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1.  E4F1 dysfunction results in autophagic cell death in myeloid leukemic cells 
Autophagy  2011;7(12):1566-1567.
The multifunctional E4F1 protein was originally identified as a cellular target of the E1A adenoviral oncoprotein. Although E4F1 is implicated in several key oncogenic pathways, its roles in tumorigenesis remain unclear. Using a genetically engineered mouse model of myeloid leukemia (histiocytic sarcomas, HS) based on the genetic inactivation of the tumor suppressor Ink4a/Arf locus, we have recently unraveled an unsuspected function of E4F1 in the survival of leukemic cells. In vivo, genetic ablation of E4F1 in established myeloid tumors results in tumor regression. E4F1 inactivation results in a cascade of alterations originating from dysfunctional mitochondria that induce increased reactive oxygen species (ROS) levels and ends in massive autophagic cell death in HS transformed, but not normal myeloid cells. E4F1 depletion also induces cell death in various human myeloid leukemic cell lines, including acute myeloid leukemic (AML) cell lines. Interestingly, the E4F1 protein is overexpressed in a large proportion of human AML samples. These data provide new insights into E4F1-associated survival functions implicated in tumorigenesis and could open the path for new therapeutic strategies.
doi:10.4161/auto.7.12.17991
PMCID: PMC3288034  PMID: 22024746
E4F1; histiocytic sarcoma; mitochondria; autophagy; cell death; leukemic cells; reactive oxygen species
2.  E4F1 dysfunction results in autophagic cell death in myeloid leukemic cells 
Autophagy  2011;7(12):1566-1567.
Summary
The multifunctional E4F1 protein was originally identified as a cellular target of the E1A adenoviral oncoprotein. Although E4F1 is implicated in several key oncogenic pathways, its roles in tumorigenesis remain unclear. Using a genetically engineered mouse model of myeloid leukemia (histiocytic sarcomas, HS) based on the genetic inactivation of the tumor suppressor Ink4a/Arf locus, we have recently unraveled an unsuspected function of E4F1 in the survival of leukemic cells. In vivo, genetic ablation of E4F1 in established myeloid tumors results in tumor regression. E4F1 inactivation results in a cascade of alterations originating from dysfunctional mitochondria that induce increased reactive oxygen species (ROS) levels and ends in massive autophagic cell death in HS transformed, but not normal myeloid cells. E4F1 depletion also induces cell death in various human myeloid leukemic cell lines, including acute myeloid leukemic (AML) cell lines. Interestingly, the E4F1 protein is overexpressed in a large proportion of human AML samples. These data provide new insights into E4F1-associated survival functions implicated in tumorigenesis and could open the path for new therapeutic strategies.
PMCID: PMC3288034  PMID: 22024746
Animals; Autophagy; Cell Survival; Cell Transformation, Neoplastic; pathology; Disease Models, Animal; Humans; Leukemia, Myeloid; metabolism; pathology; Mice; RNA, Small Interfering; metabolism; Reactive Oxygen Species; metabolism; Repressor Proteins; metabolism; E4F1; histiocytic sarcoma; mitochondria; autophagy; cell death; leukemic cells; reactive oxygen species
3.  Consistency of quality attributes for the glycosylated monoclonal antibody Humira® (adalimumab) 
mAbs  2015;7(5):805-811.
Humira® (adalimumab) is a recombinant human IgG1 monoclonal antibody (mAb) glycoprotein consisting of 1330 amino acids that is specific for human tumor necrosis factor (TNF). The biological activity and clinical profile of mAb therapeutics, including adalimumab, is influenced by their protein structure and glycosylation patterns, which can be affected by the expression system, cell culture conditions and purification process methodology. While clinical outcome cannot yet be attributed to many of the individual structural features that constitute a mAb, it is evident that detailed structural attribute analysis is necessary if structural contributions to function are to be comprehensively defined. Adalimumab product quality data generated from over a decade of manufacturing across multiple production sites and through a series of manufacturing scale changes are presented here. These data reveal a consistent and tightly controlled profile for the product.
doi:10.1080/19420862.2015.1073429
PMCID: PMC4622832  PMID: 26230301
adalimumab; biosimilars; manufacturing; oligosaccharides; quality attributes
4.  Functionalized micro-capillary film for the rapid at-line analysis of IgG aggregates in a cell culture bioreactor 
mAbs  2015;7(5):812-819.
A micro-capillary film has been developed that offers the potential for an at-line analytical tool for rapid aggregate analysis during biopharmaceutical antibody production. A non-porous walled micro-capillary film (NMCF) with cation exchange functionality was demonstrated to act as a chromatography medium that could be operated with high linear fluid velocities and was highly resistant to blockage by entrained particulates, including cells. The NMCF containing 19 parallel microcapillaries was prepared using a melt extrusion process from poly(ethylene-vinyl alcohol) copolymer (EVOH). The NMCF-EVOH was modified to have cation-exchange functionality (NMCF-EVOH-SP) and shown to differentially bind monomer and aggregated species of IgG antibody directly from a bioreactor. The use of NMCF-EVOH-SP to quantify aggregate concentrations in monoclonal antibody preparations in less than 20 minutes was demonstrated.
doi:10.1080/19420862.2015.1065365
PMCID: PMC4623336  PMID: 26176737
microcapillary film; chromatography; antibody; aggregate; monitoring; CHO
5.  DNA methylation mediates the impact of exposure to prenatal maternal stress on BMI and central adiposity in children at age 13½ years: Project Ice Storm 
Epigenetics  2015;10(8):749-761.
Prenatal maternal stress (PNMS) in animals and humans predicts obesity and metabolic dysfunction in the offspring. Epigenetic modification of gene function is considered one possible mechanism by which PNMS results in poor outcomes in offspring. Our goal was to determine the role of maternal objective exposure and subjective distress on child BMI and central adiposity at 13½ years of age, and to test the hypothesis that DNA methylation mediates the effect of PNMS on growth. Mothers were pregnant during the January 1998 Quebec ice storm. We assessed their objective exposure and subjective distress in June 1998. At age 13½ their children were weighed and measured (n = 66); a subsample provided blood samples for epigenetic studies (n = 31). Objective and subjective PNMS correlated with central adiposity (waist-to-height ratio); only objective PNMS predicted body mass index (BMI). Bootstrapping analyses showed that the methylation level of genes from established Type-1 and -2 diabetes mellitus pathways showed significant mediation of the effect of objective PNMS on both central adiposity and BMI. However, the negative mediating effects indicate that, although greater objective PNMS predicts greater BMI and adiposity, this effect is dampened by the effects of objective PNMS on DNA methylation, suggesting a protective role of the selected genes from Type-1 and -2 diabetes mellitus pathways. We provide data supporting that DNA methylation is a potential mechanism involved in the long-term adaptation and programming of the genome in response to early adverse environmental factors.
doi:10.1080/15592294.2015.1063771
PMCID: PMC4623010  PMID: 26098974
body mass index; central adiposity; DNA methylation; Ice Storm; mediating effect; prenatal maternal stress
6.  Placental 5-methylcytosine and 5-hydroxymethylcytosine patterns associate with size at birth 
Epigenetics  2015;10(8):692-697.
Altered placental function as a consequence of aberrant imprinted gene expression may be one mechanism mediating the association between low birth weight and increased cardiometabolic disease risk. Imprinted gene expression is regulated by epigenetic mechanisms, particularly DNA methylation (5mC) at differentially methylated regions (DMRs). While 5-hydroxymethylcytosine (5hmC) is also present at DMRs, many techniques do not distinguish between 5mC and 5hmC. Using human placental samples, we show that the expression of the imprinted gene CDKN1C associates with birth weight. Using specific techniques to map 5mC and 5hmC at DMRs controlling the expression of CDKN1C and the imprinted gene IGF2, we show that 5mC enrichment at KvDMR and DMR0, and 5hmC enrichment within the H19 gene body, associate positively with birth weight. Importantly, the presence of 5hmC at imprinted DMRs may complicate the interpretation of DNA methylation studies in placenta; future studies should consider using techniques that distinguish between, and permit quantification of, both modifications.
doi:10.1080/15592294.2015.1062963
PMCID: PMC4623028  PMID: 26091021
5-methylcytosine; 5-hydroxymethylcytosine; birth weight; imprinted gene; Placenta
7.  A bi-functional antibody-receptor domain fusion protein simultaneously targeting IGF-IR and VEGF for degradation 
mAbs  2015;7(5):931-945.
Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor – type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique “capture-for-degradation” mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.
doi:10.1080/19420862.2015.1055442
PMCID: PMC4623440  PMID: 26073904
angiogenesis; antibody fusion; bispecific antibody; bi-functional antibody; degradation; IGF-IR; internalization; VEGF; VEGFR1; VEGFR2
8.  Beckwith–Wiedemann syndrome prenatal diagnosis by methylation analysis in chorionic villi 
Epigenetics  2015;10(7):643-649.
Beckwith–Wiedemann syndrome (BWS) is an imprinting disorder that can be prenatally suspected or diagnosed based on established clinical guidelines. Molecular confirmation is commonly performed on amniocytes. The possibility to use fresh (CVF) and cultured (CVC) chorionic villi has never been investigated. To verify whether CVF and CVC are reliable sources of DNA to study fetal methylation, we used pyrosequencing to test the methylation level of a number of differentially methylated regions (DMRs) at several imprinted loci (ICR1, ICR2, H19, PWS/AS-ICR, GNASXL, GNAS1A, ZAC/PLAGL1, and MEST) and at non-imprinted MGMT and RASSF1A promoters. We analyzed these regions in 19 healthy pregnancies and highlighted stable methylation levels between CVF and CVC at ICR1, ICR2, GNASXL, PWS/AS-ICR, and MEST. Conversely, the methylation levels at H19 promoter, GNAS1A and ZAC/PLAGL1 were different in CVC compared to fresh CV. We also investigated ICR1 and ICR2 methylation level of CVF/CVC of 2 BWS-suspected fetuses (P1 and P2). P1 showed ICR2 hypomethylation, P2 showed normal methylation at both ICR1 and ICR2. Our findings, although limited to one case of BWS fetus with an imprinting defect, can suggest that ICR1 and ICR2, but not H19, could be reliable targets for prenatal BWS diagnosis by methylation test in CVF and CVC. In addition, PWS/AS-ICR, GNASXL, and MEST, but not GNAS1A and ZAC/PLAGL1, are steadily hemimethylated in CV from healthy pregnancies, independently from culture. Thus, prenatal investigation of genomic imprinting in CV needs to be validated in a locus-specific manner.
doi:10.1080/15592294.2015.1057383
PMCID: PMC4622958  PMID: 26061650
Beckwith-Wiedemann syndrome; chorionic villi; DNA methylation; imprinting; prenatal diagnosis
9.  Placental mitochondrial methylation and exposure to airborne particulate matter in the early life environment: An ENVIRONAGE birth cohort study 
Epigenetics  2015;10(6):536-544.
Most research to date has focused on epigenetic modifications in the nuclear genome, with little attention devoted to mitochondrial DNA (mtDNA). Placental mtDNA content has been shown to respond to environmental exposures that induce oxidative stress, including airborne particulate matter (PM). Damaged or non-functioning mitochondria are specifically degraded through mitophagy, exemplified by lower mtDNA content, and could be primed by epigenetic modifications in the mtDNA. We studied placental mtDNA methylation in the context of the early life exposome. We investigated placental tissue from 381 mother-newborn pairs that were enrolled in the ENVIRONAGE birth cohort. We determined mtDNA methylation by bisulfite-pyrosequencing in 2 regions, i.e., the D-loop control region and 12S rRNA (MT-RNR1), and measured mtDNA content by qPCR. PM2.5 exposure was calculated for each participant's home address using a dispersion model. An interquartile range (IQR) increment in PM2.5 exposure over the entire pregnancy was positively associated with mtDNA methylation (MT-RNR1: +0.91%, P = 0.01 and D-loop: +0.21%, P = 0.05) and inversely associated with mtDNA content (relative change of −15.60%, P = 0.001) in placental tissue. mtDNA methylation was estimated to mediate 54% [P = 0.01 (MT-RNR1)] and 27% [P = 0.06 (D-loop)] of the inverse association between PM2.5 exposure and mtDNA content. This study provides new insight into the mechanisms of altered mitochondrial function in the early life environment. Epigenetic modifications in the mitochondrial genome, especially in the MT-RNR1 region, substantially mediate the association between PM2.5 exposure during gestation and placental mtDNA content, which could reflect signs of mitophagy and mitochondrial death.
doi:10.1080/15592294.2015.1048412
PMCID: PMC4623402  PMID: 25996590
air pollution; DNA methylation; epigenetics; mitochondria; mitochondrial DNA content; mitochondrial DNA methylation; particulate matter; placenta
10.  Effects of particulate matter exposure on blood 5-hydroxymethylation: results from the Beijing truck driver air pollution study  
Epigenetics  2015;10(7):633-642.
Previous studies have reported epigenetic changes induced by environmental exposures. However, previous investigations did not distinguish 5-methylcytosine (5mC) from a similar oxidative form with opposite functions, 5-hydroxymethylcytosine (5hmC). Here, we measured blood DNA global 5mC and 5hmC by ELISA and used adjusted mixed-effects regression models to evaluate the effects of ambient PM10 and personal PM2.5 and its elemental components—black carbon (BC), aluminum (Al), calcium (Ca), potassium (K), iron (Fe), sulfur (S), silicon (Si), titanium (Ti), and zinc (Zn)—on blood global 5mC and 5hmC levels. The study was conducted in 60 truck drivers and 60 office workers in Beijing, China from The Beijing Truck Driver Air Pollution Study at 2 exams separated by one to 2 weeks. Blood 5hmC level (0.08%) was ∼83-fold lower than 5mC (6.61%). An inter-quartile range (IQR) increase in same-day PM10 was associated with increases in 5hmC of 26.1% in office workers (P = 0.004), 20.2% in truck drivers (P = 0.014), and 21.9% in all participants combined (P < 0.001). PM10 effects on 5hmC were increasingly stronger when averaged over 4, 7, and 14 d preceding assessment (up to 132.6% for the 14-d average in all participants, P < 0.001). PM10 effects were also significant after controlling for multiple testing (family-wise error rate; FWER < 0.05). 5hmC was not correlated with personal measures of PM2.5 and elemental components (FWER > 0.05). 5mC showed no correlations with PM10, PM2.5, and elemental components measures (FWER > 0.05). Our study suggests that exposure to ambient PM10 affects 5hmC over time, but not 5mC. This finding demonstrates the need to differentiate 5hmC and 5mC in environmental studies of DNA methylation.
doi:10.1080/15592294.2015.1050174
PMCID: PMC4623004  PMID: 25970091
DNA methylation; Epigenetics; Particulate Matter; 5-hydroxymethylcytosine; 5-methylcytosine
11.  Cross-reactive and pre-existing antibodies to therapeutic antibodies—Effects on treatment and immunogenicity 
mAbs  2015;7(4):662-671.
The potential for immunogenicity is an ever-present concern during the development of biopharmaceuticals. Therapeutic antibodies occasionally elicit an antibody response in patients, which can result in loss of response or adverse effects. However, antibodies that bind a drug are sometimes found in pre-treatment serum samples, with the amount depending on drug, assay, and patient population. This review summarizes published data on pre-existing antibodies to therapeutic antibodies, including rheumatoid factors, anti-allotype antibodies, anti-hinge antibodies, and anti-glycan antibodies. Unlike anti-idiotype antibodies elicited by the drug, pre-formed antibodies in general appear to have little consequences during treatment. In the few cases where (potential) clinical consequences were encountered, antibodies were characterized and found to bind a distinct, unusual epitope of the therapeutic. Immunogenicity testing strategies should therefore always include a proper level of antibody characterization, especially when pre-formed antibodies are present. This minimizes false-positives, particularly due to rheumatoid factors, and helps to judge the potential threat in case a genuine pre-dose antibody reactivity is identified.
doi:10.1080/19420862.2015.1048411
PMCID: PMC4623040  PMID: 25962087
pre-existing antibodies; anti-drug antibodies (ADA); glycan; idiotype; rheumatoid factor; allotype; immunogenicity
12.  Disruption of BRD4 at H3K27Ac-enriched enhancer region correlates with decreased c-Myc expression in Merkel cell carcinoma 
Epigenetics  2015;10(6):460-466.
Pathologic c-Myc expression is frequently detected in human cancers, including Merkel cell carcinoma (MCC), an aggressive skin cancer with no cure for metastatic disease. Bromodomain protein 4 (BRD4) regulates gene transcription by binding to acetylated histone H3 lysine 27 (H3K27Ac) on the chromatin. Super-enhancers of transcription are identified by enrichment of H3K27Ac. BET inhibitor JQ1 disrupts BRD4 association with super-enhancers, downregulates proto-oncogenes, such as c-Myc, and displays antitumor activity in preclinical animal models of human cancers. Here we show that an enhancer proximal to the c-Myc promoter is enriched in H3K27Ac and associated with high occupancy of BRD4, and coincides with a putative c-Myc super-enhancer in MCC cells. This observation is mirrored in tumors from MCC patients. Importantly, depleted BRD4 occupancy at the putative c-Myc super-enhancer region by JQ1 correlates with decreased c-Myc expression. Thus, our study provides initial evidence that super-enhancers regulate c-Myc expression in MCC.
doi:10.1080/15592294.2015.1034416
PMCID: PMC4622756  PMID: 25941994
BET inhibitor; BRD4; H3K27Ac; JQ1; Merkel cell carcinoma; super-enhancer
13.  Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody 
mAbs  2015;7(3):525-539.
There is a need for new analytical approaches to better characterize the nature of the concentration-dependent, reversible self-association (RSA) of monoclonal antibodies (mAbs) directly, and with high resolution, when these proteins are formulated as highly concentrated solutions. In the work reported here, hydrogen exchange mass spectrometry (HX-MS) was used to define the concentration-dependent RSA interface, and to characterize the effects of association on the backbone dynamics of an IgG1 mAb (mAb-C). Dynamic light scattering, chemical cross-linking, and solution viscosity measurements were used to determine conditions that caused the RSA of mAb-C. A novel HX-MS experimental approach was then applied to directly monitor differences in local flexibility of mAb-C due to RSA at different protein concentrations in deuterated buffers. First, a stable formulation containing lyoprotectants that permitted freeze-drying of mAb-C at both 5 and 60 mg/mL was identified. Upon reconstitution with RSA-promoting deuterated solutions, the low vs. high protein concentration samples displayed different levels of solution viscosity (i.e., approx. 1 to 75 mPa.s). The reconstituted mAb-C samples were then analyzed by HX-MS. Two specific sequences covering complementarity-determining regions CDR2H and CDR2L (in the variable heavy and light chains, respectively) showed significant protection against deuterium uptake (i.e., decreased hydrogen exchange). These results define the major protein-protein interfaces associated with the concentration-dependent RSA of mAb-C. Surprisingly, certain peptide segments in the VH domain, the constant domain (CH2), and the hinge region (CH1-CH2 interface) concomitantly showed significant increases in local flexibility at high vs. low protein concentrations. These results indicate the presence of longer-range, distant dynamic coupling effects within mAb-C occurring upon RSA.
doi:10.1080/19420862.2015.1029217
PMCID: PMC4622866  PMID: 25875351
reversible self-association; protein-protein interactions; monoclonal antibody; immunoglobulin G1; hydrogen exchange; mass spectrometry; flexibility; stability; aggregation; high protein concentration
14.  Methylation at the CpG island shore region upregulates Nr3c1 promoter activity after early-life stress  
Epigenetics  2015;10(3):247-257.
Early-life stress (ELS) induces long-lasting changes in gene expression conferring an increased risk for the development of stress-related mental disorders. Glucocorticoid receptors (GR) mediate the negative feedback actions of glucocorticoids (GC) in the paraventricular nucleus (PVN) of the hypothalamus and anterior pituitary and therefore play a key role in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis and the endocrine response to stress. We here show that ELS programs the expression of the GR gene (Nr3c1) by site-specific hypermethylation at the CpG island (CGI) shore in hypothalamic neurons that produce corticotropin-releasing hormone (Crh), thus preventing Crh upregulation under conditions of chronic stress. CpGs mapping to the Nr3c1 CGI shore region are dynamically regulated by ELS and underpin methylation-sensitive control of this region's insulation-like function via Ying Yang 1 (YY1) binding. Our results provide new insight into how a genomic element integrates experience-dependent epigenetic programming of the composite proximal Nr3c1 promoter, and assigns an insulating role to the CGI shore.
doi:10.1080/15592294.2015.1017199
PMCID: PMC4622987  PMID: 25793778
CpG island shore; DNA methylation; early-life stress; glucocorticoid receptor; insulator; Yin Yang
15.  Immunomodulatory action of the DNA methyltransferase inhibitor SGI-110 in epithelial ovarian cancer cells and xenografts  
Epigenetics  2015;10(3):237-246.
We aimed to determine the effect of SGI-110 on methylation and expression of the cancer testis antigens (CTAs) NY-ESO-1 and MAGE-A in epithelial ovarian cancer (EOC) cells in vitro and in vivo and to establish the impact of SGI-110 on expression of major histocompatibility (MHC) class I and Intracellular Adhesion Molecule 1 (ICAM-1) on EOC cells, and on recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. We also tested the impact of combined SGI-110 and NY-ESO-1-specific CD8+ T-cells on tumor growth and/or murine survival in a xenograft setting. EOC cells were treated with SGI-110 in vitro at various concentrations and as tumor xenografts with 3 distinct dose schedules. Effects on global methylation (using LINE-1), NY-ESO-1 and MAGE-A methylation, mRNA, and protein expression were determined and compared to controls. SGI-110 treated EOC cells were evaluated for expression of immune-modulatory genes using flow cytometry, and were co-cultured with NY-ESO-1 specific T-cell clones to determine immune recognition. In vivo administration of SGI-110 and CD8+ T-cells was performed to determine anti-tumor effects on EOC xenografts. SGI-110 treatment induced hypomethylation and CTA gene expression in a dose dependent manner both in vitro and in vivo, at levels generally superior to azacitidine or decitabine. SGI-110 enhanced the expression of MHC I and ICAM-1, and enhanced recognition of EOC cells by NY-ESO-1-specific CD8+ T-cells. Sequential SGI-110 and antigen-specific CD8+ cell treatment restricted EOC tumor growth and enhanced survival in a xenograft setting. SGI-110 is an effective hypomethylating agent and immune modulator and, thus, an attractive candidate for combination with CTA-directed vaccines in EOC.
doi:10.1080/15592294.2015.1017198
PMCID: PMC4623048  PMID: 25793777
cancer testis antigens; cancer germline genes; DNA methylation; DNA methyltransferase inhibitors; epithelial ovarian cancer; epigenetics; immune modulation; SGI-110
16.  Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model 
mAbs  2015;7(3):638-650.
CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (KD ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC50 typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.
doi:10.1080/19420862.2015.1016693
PMCID: PMC4623119  PMID: 25751125
CD1d; NKT cell; antibody; asthma; cytokine
17.  Insights into the molecular basis of a bispecific antibody's target selectivity  
mAbs  2015;7(3):461-469.
Bispecific antibodies constitute a valuable class of therapeutics owing to their ability to bind 2 distinct targets. Dual targeting is thought to enhance biological efficacy, limit escape mechanisms, and increase target selectivity via a strong avidity effect mediated by concurrent binding to both antigens on the surface of the same cell. However, factors that regulate the extent of target selectivity are not well understood. We show that dual targeting alone is not sufficient to promote efficient target selectivity, and report the substantial roles played by the affinity of the individual arms, overall avidity and valence. More particularly, various monovalent bispecific IgGs composed of an anti-CD70 moiety paired with variants of the anti-CD4 mAb ibalizumab were tested for preferential binding and selective depletion of CD4+/CD70+ T cells over cells expressing only one of the target antigens that resulted from antibody dependent cell-mediated cytotoxicity. Variants exhibiting reduced CD4 affinity showed a greater degree of target selectivity, while the overall efficacy of the bispecific molecule was not affected.
doi:10.1080/19420862.2015.1022695
PMCID: PMC4622944  PMID: 25730144
bispecific antibody; selective targeting; monovalent; bivalent; affinity; avidity; CD4; CD70; T cells; B cells
18.  The preclinical evaluation of the dual mTORC1/2 inhibitor INK-128 as a potential anti-colorectal cancer agent 
Cancer Biology & Therapy  2015;16(1):34-42.
The colorectal cancer is the leading contributor of cancer-related mortality. Mammalian target of rapamycin (mTOR), existing in 2 complexes (mTORC1/2), is frequently dysregulated and constitutively activated in colorectal cancers. It represents an important drug target. Here we found that INK-128, the novel ATP-competitive kinase inhibitor of mTOR, blocked both mTORC1 and mTORC2 activation in colorectal cancer cells (both primary and transformed cells). The immunoprecipitation results showed that the assembly of mTORC1 (mTOR-Raptor association) and mTORC2 (mTOR-Rictor-Sin1 association) was also disrupted by INK-128. INK-128 inhibited colorectal cancer cell growth and survival, and induced both apoptotic and non-apoptotic cancer cell death. Further, INK-128 showed no effect on Erk/MAPK activation, while MEK/Erk inhibition by MEK-162 enhanced INK-128-induced cytotoxicity in colorectal cancer cells. Meanwhile, INK-128 downregulated Fascin1 (FSCN1)/E-Cadherin expressions and inhibited HT-29 cell in vitro migration. In vivo, daily INK-128 oral administration inhibited HT-29 xenograft growth in mice, which was further enhanced by MEK-162 administration. Finally, we found that INK-128 sensitized 5-fluorouracil-(5-FU)-mediated anti-HT-29 activity in vivo and in vitro. Thus, our preclinical studies strongly suggest that INK-128 might be investigated for colorectal cancer treatment in clinical trials.
doi:10.4161/15384047.2014.972274
PMCID: PMC4623257  PMID: 25692620
cell growth and migration; colorectal cancer; INK-128; mTOR
19.  Translocation and accumulation of nicotine via distinct spatio-temporal regulation of nicotine transporters in Nicotiana tabacum 
Plant Signaling & Behavior  2015;10(7):e1035852.
In plants, secondary metabolites play important roles in adaptation to the environment. Nicotine, a pyridine alkaloid in Nicotiana tabacum, functions as chemical barrier against herbivores. Nicotine produced in the root undergoes long-distance transport and accumulates mainly in the leaves. Since production of such defensive compounds is costly, plants must regulate the allocation of the products to their tissues; however, the molecular mechanism of nicotine translocation remains unclear. Our recent studies identified a novel multidrug and toxic compound extrusion (MATE)-type nicotine transporter, JAT2 (jasmonate-inducible alkaloid transporter 2). This transporter is specifically expressed in leaves, localizes to the tonoplast, and transports nicotine as its substrate. The specific induction of JAT2 expression in leaves by methyl jasmonate (MeJA) treatment suggests that this transporter plays an important role in nicotine distribution to leaves, especially under herbivore attack, by transporting nicotine into the vacuole. Considering JAT2, together with the previously identified MATE transporters JAT1, MATE1, and MATE2, and the PUP (purine permease) transporter NUP1 (nicotine uptake permease1), we show a model of nicotine translocation and accumulation via distinct spatio-temporal regulation of nicotine transporter expression. Furthermore, we discuss the possible role of nicotine transporters in determining outcrossing rates and seed production.
doi:10.1080/15592324.2015.1035852
PMCID: PMC4622871  PMID: 26251879
alkaloid; MATE; nicotine; tobacco; translocation; transporter
20.  Twin athlete brothers with open physes operated for ACL reconstruction on the same day, but with different elapsed times after injury: a 5-year follow-up 
BMJ Case Reports  2014;2014:bcr2013202650.
We present the case of twin brothers with open physes who practiced judo to a high level and were operated on the same day for anterior cruciate ligament reconstruction. One of them was injured a year before surgery, and the other was injured a month before the procedure. The brother who chose to undergo a conservative treatment sustained meniscus injury afterwards and showed lower objective results when evaluated 5 years after surgery.
doi:10.1136/bcr-2013-202650
PMCID: PMC3918611  PMID: 24510695
21.  Excellent postoperative analgesia with the addition of hyaluronidase to lignocaine for subcostal TAP block used in conjunction with systemic analgesia for laparoscopic cholecystectomy 
BMJ Case Reports  2014;2014:bcr2013202911.
Subcostal transversus abdominis plane (TAP) blocks provide good postoperative analgesia for laparoscopic cholecystectomies. We hypothesised that adding hyaluronidase may improve the efficacy of this technique by increasing spread of the local anaesthetic (LA). In this case, we performed a bilateral ultrasound-guided subcostal TAP block using lignocaine (40 mL 1%) with hyaluronidase (75 IU/mL) for postoperative analgesia following elective laparoscopic cholecystectomy. It was used in combination with intraoperative morphine, diclofenac and paracetamol. Regular paracetamol was administered postoperatively. We monitored serial serum lignocaine levels and recorded the patient's visual analogue scale (VAS) pain scores postoperatively. We found that the patient experienced excellent analgesia throughout the postoperative period and that the serum lignocaine levels did not exceed the therapeutic range.
doi:10.1136/bcr-2013-202911
PMCID: PMC3918615  PMID: 24510699
22.  Bevacizumab in vitreous haemorrhage secondary to radiation retinopathy 
BMJ Case Reports  2014;2014:bcr2013203177.
Radiation retinopathy is a delayed-onset side effect of radiation exposure caused by retinal ischaemia that may induce proliferative retinopathy with neovascularisation, vitreous haemorrhage and macular oedema. An otherwise healthy, 51-year-old male patient who had been diagnosed with olfactory neuroblastoma and undergone complete surgical removal of the lesion followed by cranial irradiation developed bilateral cataracts and radiation retinopathy. The patient was treated by panretinal photocoagulation (PRP), followed by three-port pars-plana vitrectomy. Recurrent episodes of vitreous haemorrhages occurred following surgery and the patient was successfully treated by one intravitreal injection of bevacizumab with resolution of vitreous blood. Vitreous haemorrhage recurred 6 months later and a scheduled treatment with intravitreal bevacizumab every 4 months was established, preventing further haemorrhagic episodes. Six months after the last injection, a new episode of vitreous haemorrhage occurred.
Scheduled intravitreal bevacizumab injections may help prevent recurrent vitreous haemorrhages in vitrectomised patients with radiation retinopathy.
doi:10.1136/bcr-2013-203177
PMCID: PMC3918616  PMID: 24510700
23.  Widespread bullous fixed drug eruption 
BMJ Case Reports  2014;2014:bcr2013200584.
A 53-year-old man developed a widespread erythematous eruption which rapidly evolved into fluid-filled bulla mostly involving the distal areas of all four limbs and erosions on the oral as well as anogenital mucosa. Based on clinical presentation, chronology of drug exposure, past events and histopathology as diagnosis of widespread bullous fixed drug eruption was made over Steven Johnson-toxic epidermal necrolysis syndrome. Steroids were deferred and the lesions healed with minimal pigmentation within a week. Differentiating between the two entities has been historically difficult, and yet can have significant therapeutic and prognostic implications.
doi:10.1136/bcr-2013-200584
PMCID: PMC3918620  PMID: 24510691
24.  A rare case of prosthetic endocarditis and dehiscence in a mechanical valved conduit 
BMJ Case Reports  2014;2014:bcr2013200720.
A middle-aged adult patient with a history of aortic root replacement with a mechanical valved conduit and remote chest trauma was referred to our institution with prosthetic endocarditis. Transoesophageal echocardiogram at our institution confirmed a near-complete dehiscence of the prosthetic aortic valve from the conduit, with significant perivalvular flow forming a pseudoaneurysm. The patient underwent a high-risk re-operation, involving redo aortic root replacement with a homograft after extensive debridement of the infected tissue. The patient was discharged to an outside facility after an uncomplicated hospital course, and remains stable.
doi:10.1136/bcr-2013-200720
PMCID: PMC3918631  PMID: 24510692
25.  A rare case of massive cutaneous metastases in breast carcinoma 
BMJ Case Reports  2014;2014:bcr2013202839.
doi:10.1136/bcr-2013-202839
PMCID: PMC3918612  PMID: 24503663

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