two rations (1.0% and 0.25% of the total fish biomass) and two different initial stocking densities (7 and 30 kg m(-2)) was performed throughout a 60 days culture period. Soles fed 1.0% showed a higher specific growth rate (SGR) than those fed 0.25% (3.3-fold). No differences in SGR at 60 days were found between densities in spite of reduced values were detected at high density after 20 days (soles fed 0.25%) and 40 days (soles fed 1%) suggesting a compensatory growth. Physiologically, plasma cortisol levels were elevated in soles at high density (45-fold higher than at 7 kg m(-2)) whereas no differences associated to the feeding ration were observed. To assess the effects at a molecular level, the mRNA levels of genes involved in cellular stress (heat shock proteins HSP70 and HSP90), growth (insulin-like growth factors IGF-I, the spliced variants IGF-Ia and IGFI-b, and IGF-II) and innate immune system (g-type lysozyme and hepcidin (HAMP1)) were quantified. No differences in HSP90 expression were detected between densities or rations. In contrast, IGF-I, IGF-Ia and IGF-II showed reduced transcript levels in liver and HSP70 in liver and kidney at high density. Finally, g-type lysozyme and HAMP1 expression was greatly affected by both factors exhibiting an important reduction in the transcript levels at high density and low ration. Overall, our results show that S. senegalensis juveniles might exhibit satisfactory SGR at high density although the high plasma cortisol levels indicate a crowding stress that could negatively affect the expression levels of some of the genes studied.

Model-Informed Dose Selection for Xentuzumab, a Dual Insulin-Like Growth Factor-I/II-Neutralizing Antibody.

Over the past decade, the insulin-like growth factor (IGF)-signaling pathway has gained substantial interest as potential therapeutic target in oncology. Xentuzumab, a humanized IgG1 monoclonal antibody, binds to IGF-I and IGF-II thereby inhibiting the downstream signaling essential for survival and tumor growth. This pathway is further regulated by circulating IGF binding proteins (IGFBPs). In this work, a mechanistic model characterizing the dynamics and interactions of IGFs, IGFBPs, and Xentuzumab has been developed to guide dose selection. Therefore, in vitro and in vivo literature information was combined with temporal IGF-I, IGF-II, and IGFBP-3 total plasma concentrations from two phase I studies. Based on the established quantitative framework, the time-course of free IGFs as ultimate drug targets not measured in clinics was predicted. Finally, a dose of 1000 mg/week-predicted to reduce free IGF-I and free IGF-II at steady-state by at least 90% and 64%, respectively-was suggested for phase II.

Long non-coding RNA SNHG16 promotes lipopolysaccharides-induced acute pneumonia in A549 cells via targeting miR-370-3p/IGF2 axis.

BACKGROUND: Pneumonia is an infectious lung inflammation in children with high mortality and morbidity rates. Small nucleolar RNA host gene 16 (SNHG16) has been verified to accelerate the progression of acute pneumonia. However, the role of SNHG16 in acute pneumonia has not yet been fully elucidated. The study was aimed to explore the regulatory mechanism of SNHG16 in LPS-induced acute pneumonia in A549 cells. METHODS: The levels of SNHG16, miR-370-3p and IGF2 in serum samples and LPS-induced A549 cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The cell viability and apoptosis of A549 cells were examined by Cell Counting Kit-8 (CCK-8) assay and flow cytometer, respectively. The levels of interleukin 1ß (IL-1ß), interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA). The binding relationships among SNHG16, miR-370-3p and IGF2 were predicted by online database and verified by Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. The protein levels of IGF2 were tested by Western blot. RESULTS: SNHG16 and IGF2 were upregulated while miR-370-3p was downregulated in serum of acute pneumonia patients and LPS-induced A549 cells. SNHG16 regulated proliferation, apoptosis and inflammatory cytokines by inhibiting miR-370-3p in LPS-induced A549 cells. MiR-370-3p targeted IGF2 and inhibited LPS-induced inflammatory injury via IGF2 in A549 cells. Furthermore, SNHG16 was verified to promote IGF2 expression by sponging miR-370-3p in A549 cells. CONCLUSION: SNHG16 impeded cell viability and promoted apoptosis, inflammatory injury by targeting IGF2 mediated by miR-370-3p in LPS-induced A549 cells.

HIF-1 alpha: a key survival factor for serum-deprived prostate cancer cells.

BACKGROUND: Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is commonly overexpressed in prostate cancer (PCa) cells. As PCa cells are known to survive serum deprivation, we investigated the effect of prolonged serum deprivation on HIF-1 alpha expression, and the function of HIF-1 alpha in regulating the survival of normoxic serum-deprived PCa cells. METHODS: HIF-1 alpha protein was assessed by immunoblots. Cell viability and proliferation were assessed by trypan blue assay and flow cytometric analysis. Transcriptional activity was assessed by luciferase reporter assay and RT-PCR. HIF-1 alpha expression was suppressed with siRNA. Activities of HIF-1 alpha-target genes were inhibited with neutralizing antibody. RESULTS: Prolonged serum deprivation is a potent inducer of HIF-1 alpha in PC-3 and LNCaP PCa cells, despite normal oxygen conditions. In contrast, cells grown in the presence of serum did not show HIF-1 alpha protein accumulation. Moreover, HIF-1 alpha protein increase during serum deprivation correlated with increased cell survival, while suppression of HIF-1 alpha expression significantly decreased PCa cell viability. Our results further demonstrate that HIF-1 alpha protein increase is due to increased HIF-1 alpha protein synthesis. First, there was no significant increase in HIF-1 alpha mRNA. Secondly, cycloheximide, a protein synthesis inhibitor, prevented HIF-1 alpha protein increase in serum-deprived PCa cells. Moreover, the expression of HIF-1 alpha-target genes, VEGF and IGF-2, was concomitantly increased in serum-deprived PCa cells, while suppression of HIF-1 alpha expression significantly inhibited their induction. Furthermore, inhibition of IGF-2 activity resulted in a significant decline in PCa cell survival. CONCLUSION: PCa cells counteract the stress of prolonged serum deprivation by upregulating HIF-1 alpha protein which increases IGF-2 expression to promote cell survival.

Autoimmune regulator initiates the expression of promiscuous genes in thymic epithelial cells.

The expression of peripheral antigens in the thymus, known as promiscuous gene expression, has been implicated in T cell tolerance and autoimmunity. Here we identified thymic epithelial cells (TECs) as the main cell type that expresses a diverse range of tissue-restricted antigens (TRAs). The TECs of a common autoimmune (non-obese diabetic [NOD]) mouse model express much lower levels of an autoimmune regulator (Aire) and TRAs than normal (Balb/c) TECs. Transfection of an Aire plasmid led to increased levels of TRA expression in cultured TECs from Balb/c and NOD mice; an increase that was enhanced by the presence of thymocytes. These data show that Aire initiates promiscuous gene expression in TECs, and that this function might be under thymocyte control.

Single-Cell Analysis Identifies Thymic Maturation Delay in Growth-Restricted Neonatal Mice.

Fetal growth restriction (FGR) causes a wide variety of defects in the neonate which can lead to increased risk of heart disease, diabetes, anxiety and other disorders later in life. However, the effect of FGR on the immune system, is poorly understood. We used a well-characterized mouse model of FGR in which placental Igf-2 production is lost due to deletion of the placental specific Igf-2 P0 promotor. The thymi in such animals were reduced in mass with a ~70% reduction in cellularity. We used single cell RNA sequencing (Drop-Seq) to analyze 7,264 thymus cells collected at postnatal day 6. We identified considerable heterogeneity among the Cd8/Cd4 double positive cells with one subcluster showing marked upregulation of transcripts encoding a sub-set of proteins that contribute to the surface of the ribosome. The cells from the FGR animals were underrepresented in this cluster. Furthermore, the distribution of cells from the FGR animals was skewed with a higher proportion of immature double negative cells and fewer mature T-cells. Cell cycle regulator transcripts also varied across clusters. The T-cell deficit in FGR mice persisted into adulthood, even when body and organ weights approached normal levels due to catch-up growth. This finding complements the altered immunity found in growth restricted human infants. This reduction in T-cellularity may have implications for adult immunity, adding to the list of adult conditions in which the in utero environment is a contributory factor.

Novel human monoclonal antibodies to insulin-like growth factor (IGF)-II that potently inhibit the IGF receptor type I signal transduction function.

The insulin-like growth factor (IGF) system plays an important role in a variety of physiologic processes and in diseases such as cancer. Although the role of the IGF system in cancer has been recognized many years ago, components of the system have only recently been targeted and shown to affect cell transformation, proliferation, survival, motility, and migration in tissue cultures and in mouse models of cancer. We have been hypothesizing that targeting IGF-II in addition to blocking its interaction with the IGF receptor type I (IGF-IR) would also allow to block that portion of the signal transduction through the insulin receptor that is due to its interaction with IGF-II. Lowering its level may also not induce up-regulation of its production as for IGF-I. Finally, targeting a diffusable ligand as IGF-II may not require penetration of the antibody inside tumors but could shift the equilibrium to IGF-II complexed with antibody so the ligand concentration would decrease in the tumor environment without the need for the antibody to penetrate the tumor. Here, we describe the identification and characterization of three novel anti-IGF-II fully human monoclonal antibodies. They bound with high (subnanomolar) affinity to IGF-II, did not cross-react with IGF-I and insulin, and potently inhibited signal transduction mediated by the IGF-IR interaction with IGF-II. The most potent neutralizer, IgG1 m610, inhibited phosphorylation of the IGF-IR and the insulin receptor, as well as phosphorylation of the downstream kinases Akt and mitogen-activated protein kinase with an IC(50) of the order of 1 nmol/L at IGF-II concentration of 10 nmol/L. It also inhibited growth of the prostate cancer cell line DU145 and migration of the breast cancer line cells MCF-7. These results indicate an immunotherapeutic potential of IgG1 m610 likely in combination with other antibodies and anticancer drugs but only further experiments in mouse models of cancer and human clinical trials could evaluate this possibility.