Roche (Belgium)

To investigate the role of IL-4 in vivo in allergic asthma, we developed a murine model of allergen-induced airway inflammation. Repeated daily exposures of actively immunized C57BL/6 mice to aerosolized ovalbumin (OVA) induced a peribronchial inflammation and an increase in eosinophils and lymphocytes in bronchoalveolar-lavage (BAL) fluid. In IL-4 deficient (IL4-/-) mice, treated in the same way, there were substantially fewer eosinophils in BAL and much less peribronchial inflammation compared with wild type mice. In this model, mast cell deficient (W/Wv) mice developed a similar degree of BAL eosinophilia and peribronchial inflammation as wild type mice, demonstrating that the mast cell is not required for the induction of chronic airway inflammation. In contrast, BAL eosinophilia and airway inflammation were absent in OVA-treated MHC ClassII deficient (B6.Aa-/-) mice which lack mature CD4+ T lymphocytes. In conclusion, these results indicate that IL-4 is a central mediator of allergic airway inflammation, regulating antigen-induced eosinophil recruitment into the airways by a T cell dependent mechanism.

The leptin receptor (OB-R) mediates the weight regulatory effects of the adipocyte secreted hormone leptin (OB). Previously we have shown that the long form of OB-R, expressed predominantly in the hypothalamus, can mediate ligand-induced activation of signal transducer and activator of transcription factors 1, 3, and 5 and stimulate transcription via interleukin-6 and hematopoietin receptor responsive gene elements. Here we report that deletion and tyrosine substitution mutagenesis of OB-R identifies two distinct regions of the intracellular domain important for signaling. In addition, granulocyte-colony stimulatory factor receptor/OB-R and OB-R/granulocyte-colony stimulatory factor receptor chimeras are signaling competent and provide evidence that aggregation of two OB-R intracellular domains is sufficient for ligand-induced receptor activation. However, signaling by full-length OB-R appears to be relatively resistant to dominant negative repression by signaling-incompetent OB-R, suggesting that mechanisms exist to permit signaling by the long form of OB-R even in the presence [corrected] of excess naturally occurring short form of OB-R.

The advent of immune-checkpoint inhibitors (ICI) in modern oncology has significantly improved survival in several cancer settings. A subgroup of women with breast cancer (BC) has immunogenic infiltration of lymphocytes with expression of programmed death-ligand 1 (PD-L1). These patients may potentially benefit from ICI targeting the programmed death 1 (PD-1)/PD-L1 signaling axis. The use of tumor-infiltrating lymphocytes (TILs) as predictive and prognostic biomarkers has been under intense examination. Emerging data suggest that TILs are associated with response to both cytotoxic treatments and immunotherapy, particularly for patients with triple-negative BC. In this review from The International Immuno-Oncology Biomarker Working Group, we discuss (a) the biological understanding of TILs, (b) their analytical and clinical validity and efforts toward the clinical utility in BC, and (c) the current status of PD-L1 and TIL testing across different continents, including experiences from low-to-middle-income countries, incorporating also the view of a patient advocate. This information will help set the stage for future approaches to optimize the understanding and clinical utilization of TIL analysis in patients with BC.

Stromal tumor-infiltrating lymphocytes (sTILs) are important prognostic and predictive biomarkers in triple-negative (TNBC) and HER2-positive breast cancer. Incorporating sTILs into clinical practice necessitates reproducible assessment. Previously developed standardized scoring guidelines have been widely embraced by the clinical and research communities. We evaluated sources of variability in sTIL assessment by pathologists in three previous sTIL ring studies. We identify common challenges and evaluate impact of discrepancies on outcome estimates in early TNBC using a newly-developed prognostic tool. Discordant sTIL assessment is driven by heterogeneity in lymphocyte distribution. Additional factors include: technical slide-related issues; scoring outside the tumor boundary; tumors with minimal assessable stroma; including lymphocytes associated with other structures; and including other inflammatory cells. Small variations in sTIL assessment modestly alter risk estimation in early TNBC but have the potential to affect treatment selection if cutpoints are employed. Scoring and averaging multiple areas, as well as use of reference images, improve consistency of sTIL evaluation. Moreover, to assist in avoiding the pitfalls identified in this analysis, we developed an educational resource available at www.tilsinbreastcancer.org/pitfalls.

Tumor necrosis factor (TNF) is a central mediator of a number of important pathologies such as the systemic inflammatory response syndrome. Administration of high TNF doses induces acute anorexia, metabolic derangement, inflammation, and eventually shock and death. The in vivo effects of TNF are largely mediated by a complex network of TNF-induced cytokines and hormones acting together or antagonistically. Since TNF also induces leptin, a hormone secreted by adipocytes that modulates food intake and metabolism, we questioned the role of leptin in TNF-induced pathology. To address this question, we tested mouse strains that were defective either in leptin gene (ob/ob) or in functional leptin receptor gene (db/db), and made use of a receptor antagonist of leptin. Ob/ob and db/db mice, as well as normal mice treated with antagonist, exhibited increased sensitivity to the lethal effect of TNF. Exogenous leptin afforded protection to TNF in ob/ob mice, but failed to enhance the protective effect of endogenous leptin in normal mice. We conclude that leptin is involved in the protective mechanisms that allow an organism to cope with the potentially autoaggressive effects of its immune system.

Assessment of tumor-infiltrating lymphocytes (TILs) is increasingly recognized as an integral part of the prognostic workflow in triple-negative (TNBC) and HER2-positive breast cancer, as well as many other solid tumors. This recognition has come about thanks to standardized visual reporting guidelines, which helped to reduce inter-reader variability. Now, there are ripe opportunities to employ computational methods that extract spatio-morphologic predictive features, enabling computer-aided diagnostics. We detail the benefits of computational TILs assessment, the readiness of TILs scoring for computational assessment, and outline considerations for overcoming key barriers to clinical translation in this arena. Specifically, we discuss: 1. ensuring computational workflows closely capture visual guidelines and standards; 2. challenges and thoughts standards for assessment of algorithms including training, preanalytical, analytical, and clinical validation; 3. perspectives on how to realize the potential of machine learning models and to overcome the perceptual and practical limits of visual scoring.

The leptin receptor is a class I transmembrane protein with either a short or a long cytoplasmic domain. Using chemical cross-linking we have analyzed the binding of leptin to its receptor. Cross-linking of radiolabeled leptin to different isoforms of the leptin receptor expressed on COS-1 cells reveals leptin receptor monomer, homodimer, and oligomer complexes. Cotransfection of the long and short form of the leptin receptor did not provide any evidence for the formation of heterodimer complexes. Soluble forms consisting of either the entire extracellular domain or the two cytokine receptor homologous domains of the leptin receptor were purified to homogeneity from recombinant baculovirus-infected insect cells by leptin affinity chromatography. Gel filtration chromatography showed that these proteins exist in a dimeric form. Analysis of the complex formed between soluble leptin receptor and leptin by native polyacrylamide gel electrophoresis, and data obtained from the amino acid composition of the complex provide direct evidence that the extracellular domain of the leptin receptor binds leptin in a 1:1 ratio.

By use of a 3' extension PCR strategy, cDNA clones were isolated spanning the transmembrane region and a complete cytoplasmic domain of the human interleukin 5 receptor alpha subunit (hIL5R alpha). These cDNAs differ from previously isolated clones encoding a soluble hIL5R alpha form by a sequence switch at position 1243. When expressed in COS-1 cells, only low-affinity binding of 125I-labeled human interleukin 5 was observed. Coexpression of the hIL5R beta chain led to a 2-fold increase in binding affinity. In addition, this same cloning strategy allowed us to identify a putative second soluble isoform of hIL5R alpha. Genomic data revealed that the two soluble variants arise from either a "normal" splicing event or from the absence of splicing, whereas synthesis of the membrane-anchored form requires alternative splicing.

BACKGROUND: The establishment of a rationale for therapeutic drug monitoring for mycophenolic acid (MPA) and outlining a therapeutic window remains a challenging task in renal transplantation. Furthermore, the pharmacokinetic characteristics of free and total MPA and its glucuronides depend directly or indirectly on graft function and the type of co-administered calcineurin-inhibitor. METHODS: The authors conducted a prospective 12-month multicenter pharmacokinetic study on MPA (MPA, free MPA, free fraction MPA) and its metabolites (MPAG, Acyl-MPAG). The aim of this study was to examine the long-term pharmacokinetic characteristics of MMF when combined with tacrolimus in renal allograft recipients and to identify a possible relationship between these pharmacokinetic parameters and clinical outcome parameters. RESULTS: They have demonstrated that in renal transplant recipients MPA, free MPA, Acyl-MPAG and MPAG have a particular pharmacokinetic profile when combined with tacrolimus which differs from the combination with CsA. They could not establish a relationship between pre-dose trough concentration of MPA and its metabolites and clinical efficacy endpoints and drug-related adverse events, except for anemia. CONCLUSIONS: These findings suggest that trough plasma concentration monitoring of MPA and its metabolites might not provide a useful clinical tool for guiding MMF dose adjustments to avoid drug-related toxicity. More extensive pharmacokinetic measurements like area under the concentration curves might be necessary for routine therapeutic drug monitoring of MMF.

Limiting numbers of peripheral-blood mononuclear cells (PBMC) from melanoma patients were stimulated with irradiated autologous tumor cells in the presence of interleukins-2 and -4 and in the absence of feeder cells. The responder cells were restimulated every week. After 2 to 4 weeks, the microcultures were tested for their lytic activity against the autologous tumor cells. Significant lysis of the tumor cells was observed with a fraction of these microcultures, whereas no lysis was observed with control microcultures seeded without stimulator melanoma cells. Because our aim was to measure the precursor frequency of CTL showing specificity for the tumor, and not that of NK-like effectors that were also capable of lysing the melanoma cells, we used cold-target inhibition with an excess of NK target K562 to inhibit the NK-like activity. Microcultures whose lysis on the tumor cells was not abolished by K562 competition were observed. The specificity of these CTL clones was confirmed by the absence of lytic activity on autologous T-cell blasts. The numbers of microcultures with anti-tumor CTL activity fitted the zero-order of the Poisson distribution equation, indicating that they resulted from the activity of single T-cell clones. The frequency of anti-tumor CTL precursor cells (CTL-P) of 7 melanoma patients ranged from 1/900 to 1/33,000. Frequencies of anti-tumoral CTL-P were higher and NK-like effectors were less frequent when sorted CD8+ T lymphocytes were used as responder cells.

Binding studies were conducted to identify the anatomical location of brain target sites for OB protein, the ob gene product. 125I-labeled recombinant mouse OB protein or alkaline phosphatase-OB fusion proteins were used for in vitro and in vivo binding studies. Coronal brain sections or fresh tissue from lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats were probed to identify potential central OB protein-binding sites. We report here that recombinant OB protein binds specifically to the choroid plexus. The binding of OB protein (either radiolabeled or the alkaline phosphatase-OB fusion protein) and its displacement by unlabeled OB protein was similar in lean, obese ob/ob, and obese db/db mice as well as lean and obese Zucker rats. These findings suggest that OB protein binds with high affinity to a specific receptor in the choroid plexus. After binding to the choroid plexus receptor, OB protein may then be transported across the blood-brain barrier into the cerebrospinal fluid. Alternatively, binding of OB protein to a specific receptor in the choroid plexus may activate afferent neural inputs to the neural network that regulates feeding behavior and energy balance or may result in the clearance or degradation of OB protein. The identification of the choroid plexus as a brain binding site for OB protein will provide the basis for the construction of expression libraries and facilitate the rapid cloning of the choroid plexus OB receptor.

A detailed structure-function analysis of human interleukin 5 (hIL5) has been performed. The hIL5 receptor is composed of two different polypeptide chains, the alpha and beta subunits. The alpha subunit alone is sufficient for ligand binding, but association with the beta subunit leads to a 2- to 3-fold increase in binding affinity. The beta chain is shared with the receptors for IL3 and granulocyte/macrophage-colony-stimulating factor--hence the descriptor beta C (C for common). All hIL5 mutants were analyzed in a solid-phase binding assay for hIL5R alpha interaction and in a proliferation assay using IL5-dependent cell lines for receptor-complex activation. Most residues affecting binding to the receptor alpha subunit were clustered in a loop connecting beta-strand 1 and helix B (mutants H38A, K39A, and H41A), in beta-strand 2 (E89A and R91A; weaker effect for E90A) and close to the C terminus (T109A, E110A, W111S, and I112A). Mutations at one position, E13 (Glu13), caused a reduced activation of the hIL5 receptor complex. In the case of E13Q, only 0.05% bioactivity was detected on a hIL5-responsive subclone of the mouse promyelocytic cell line FDC-P1. Moreover, on hIL5-responsive TF1 cells, the same mutant was completely inactive and proved to have antagonistic properties. Interactions of this mutant with both receptor subunits were nevertheless indistinguishable from those of nonmutated hIL5 by crosslinking and Scatchard plot analysis of transfected COS-1 cells.

Recombinant soluble human interleukin-5 receptor alpha (shIL-5R alpha) has been expressed in COS-1 cells and in baculovirus-infected cells. The protein was purified from the supernatant by chromatography on concanavalin A-Sepharose, MonoQ, and a final gel filtration step. A chimeric fusion receptor protein (hIL-5R alpha-h gamma 3) was constructed by fusion of the cDNA corresponding to the shIL-5R alpha to the cDNA corresponding to the Fc part of the human IgG C gamma 3 chain, and was expressed in baculovirus-infected insect cells. The chimeric receptor was secreted as a disulfide-linked homodimer, and was purified by protein G affinity chromatography. In a solid-phase binding assay the shIL-5R alpha and the bivalent hIL-5R alpha-h gamma 3 were found to bind hIL-5 with a similar affinity, corresponding to the membrane-bound, low affinity hIL-5R alpha. SDS-polyacrylamide gel electrophoresis of shIL-5R alpha cross-linked to radiolabeled hIL-5, suggested that one shIL-5R alpha molecule binds to one hIL-5 homodimer molecule. Gel filtration studies of the complex formed between the shIL-5R alpha and hIL-5 pointed toward the same stoichiometry of binding. The formation of such a complex could be observed by electrophoresis in native gels. Immunoaffinity chromatography using a non-neutralizing monoclonal antibody directed against hIL-5, followed by size column chromatography, allowed the purification of the complex. The data obtained from the amino acid analysis of the constituents of the complex blotted from an SDS-polyacrylamide gel, and from the amino acid composition of the complex blotted from a native polyacrylamide gel, provided direct evidence that the shIL-5R alpha binds the hIL-5 dimer in a 1:1 ratio.

The T cell product interleukin 5 (IL-5) has been shown to be a key factor in the development and the maturation of the eosinophilic cell lineage. We report here on the detection of human IL-5 receptors on eosinophilic sublines of the promyelocytic leukemia HL-60. Sodium butyrate, which initiates differentiation to mature eosinophils, also induces the appearance of high affinity (Kd 1-5 X 10(-11) M) IL-5 binding sites on these cells. The receptors are specific for IL-5, since binding of radiolabeled ligand can only be inhibited with homologous or murine IL-5 and not by other cytokines. We further show that the receptors are functional, since IL-5 can stimulate the proliferation of these cells. Affinity crosslinking of surface-bound 125I human IL-5 or 35S mouse IL-5 identified two membrane polypeptides of approximately 60 and approximately 130 kD to which IL-5 is closely associated. The presence of granulocyte/macrophage-colony-stimulating factor or tumor necrosis factor during butyrate induction decreased the expression of IL-5 binding sites compared with control cultures. The identification and characterization of human IL-5 receptors on HL-60 sublines should provide new insight into the role of this cytokine in eosinophil differentiation.

The mechanism by which tumour necrosis factors (TNF and lymphotoxin, also called TNF alpha and TNF beta respectively) exert their cytotoxic activity on many malignant cells, remains largely unknown. Furthermore, the broad array of differentiation (gene induction) and mitogenic activities towards many primary cells is still a subject of intensive investigation. TNF is an important mediator in inflammation, immune responses and infection-related phenomena and these activities contribute to the severe toxicity seen when TNF is used as an anticancer agent. The first step in the mechanism of action is the specific binding of the ligand to its receptors and dissection of the molecular mechanism involved in this interaction is the subject of this review. The reasons for the interest in this aspect are obvious: first, the development of strong antagonistic TNF analogues can be useful in dampening the potentially lethal or debilitating effects of an overproduction of the cytokine (as in septic shock or rheumatoid arthritis). Secondly, since two distinct TNF receptors exist, construction of TNF muteins that distinguish between both types may lead to derivatives of this pleiotropic agent with a more restricted biological activity pattern. Ideally, one would like to develop a TNF mutant that has retained its cytotoxic action on tumour cells without inducing the deleterious systemic toxicity. Such an optimized TNF molecule could become a potent anticancer agent.

T-helper 2 (Th2)-like cells are thought to play a crucial role in the pathogenesis of the eosinophilic airway inflammation observed in asthma. In a murine model of allergen-induced airway eosinophilia and bronchial hyperresponsiveness (BHR), we have shown that interleukin (IL)-12 can suppress antigen-induced airway changes despite the presence of circulating specific IgE. In the present study, we investigated the role of interferon-gamma (IFN-gamma) in the inhibitory effects of IL-12 on allergic airway inflammation. Repeated daily exposure of actively immunized mice to aerosolized ovalbumin (OVA), as compared with aerosolized saline (SAL), induced a significant increase in bronchoalveolar lavage fluid (BALF) eosinophilia and OVA-specific serum IgE in both IFN-gamma-receptor-deficient (IFN-gammaR KO) and wild-type mice. As compared with placebo (PLAC), administration of recombinant murine IL-12 (rmIL-12) during the daily aerosol exposure (but not at the time of immunization) significantly inhibited BALF eosinophilia in both IFN-gammaR KO mice and wild-type controls, without influencing the production of specific IgE. In contrast, administration of rmIL-12 during the active immunization inhibited both BALF eosinophilia and specific IgE in wild-type mice as compared with littermates given PLAC; however, treatment with rmIL-12 during immunization, in comparison with PLAC, caused a significant increase in BALF eosinophilia and specific IgE in IFN-gammaR KO mice. These results demonstrate that inhibition of the allergen-induced eosinophil influx in murine airways by IL-12 is IFN-gamma-dependent during the initial sensitization, but becomes IFN-gamma-independent during the secondary response.

T helper 2 (Th2)-like cells are thought to play a crucial role in the pathogenesis of atopic asthma. In this study, we attempted to evaluate the in vivo effect of suppressing Th2 cell development on allergen-induced airway changes. Repeated exposure of actively sensitized C57Bl/6 mice to aerosolized ovalbumin (OA) causes, in comparison to saline-exposed control animals, synthesis of specific IgE, increase of eosinophils in bronchoalveolar lavage fluid and airway hyperresponsiveness. These effects are not observed in OA-exposed, sensitized IL-4-knockout mice. Likewise, these effects are inhibited in OA-exposed C57Bl/6 mice treated with IL-12 during initial antigen exposure. These results suggest that suppressing Th2 cell development in vivo might have profound inhibitory effects on allergen-induced airway changes.

Leptin, a fat secreted hormone, regulates ingestive behaviour and energy balance by binding to a specific receptor. Using site-directed mutagenesis, we screened for single amino acid residues in human leptin which are critical for receptor binding and biological activity. Here we report that one of these mutants has in vivo antagonistic properties. An Arg to Gln substitution at position 128 of human leptin does not affect receptor binding but knocks out biological activity. Repeated injection of R128Q in normal C57BL/6J mice results in a progressive increase in body weight. This demonstrates that R128Q is able to interfere with the negative feedback control of endogenous leptin. This mutant could be of therapeutic use for wasting disorders, such as anorexia and cachexia, where weight gain would be beneficial.

OBJECTIVES: Obesity is a common condition in type 2 diabetic patients. Treating obesity may enhance hypoglycemic treatment and contribute to the reduction of long-term microvascular and macrovascular complications. Orlistat reduces cardiovascular risk factors in obese type 2 diabetic patients. The objectives of this study were to estimate the long-term clinical consequences of this weight loss and the resulting cost-effectiveness of treating obese type 2 diabetic patients with orlistat. RESEARCH DESIGN AND METHODS: A Markov model was developed to predict, over a 10-year period, the complication rates and mortality with and without a 2-year orlistat treatment, assuming a 5-year catch-up period after treatment. A stepwise approach was used to obtain the clinical data. First, the impact of weight loss with orlistat on HbA(1c), blood pressure, and cholesterol was assessed; then, the impact on mortality and micro- and macrovascular complications of decreasing these risk factors was applied. Four subgroups were studied based on the presence of risk factors. RESULTS: Cost-effectiveness varies between 3,462 Euro/life-year gained (LYG) for obese diabetic patients with hypertension and hypercholesterolemia and 19,986 Euro/LYG for obese diabetic patients without other risk factors. The latter result is not robust according to sensitivity analyses. CONCLUSIONS: Our results suggest that orlistat is cost-effective in the management of obese type 2 diabetic patients, especially in those with the presence of hypercholesterolemia and/or hypertension. Evidence on longer-term benefits of orlistat (>2 years) will be of importance for future decision-making.

Tumor necrosis factor (TNF) is a central mediator of a number of important pathologies such as the systemic inflammatory response syndrome. Administration of high TNF doses induces acute anorexia, metabolic derangement, inflammation, and eventually shock and death. The in vivo effects of TNF are largely mediated by a complex network of TNF-induced cytokines and hormones acting together or antagonistically. Since TNF also induces leptin, a hormone secreted by adipocytes that modulates food intake and metabolism, we questioned the role of leptin in TNF-induced pathology. To address this question, we tested mouse strains that were defective either in leptin gene (ob/ob) or in functional leptin receptor gene (db/db), and made use of a receptor antagonist of leptin. Ob/ob and db/db mice, as well as normal mice treated with antagonist, exhibited increased sensitivity to the lethal effect of TNF. Exogenous leptin afforded protection to TNF in ob/ob mice, but failed to enhance the protective effect of endogenous leptin in normal mice. We conclude that leptin is involved in the protective mechanisms that allow an organism to cope with the potentially autoaggressive effects of its immune system.