IL-5 up-regulates cysteinyl leukotriene 1 receptor expression in HL-60 cells differentiated into eosinophils

The cysteinyl leukotrienes, leukotriene (LT) C(4), LTD(4), and LTE(4), are lipid mediators that have been implicated in the pathogenesis of several inflammatory processes, including asthma. The human LTD(4) receptor, CysLT(1)R, was recently cloned and characterized. We had previously shown that HL-60 cells differentiated toward the eosinophilic lineage (HL-60/eos) developed specific functional LTD(4) receptors. The present work was undertaken to study the potential modulation of CysLT(1)R expression in HL-60/eos by IL-5, an important regulator of eosinophil function. Here, we report that IL-5 rapidly up-regulates CysLT(1)R mRNA expression, with consequently enhanced CysLT(1)R protein expression and function in HL-60/eos. CysLT(1)R mRNA expression was augmented 2- to 15-fold following treatment with IL-5 (1-20 ng/ml). The effect was seen after 2 h, was maximal by 4 h, and maintained at 8 h. Although CysLT(1)R mRNA was constitutively expressed in undifferentiated HL-60 cells, its expression was not modulated by IL-5 in the absence of differentiation. Differentiated HL-60/eos cells pretreated with IL-5 (10 ng/ml) for 24 h showed enhanced CysLT(1)R expression on the cell surface, as assessed by flow cytometry using a polyclonal anti-CysLT(1)R Ab. They also showed enhanced responsiveness to LTD(4), but not to LTB(4) or platelet-activating factor, in terms of Ca(2+) mobilization, and augmented the chemotactic response to LTD(4). Our findings suggest a possible mechanism by which IL-5 can modulate eosinophil functions and particularly their responsiveness to LTD(4), and thus contribute to the pathogenesis of asthma and allergic diseases.     

Controlled modulation of serum protein binding and biodistribution of asymmetric cyanine dyes by variation of the number of sulfonate groups

To assess the suitability of asymmetric cyanine dyes for in vivo fluoro-optical molecular imaging, a comprehensive study on the influence of the number of negatively charged sulfonate groups governing the hydrophilicity of the DY-67x family of asymmetric cyanines was performed. Special attention was devoted to the plasma protein binding capacity and related pharmacokinetic properties. Four members of the DY-67x cyanine family composed of the same main chromophore, but substituted with a sequentially increasing number of sulfonate groups (n = 1-4; DY-675, DY-676, DY-677, DY-678, respectively), were incubated with plasma proteins dissolved in phosphate-buffered saline. Protein binding was assessed by absorption spectroscopy, gel electrophoresis, ultrafiltration, and dialysis. Distribution of dye in organs was studied by intraveneous injection of 62 nmol dye/kg body weight into mice (n = 12; up to 180 minutes postinjection) using whole-body near-infrared fluorescence imaging. Spectroscopic studies, gel electrophoresis, and dialysis demonstrated reduced protein binding with increasing number of sulfonate groups. The bovine serum albumin binding constant of the most hydrophobic dye, DY-675, is 18 times higher than that of the most hydrophilic fluorophore, DY-678. In vivo biodistribution analysis underlined a considerable influence of dye hydrophilicity on biodistribution and excretion pathways, with the more hydrophobic dyes, DY-675 and DY-676, accumulating in the liver, followed by strong fluorescence signals in bile and gut owing to accumulation in feces and comparatively hydrophilic DY-678-COOH accumulating in the bladder. Our results demonstrate the possibility of selectively controlling dye-protein interactions and, thus, biodistribution and excretion pathways via proper choice of the fluorophore's substitution pattern. This underlines the importance of structure-property relationships for fluorescent labels. Moreover, our data could provide the basis for the rationalization of future contrast agent developments.     

Up-regulation of cysteinyl leukotriene 1 receptor by IL-13 enables human lung fibroblasts to respond to leukotriene C4 and produce eotaxin

Cysteinyl leukotrienes (CysLTs) play an important role in eosinophilic airway inflammation. In addition to their direct chemotactic effects on eosinophils, indirect effects have been reported. Eotaxin is a potent eosinophil-specific chemotactic factor produced mainly by fibroblasts. We investigated whether CysLTs augment eosinophilic inflammation via eotaxin production by fibroblasts. Leukotriene (LT)C(4) alone had no effect on eotaxin production by human fetal lung fibroblasts (HFL-1). However, LTC(4) stimulated eotaxin production by IL-13-treated fibroblasts, thereby indirectly inducing eosinophil sequestration. Unstimulated fibroblasts did not respond to LTC(4), but coincubation or preincubation of fibroblasts with IL-13 altered the response to LTC(4). To examine the mechanism(s) involved, the expression of CysLT1R in HFL-1 was investigated by quantitative real-time PCR and flow cytometry. Only low levels of CysLT1R mRNA and no CysLT1R protein were expressed in unstimulated HFL-1. In contrast, stimulation with IL-13 at a concentration of 10 ng/ml for 24 h significantly up-regulated both CysLT1R mRNA and protein expression in HFL-1. The synergistic effect of LTC(4) and IL-13 on eotaxin production was abolished by CysLT1R antagonists pranlukast and montelukast. These findings suggest that IL-13 up-regulates CysLT1R expression, which may contribute to the synergistic effect of LTC(4) and IL-13 on eotaxin production by lung fibroblasts. In the Th2 cytokine-rich milieu, such as that in bronchial asthma, CysLT1R expression on fibroblasts might be up-regulated, thereby allowing CysLTs to act effectively and increase eosinophilic inflammation.     

Regulation of cysteinyl leukotriene type 1 receptor internalization and signaling

Cysteinyl leukotrienes activate the cysteinyl leukotriene type 1 receptor (CysLT1R) to regulate numerous cell functions important in inflammatory processes and diseases such as asthma. Despite its physiologic importance, no studies to date have examined the regulation of CysLT1R signaling or trafficking. We have established model systems for analyzing recombinant human CysLT1R and found regulation of internalization and signaling of the CysLT1R to be unique among G protein-coupled receptors. Rapid and profound LTD4-stimulated internalization was observed for the wild type (WT) CysLT1R, whereas a C-terminal truncation mutant exhibited impaired internalization yet signaled robustly, suggesting a region within amino acids 310-321 as critical to internalization. Although overexpression of WT arrestins significantly increased WT CysLT1R internalization, expression of dominant-negative arrestins had minimal effects, and WT CysLT1R internalized in murine embryonic fibroblasts lacking both arrestin-2 and arrestin-3, suggesting that arrestins are not the primary physiologic regulators of CysLT1Rs. Instead, pharmacologic inhibition of protein kinase C (PKC) was shown to profoundly inhibit CysLT1R internalization while greatly increasing both phosphoinositide (PI) production and calcium mobilization stimulated by LTD4 yet had almost no effect on H1 histamine receptor internalization or signaling. Moreover, mutation of putative PKC phosphorylation sites within the CysLT1R C-tail (CysLT1RS(313-316)A) reduced receptor internalization, increased PI production and calcium mobilization by LTD4, and significantly attenuated the effects of PKC inhibition. These findings characterized the CysLT1R as the first G protein-coupled receptor identified to date in which PKC is the principal regulator of both rapid agonist-dependent internalization and rapid agonist-dependent desensitization.     

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.     

Development of Antibody to Human GM3 Synthase and Immunodetection of the Enzyme in Human Tissues

Polyclonal antibody was raised to a cloned fragment of human GM3 synthase. Affinity purified R27C1 antibody to the tagged recombinant protein inhibited GM3 synthase activity in human liver and HL-60 cells in a dose-dependent manner. However, the R27C1 antibody did not affect liver sialyltransferase activity towards asialofetuin. We are the first to measure GM3 synthase activity in human liver (194 +/- 60 pmol NeuAc/h per mg protein), which was about 10-fold lower than in phorbol myristate acetate-stimulated HL-60 cells (1353 +/- 573 pmol NeuAc/h per mg protein). On immunoblotting the R27C1 antibody recognized a common protein band in a number of human tissues (liver, brain, atherosclerotic aortic intima, HL-60 cells) with molecular mass of about 60 kD, which is similar to that of the purified GM3 synthase from rat liver. In human liver and aortic intima, the 60-kD band was almost a single band, which makes possible the use of the R27C1 antibody for immunohistochemical studies in these tissues.     

Targeted gene disruption reveals the role of cysteinyl leukotriene 1 receptor in the enhanced vascular permeability of mice undergoing acute inflammatory responses

The cysteinyl leukotrienes (cysLTs), leukotriene (LT) C(4), LTD(4), and LTE(4), are proinflammatory lipid mediators generated in the mouse by hematopoietic cells such as macrophages and mast cells. There are two mouse receptors for the cysLTs, CysLT(1) receptor (CysLT(1)R) and CysLT(2)R, which are 38% homologous and are located on mouse chromosomes X and 14, respectively. To clarify the different roles of the CysLT(1)R and CysLT(2)R in inflammatory responses in vivo, we generated CysLT(1)R-deficient mice by targeted gene disruption. These mice developed normally and were fertile. In an intracellular calcium mobilization assay with fura-2 acetoxymethyl ester, peritoneal macrophages from wild-type littermates, which express both CysLT(1)R and CysLT(2)R, responded substantially to 1 x 10(-6) m LTD(4) and slightly to 1 x 10(-6) m LTC(4), whereas the macrophages from CysLT(1)R-deficient mice did not respond to either LTD(4) or LTC(4). Plasma protein extravasation, but not neutrophil infiltration, was significantly reduced in CysLT(1)R-deficient mice subjected to zymosan A-induced peritoneal inflammation. Plasma protein extravasation was also significantly diminished in CysLT(1)R-deficient mice undergoing IgE-mediated passive cutaneous anaphylaxis as compared with the wild-type mice. Thus, the cysLTs generated in vivo by either monocytes/macrophages or mast cells utilize CysLT(1)R for the response of the microvasculature in acute inflammation.     

Quantifying Cell-Surface Biomarker Expression in Thick Tissues with Ratiometric Three-Dimensional Microscopy

The burgeoning fields of in vivo three-dimensional (3D) microscopy and endomicroscopy, as well as ex vivo tissue cytometry have introduced new challenges for tissue preparation and staining with exogenous molecular contrast agents. These challenges include effective delivery of the agents, and once delivered, distinguishing between bound verses unbound molecular probes. If applied topically, there are additional issues with rinsing off unbound probe, which can be nonuniform and inefficient in thick tissues, thus leading to ambiguous contrast and a large nonspecific background that may obscure molecule-specific staining. Therefore, we have developed a ratiometric 3D microscopy scheme that not only reduces the effects of nonspecific sources of contrast, but also enables quantification of the relative binding affinity of imaging probes to their biomarker targets. Here we demonstrate this ratiometric approach by simultaneously imaging a HER2/neu (erbB2)-targeted monoclonal antibody labeled with one fluorophore and an isotype-matched negative control antibody labeled with another fluorophore. By taking a pixel-by-pixel calibrated ratio between the signals from each fluorescent image channel, accurate quantification of specific versus nonspecific binding affinity is achieved with cultured cells, yielding data that are in agreement with analyses via flow cytometry. We also demonstrate quantitative 3D microscopic imaging of biomarker expression in tissue models and in thick human biopsy samples of normal, HER2-negative, and HER2-positive breast tumors. This strategy enables rapid, quantitative, and unambiguous volumetric microscopy of biomarker expression in thick tissues, including whole biopsies, and will enable real-time optical assessment of disease markers in the living body.     

Antiasthmatic drugs targeting the cysteinyl leukotriene receptor 1 alleviate central nervous system inflammatory cell infiltration and pathogenesis of experimental autoimmune encephalomyelitis

Cysteinyl leukotrienes (CysLTs) are potent proinflammatory mediators and are considered to play a key role in inflammatory diseases such as asthma. Antagonists targeting the receptor of CysLTs (CysLT1) are currently used as antiasthmatic drugs. CysLTs have also been implicated in other inflammatory reactions. In this study, we report that in experimental autoimmune encephalomyelitis animals, CysLT1 is upregulated in immune tissue and the spinal cord, and CysLT levels in the blood and cerebrospinal fluid are also higher than in normal mice. Two clinically used antiasthma drugs, montelukast and zafirlukast, both targeting CysLT1, effectively block the CNS infiltration of inflammatory cells and thus reduce the incidence, peak severity, and cumulative clinical scores. Further study indicated that CysLT1 signaling does not affect the differentiation of pathogenic T helper cells. It might affect the pathogenesis of experimental autoimmune encephalomyelitis by increasing the secretion of IL-17 from myelin oligodendrocyte glycoprotein-specific T cells, increasing the permeability of the blood-brain barrier and inducing chemotaxis of T cells. These effects can be blocked by CysLT1 antagonists. Our findings indicate that the antiasthmatic drugs against CysLT1 can also be used to treat multiple sclerosis.     

Transfer to wheat (Triticum aestivum) of small chromosome segments from rye (Secale cereale) carrying disease resistance genes

One hundred wheat lines, derived from monosomic additions of chromosome 1R of rye inbred line R12 (Chinese rye), were detected by PCR amplification using rye-specific primer pairs. Only 5 wheat lines, 1R296, 1R330, 1R314, 1R725, and 1R734, were determined to contain rye chromatin. While 1R296 and 1R330 were highly susceptible to stripe rust and powdery mildew, 1R314, 1R725 and 1R734 were highly resistant to both diseases. Acid-polyacrylamide gel electrophoresis showed that the ω-secalin bands were absent in 1R314, but present in the other 4 wheat lines. Genomicin situ hybridization indicated that 1R296, 1R330, and 1R725 contained translocations involving the whole short arm of chromosome 1R. However, 1R314 and 1R734 contained a pair of wheat chromosomes with small, terminal, rye-derived chromosome segments. The results suggest that the translocation breakpoint of 1RS in 1R314 was located between theSec-1 locus and the disease-resistance loci, while in line 1R734, the breakpoint was located between theSec-1 locus and the centromere. Taking account of the improved disease resistance of 1R725, 1R314 and 1R734, the chromosome arm 1RS of R12 may represent new and valuable disease resistance resources for wheat improvement.     

Leukotriene D4 induces brain edema and enhances CysLT2 receptor-mediated aquaporin 4 expression

Cysteinyl leukotrienes (including LTC(4), LTD(4), and LTE(4)), potent inflammatory mediators, can induce brain-blood barrier (BBB) disruption and brain edema. These reactions are mediated by their receptors, CysLT(1) and CysLT(2) receptors. On the other hand, aquaporin 4 (AQP4) primarily modulates brain water homeostasis and edema after various injuries. Here, we aimed to determine whether AQP4 is involved in LTD(4)-induced brain edema. LTD(4) (1ng in 0.5mul PBS) microinjection into the cortex increased endogenous IgG exudation (BBB disruption) and water content (brain edema), and enhanced AQP4 expression in mouse brain. The selective CysLT(1) receptor antagonist pranlukast inhibited the IgG exudation, but not the increased water content and AQP4 expression induced by LTD(4). In the cultured rat astrocytes, LTD(4) (10(-9)-10(-7)M, for 24h) similarly enhanced AQP4 expression. The enhanced AQP4 expression was inhibited by Bay u9773, a non-selective CysLT(1)/CysLT(2) receptor antagonist, but not by pranlukast. LTD(4) (10(-9)-10(-7)M) also induced the mRNA expression of CysLT(2) (not CysLT(1)) receptor in astrocytes. These results indicate that LTD(4) modulates brain edema; CysLT(1) receptor mediates vasogenic edema while CysLT(2) receptor may mediate cytotoxic edema via up-regulating AQP4 expression.     

XGFR*, a novel affinity-matured bispecific antibody targeting IGF-1R and EGFR with combined signaling inhibition and enhanced immune activation for the treatment of pancreatic cancer

The epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) play critical roles in tumor growth, providing a strong rationale for the combined inhibition of IGF-1R and EGFR signaling in cancer therapy. We describe the design, affinity maturation, in vitro and in vivo characterization of the bispecific anti-IGF-1R/EGFR antibody XGFR*. XGFR* is based on the bispecific IgG antibody XGFR, which enabled heterodimerization of an IGF-1R binding scFab heavy chain with an EGFR-binding light and heavy chain by the “knobs-into-holes” technology. XGFR* is optimized for monovalent binding of human EGFR and IGF-1R with increased binding affinity for IGF-1R due to affinity maturation and highly improved protein stability to oxidative and thermal stress. It bears an afucosylated Fc-portion for optimal induction of antibody-dependent cell-mediated cytotoxicity (ADCC). Stable Chinese hamster ovary cell clones with production yields of 2–3 g/L were generated, allowing for large scale production of the bispecific antibody. XGFR* potently inhibits EGFR- and IGF-1R-dependent receptor phosphorylation, reduces tumor cell proliferation in cells with heterogeneous levels of IGF-1R and EGFR receptor expression and induces strong ADCC in vitro. A comparison of pancreatic and colorectal cancer lines demonstrated superior responsiveness to XGFR*-mediated signaling and tumor growth inhibition in pancreatic cancers that frequently show a high degree of IGF-1R/EGFR co-expression. XGFR* showed potent anti-tumoral efficacy in the orthotopic MiaPaCa-2 pancreatic xenograft model, resulting in nearly complete tumor growth inhibition with significant number of tumor remissions. In summary, the bispecific anti-IGF-1R/EGFR antibody XGFR* combines potent signaling and tumor growth inhibition with enhanced ADCC induction and represents a clinical development candidate for the treatment of pancreatic cancer.     

Further evidence for heterogeneity of Fc gamma-receptors on guinea pig splenic B and T lymphocytes: analysis using monoclonal antibodies to two distinct types of Fc gamma-receptor

In our previous paper, we reported that guinea pig splenic lymphocytes expressed two distinct Fc-receptors for homologous IgG (Fc gamma Rs), one monospecific for IgG2 (Fc gamma 2R) and the other bispecific for IgG1 and IgG2 (Fc gamma 1/gamma 2R), when analyzed by EA-rosette assay. These Fc gamma Rs on the cells were further studied by using two monoclonal antibodies toward the Fc gamma Rs on guinea pig peritoneal macrophages (anti-Fc gamma 1/gamma 2R and anti-Fc gamma 2R antibody). The anti-Fc gamma 1/gamma 2R antibody completely inhibited the rosette formation of splenic lymphocytes with IgG1-sensitized sheep erythrocytes [EA(IgG1)]. On the other hand, EA(IgG2)-rosette formation was inhibited partially by anti-Fc gamma 2R but not by anti-Fc gamma 1/gamma 2R antibody. Complete inhibition of the EA (IgG2)-rosette formation was achieved by simultaneous additions of both anti-Fc gamma 2R and anti-Fc gamma 1/gamma 2R antibodies. The binding of IgG2 antibody complexed with ovalbumin to the cells was partially inhibited by either anti-Fc gamma R antibody, and complete inhibition occurred in the presence of both the antibodies, indicating that two types of Fc gamma R, Fc gamma 1/gamma 2R, and Fc gamma 2R, are expressed on the cells. The determination of these Fc gamma Rs on B and T lymphocytes by two-color flow cytometry showed that about 52% of B lymphocytes expressed Fc gamma 1/gamma 2R alone and 32% of the cells expressed both the Fc gamma Rs. On the other hand, about 12% of T lymphocytes was found to express Fc gamma 2R alone and the cells expressing Fc gamma 1/gamma 2R were in the minority (3.8%). T lymphocytes expressing both the Fc gamma Rs were not detected. These results show that guinea pig B lymphocytes bear two types of Fc gamma Rs and are heterogeneous with regard to their Fc gamma Rs and that T lymphocytes express Fc gamma 2R mainly.     

Biodistribution Analyses of a Near-Infrared,Fluorescently Labeled,Bispecific Monoclonal Antibody Using Optical Imaging

In recent years, biodistribution analyses of pharmaceutical compounds in preclinical animal models have become an integral part of drug development. Here we report on the use of optical imaging biodistribution analyses in a mouse xenograft model to identify tissues that nonspecifically retained a bispecific antibody under development. Although our bispecific antibody bound both the epidermal growth factor receptor and insulin growth factor 1 receptor are expressed on H358, nonsmall-cell lung carcinoma cells, the fluorescence from labeled bispecific antibody was less intense than expected in xenografted tumors. Imaging analyses of live mice and major organs revealed that the majority of the Alexa Fluor 750 labeled bispecific antibody was sequestered in the liver within 2 h of injection. However, results varied depending on which near-infrared fluorophore was used, and fluorescence from the livers of mice injected with bispecific antibody labeled with Alexa Fluor 680 was less pronounced than those labeled with Alexa Fluor 750. The tissue distribution of control antibodies remained unaffected by label and suggests that the retention of fluorophores in the liver may differ. Given these precautions, these results support the incorporation of optical imaging biodistribution analyses in biotherapeutic development strategies.Abbreviations: EGFR, epidermal growth factor receptor; EIBS, EGFR–IGF1R bispecific monoclonal antibody; FMT, fluorescence molecular tomography; IGF1R, insulin-like growth factor 1 receptor; NIRF, near-infrared fluorophore; PET, positron emission tomographyOver the past 25 y, bispecific antibodies have been added to the arsenal of humanized antibodies that have been developed to treat cancer, infectious diseases, and inflammatory diseases. Bispecific biotherapeutics simultaneously recognize 2 different epitopes on the same or different cells or bacteria. This multispecificity offers several advantages over their monospecific counterparts in that cell or tissue specificity can be increased and thus toxicity reduced, 2 (or more) receptor pathways can be targeted simultaneously, and immune-mediated responses at clinically affected sites may be activated (cancer) or deactivated (inflammatory disease).^13,18 As the size and structure of monoclonal antibodies are altered from their natural state to create biotherapeutics, such as bispecific antibodies,⁸ the pharmacokinetic properties of the products may differ from that of their parental (natural) antibodies. Aside from the potential to increase immunogenicity or aggregate, very little is known about how these structural differences affect pharmacokinetics and biodistribution. Receptor-mediated uptake and cellular processing both are likely to play a role,²⁰ but identifying the tissues or cell types involved in recognizing these altered proteins is a necessary first step toward developing engineering strategies that can circumvent these impediments.At the organ or tissue level, the use of radiolabeling and imaging in clinical and preclinical studies has greatly facilitated our understanding of the pharmacokinetics and tissue distribution of biotherapeutics.^7,21 This advance is in part due to the ability to track and monitor the distribution of labeled compounds in the same animal over time. The increased sensitivity of positron emission tomography (PET) and the ability of radioisotope signals to travel through tissues without being significantly attenuated or diffracted has made PET the leading modality for preclinical and clinical biodistribution analyses. Disadvantages of PET are that its application is more expensive and requires skill in chemical conjugation, imaging technology, and radiation safety. Given that these resources are often limited or unavailable at several institutions (ours included), near-infrared optical imaging with improved tissue penetrance and quantitative imaging algorithms are demonstrating value when compared with ‘high-end’ imaging modalities.^19,33 The accessibility of a variety commercially available fluorescently labeled targeting proteins and peptides, activatable probes, and fluorescent protein labeling kits has made this technology more readily available. In addition, because multiple fluorophores of nonoverlapping emission spectrums can be administered simultaneously, colocalization studies and direct comparisons of 2 or more labeled compounds can be performed in the same animal. Here we show how fluorescence-based optical imaging was used to identify potential tissue depots or sinks and characterize the biodistribution profile of a bispecific antibody under preclinical development.     

Production and characterization of monoclonal antibodies to rat liver arginase

Rat liver arginase was purified and five monoclonal antibodies were produced by fusion of spleen cells from a Balb/c mouse and the myeloma cell line P3-X36-Ag-U1. One, R2D19, of five antibodies belonged to the IgG2a subclass, the other four, R1D81, R1G11, R2E10, and R2G51, were of the IgG1 type. The R1D81 cross-reacted with human liver arginase. This antibody inhibited the arginase activity, competing with arginine. These results suggest that R1D81 binds to the catalytic site of arginase. The R2D19 also inhibited the enzyme activity but acted as a noncompetitive inhibitor. With the use of R1D81 and a polyclonal anti-human liver arginase antibody conjugated with alkaline phosphatase, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of human arginase. Specificity of monoclonal antibodies for rat liver arginase was examined by means of the sandwich ELISA. Eight pairs of monoclonal antibodies could form a sandwich with the arginase. Only the R2E10 could be used for both the first and the second antibody in the sandwich system. In other cases, monoclonal antibodies could not be interchanged between solid and liquid phase.     

Identification and functional characterization of natural human melanocortin 1 receptor mutant alleles in Pakistani population

Melanocortin 1 receptor (MC1R), a Gs protein‐coupled receptor of the melanocyte's plasma membrane, is a major determinant of skin pigmentation and phototype. Upon activation by α‐melanocyte stimulating hormone, MC1R triggers the cAMP cascade to stimulate eumelanogenesis. We used whole‐exome sequencing to identify causative alleles in Pakistani families with skin and hair hypopigmentation. Six MC1R mutations segregated with the phenotype in seven families, including a p.Val174del in‐frame deletion and a p.Tyr298* nonsense mutation, that were analyzed for function in heterologous HEK293 cells. p.Tyr298* MC1R showed no agonist‐induced signaling to the cAMP or ERK pathways, nor detectable agonist binding. Conversely, signaling was comparable for p.Val174del and wild‐type in HEK cells overexpressing the proteins, but binding analysis suggested impaired cell surface expression. Flow cytometry and confocal imaging studies revealed reduced plasma membrane expression of p.Val174del and p.Tyr298*. Therefore, p.Tyr298* was a total loss‐of‐function (LOF) allele, while p.Val174del displayed a partial LOF attribute.     

IL-10R Blockade during Chronic Schistosomiasis Mansoni Results in the Loss of B Cells from the Liver and the Development of Severe Pulmonary Disease

In schistosomiasis patients, parasite eggs trapped in hepatic sinusoids become foci for CD4⁺ T cell-orchestrated granulomatous cellular infiltrates. Since the immune response is unable to clear the infection, the liver is subjected to ongoing cycles of focal inflammation and healing that lead to vascular obstruction and tissue fibrosis. This is mitigated by regulatory mechanisms that develop over time and which minimize the inflammatory response to newly deposited eggs. Exploring changes in the hepatic inflammatory infiltrate over time in infected mice, we found an accumulation of schistosome egg antigen-specific IgG1-secreting plasma cells during chronic infection. This population was significantly diminished by blockade of the receptor for IL-10, a cytokine implicated in plasma cell development. Strikingly, IL-10R blockade precipitated the development of portal hypertension and the accumulation of parasite eggs in the lungs and heart. This did not reflect more aggressive Th2 cell responsiveness, increased hepatic fibrosis, or the emergence of Th1 or Th17 responses. Rather, a role for antibody in the prevention of severe disease was suggested by the finding that pulmonary involvement was also apparent in mice unable to secrete class switched antibody. A major effect of anti-IL-10R treatment was the loss of a myeloid population that stained positively for surface IgG1, and which exhibited characteristics of regulatory/anti-inflammatory macrophages. This finding suggests that antibody may promote protective effects within the liver through local interactions with macrophages. In summary, our data describe a role for IL-10-dependent B cell responses in the regulation of tissue damage during a chronic helminth infection.     

Distinct roles of CysLT1 and CysLT2 receptors in oxygen glucose deprivation-induced PC12 cell death

Cysteinyl leukotrienes are involved in ischemic brain injury, and their receptors (CysLT(1) and CysLT(2)) have been cloned. To clarify which subtype mediates the ischemic neuronal injury, we performed permanent transfection to increase CysLT(1) and CysLT(2) receptor expressions in PC12 cells. Oxygen glucose deprivation (OGD)-induced cell death was detected by Hoechst 33258 and propidium iodide fluorescent staining as well as by flow cytometry. OGD induced late phase apoptosis mainly and necrosis minimally. Over-expression of CysLT(1) receptor decreased and over-expression of CysLT(2) receptor increased OGD-induced cell death. An agonist LTD(4) (10(-7)M) also induced apoptosis, especially in CysLT(2) receptor over-expressing cells. A selective CysLT(1) receptor antagonist montelukast did not affect OGD-induced apoptosis; while non-selective CysLT receptor antagonist Bay u9773 inhibited OGD-induced apoptosis, especially in CysLT(2) receptor over-expressing cells. Thus, CysLT(1) and CysLT(2) receptors play distinct roles in OGD-induced PC12 cell death; CysLT(1) attenuates while CysLT(2) facilitates the cell death.     

Scintigraphic detection in mice of inflammatory lesions and tumours by an indium-labelled monoclonal antibody directed against Mac-1 antigen

Summary The monoclonal antibody, 3A33, directed against Mac-1 antigen which is expressed essentially on macrophages and polymorphonuclear cells, was injected i.v. into mice, as part of an attempt to visualize inflammatory lesions and tumours by external scintigraphy. The monoclonal antibody, a rat IgG2a, was conjugated with a bifunctional chelating agent, diethylenetriaminepentaacetic acid at a 1:1 molecular ratio and complexed with ¹¹¹-indium, a procedure which apparently did not alter its binding to peritoneal macrophages and provided relatively stable cell labelling. An unrelated rat IgG2a of unknown specificity radiolabelled in the same manner as 3A33 served as a control. The uptake of i.v. injected 3A33 by peritoneal macrophages was up to 50 times that of unrelated IgG2a. After i.v. inoculation, the antibody accumulated in the liver, spleen, lung, in foot-pad inflammatory reactions induced by injection of Freund's adjuvant and in experimentally grafted tumours. The 3A33: non-specific IgG2a uptake ratio in inflammatory lesions and tumours, however, was much lower than for peritoneal macrophages and was generally close to 2. This was sufficient to obtain scintigraphic images of inflammations and tumours. The images obtained after injection of 3A33 were clearly of better quality than those given by the non-specific immunoglobulin. They could be improved by subtraction of the vascular images obtained after injection of ^99m-technetium serum albumin. The labelling of Mac-1-positive blood mononuclear cells by in vitro incubation with radioactive 3A33 was not intense enough to allow scintigraphic imaging after in vivo re-infusion but seemed more selective than the injection of whole antibody in detecting inflammatory reactions. These results seem interesting in view of the potential human application to the detection inflammatory lesions and the appreciation of tumour inflammatory components. Possible improvements in the technique are discussed.This work was supported by the Fédération Nationale des Centres de Lutte contre le Cancer and the Association pour la Recherche contre le Cancer