Giulia Petroni

Allergological in vitro and in vivo evaluation of patients with hypersensitivity reactions to infliximab.

The administration of biological agents is potentially affected by IgE‐mediated infusion reactions.

Drug-Specific Th2 Cells and IgE Antibodies in a Patient with Anaphylaxis to Rituximab

Rituximab (RTX) is currently used in the treatment of lymphoproliferative diseases and of several rheumatologic disorders and is a frequent cause of acute infusion reactions, usually classified as cytokine release syndrome (CRS). Some infusion reactions to RTX raise concern for immediate type I hypersensitivity, even if to date RTX-specific IgE antibodies have not been reported. To improve knowledge of the mechanisms of reactions to RTX, we investigated humoral and cellular immune responses to this drug in a patient suffering from rheumatoid arthritis who displayed two immediate infusion-related reactions. RTX-exposed tolerant patients and healthy untreated subjects were used as controls. Non-isotype-specific and IgE anti-RTX antibodies were positive in the serum samples collected from the reactive patient but not in those from the control groups. Only the reactive patient also displayed skin testing positivity with RTX. More importantly, RTX-stimulated peripheral blood mononuclear cells from the reactive patient, but not from the controls, displayed a dose-dependent proliferative response associated with a Th2 cytokine production profile. Our results show the presence of RTX-specific Th2-type cells and IgE antibodies, thus suggesting that type I hypersensitivity may be an additional mechanism to CRS in the development of RTX reactions.

The conformational state of hERG1 channels determines integrin association, downstream signaling, and cancer progression

Whether hERG1 potassium channels promote proliferation or metastasis in breast cancer cells depends on channel conformation. Channeling proliferation or metastasis The hERG1 potassium channel is best known for its role in repolarizing excitable cells such as cardiomyocytes, but the abundance of this cardiac channel is aberrantly high in cancer cells. Becchetti et al. investigated the interaction of hERG1 with the β1 integrin subunit, a member of a family of adhesion molecules. Forms of hERG1 with mutations that fixed the channel in the open conformation more weakly interacted with β1 integrin in cells and enhanced proliferation when expressed in breast cancer cells injected into mice. However, K+ flow through open hERG1 channels enhanced the activation of FAK downstream of β1 integrin and promoted metastasis in breast cancer cells injected into mice. Thus, whether hERG1 promotes proliferation or metastasis in cancer cells depends on the conformation of the channel and suggests that hERG1 inhibitors that are tailored to the channel conformation could be used to prevent different aspects of tumor progression. Ion channels regulate cell proliferation, differentiation, and migration in normal and neoplastic cells through cell-cell and cell–extracellular matrix (ECM) transmembrane receptors called integrins. K+ flux through the human ether-à-go-go–related gene 1 (hERG1) channel shapes action potential firing in excitable cells such as cardiomyocytes. Its abundance is often aberrantly high in tumors, where it modulates integrin-mediated signaling. We found that hERG1 interacted with the β1 integrin subunit at the plasma membrane of human cancer cells. This interaction was not detected in cardiomyocytes because of the presence of the hERG1 auxiliary subunit KCNE1 (potassium voltage-gated channel subfamily E regulatory subunit 1), which blocked the β1 integrin–hERG1 interaction. Although open hERG1 channels did not interact as strongly with β1 integrins as did closed channels, current flow through hERG1 channels was necessary to activate the integrin-dependent phosphorylation of Tyr397 in focal adhesion kinase (FAK) in both normal and cancer cells. In immunodeficient mice, proliferation was inhibited in breast cancer cells expressing forms of hERG1 with impaired K+ flow, whereas metastasis of breast cancer cells was reduced when the hERG1/β1 integrin interaction was disrupted. We conclude that the interaction of β1 integrins with hERG1 channels in cancer cells stimulated distinct signaling pathways that depended on the conformational state of hERG1 and affected different aspects of tumor progression.

Circulating T cells to infliximab are detectable mainly in treated patients developing anti-drug antibodies and hypersensitivity reactions

Antibodies recognizing infliximab (IFX) may develop in a proportion of treated patients, leading to loss of response or hypersensitivity reactions (HRs). T cell response to IFX has been poorly investigated. This paper was addressed to detect IFX‐specific T cells in treated patients with inflammatory diseases developing, or not, anti‐drug antibodies (ADA) and to correlate the presence of specific T cells with the clinical outcomes of the treatment. A co‐culture system of IFX‐loaded dendritic cells and purified autologous CD4+ T cells was used to detect memory T cells in 32 ADA+ and 39 ADA– IFX‐treated patients and control groups. The cytokine profile of IFX‐specific T cells was also studied in culture supernatants. IFX‐specific cell proliferation was detected mainly in cells from ADA+ patients, irrespective of their different diseases. HR patients displayed higher T cell proliferation than non‐responder and tolerant patients. A mixed [interferon (IFN)‐γ, interleukin (IL)‐13, IL‐10] cytokine profile was shown in cells from ADA+ patients, while IL‐10 was the most frequently detected cytokine in the supernatants of cultures from ADA‐ patients. Immunoglobulin (Ig)E+ADA+ patients with previous HRs exhibited a more pronounced type 2 profile than IgE–ADA+ patients. This work provides evidence that IFX‐specific circulating T cells are detectable mainly in ADA+ patients with HRs, regardless of their disease. The IFX‐induced cytokine pattern partially correlates with the ADA isotype.

New gold carbene complexes as candidate anticancer agents

Three structurally related gold(I) carbene complexes with bulky hydrophobic ligands i.e. 1–3 were investigated in solution for further consideration as candidate anticancer agents. Cytotoxic assays were subsequently conducted on bone marrow-derived preosteoclast cell line of human origin (FLG 29.1) and human colon cancer cells (HCT-116). A far greater cytotoxic activity was measured for compound 1 against HCT-116 cells compared to 2 and 3; conversely, all compounds were highly and similarly active against FLG 29.1 cells. Results obtained for the reaction of complexes 1 and 2 with RNase A documented the occurrence of a weak interaction with this model protein and the formation of a tiny amount of the corresponding adduct. Moreover, a certain reactivity of the complex 2 was also detected toward GSH. The general implications of the obtained results are discussed.

Assays and strategies for immunogenicity assessment of biological agents.

Enabling Technology, Genomics, Proteomics Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics

Circulating T cells to infliximab are mainly detectable in treated patients developing anti-drug antibodies and hypersensitivity reactions.

Antibodies recognizing infliximab (IFX) may develop in a proportion of treated patients, leading to loss of response or hypersensitivity reactions (HRs). T cell response to IFX has been poorly investigated. This paper was addressed to detect IFX‐specific T cells in treated patients with inflammatory diseases developing, or not, anti‐drug antibodies (ADA) and to correlate the presence of specific T cells with the clinical outcomes of the treatment. A co‐culture system of IFX‐loaded dendritic cells and purified autologous CD4+ T cells was used to detect memory T cells in 32 ADA+ and 39 ADA– IFX‐treated patients and control groups. The cytokine profile of IFX‐specific T cells was also studied in culture supernatants. IFX‐specific cell proliferation was detected mainly in cells from ADA+ patients, irrespective of their different diseases. HR patients displayed higher T cell proliferation than non‐responder and tolerant patients. A mixed [interferon (IFN)‐γ, interleukin (IL)‐13, IL‐10] cytokine profile was shown in cells from ADA+ patients, while IL‐10 was the most frequently detected cytokine in the supernatants of cultures from ADA‐ patients. Immunoglobulin (Ig)E+ADA+ patients with previous HRs exhibited a more pronounced type 2 profile than IgE–ADA+ patients. This work provides evidence that IFX‐specific circulating T cells are detectable mainly in ADA+ patients with HRs, regardless of their disease. The IFX‐induced cytokine pattern partially correlates with the ADA isotype.

A novel allergen-adjuvant conjugate suitable for specific immunotherapy of respiratory allergy.

BACKGROUND Several approaches to find a better adjuvant, focus immunomodulation, and reduce allergenicity are under investigation to improve the efficacy and safety of specific immunotherapy. OBJECTIVE We performed an investigation of the in vitro and in vivo effects of a purified allergen chemically conjugated to a novel 8-OH modified adenine as an adjuvant. METHODS Purified group 2 major allergen from house dust mite chemically conjugated to 4-(6-amino-9-benzyl-8-hydroxy-9H-purin-2-ylsulfanyl)-butyric acid succinimidyl ester was analyzed by using mass spectrometry. The adduct (nDer p 2-Conj) was assayed for Toll-like receptor activation on transfected HEK293 cells, stimulation of innate cells, and effects on the functional phenotype of specific T-cell lines and clones by means of flow cytometry, real-time PCR, and expression of TH-related transcription factors. Lung cells and sera of nDer p 2-Conj-sensitized C57Bl/6 mice were studied by means of cytology, histology, real-time PCR, and ELISA. RESULTS nDer p 2-Conj stimulated IL-12 and IFN-α production from monocytes and plasmacytoid dendritic cells, respectively, retaining the ability to trigger Toll-like receptor 7 exclusively, and expanded human allergen-specific lymphocytes with reduced ability to produce T(H)2-related cytokines and increased IFN-γ levels, as based on GATA-3/T-bet expression. In vivo adduct-sensitized mice exhibited reduced eosinophil infiltration and IL-13 expression in the airways, IFN-γ upregulation together with IgE downregulation, and an increase in allergen-specific IgG(2a) levels in sera. The conjugate exhibited reduced ability to activate human FcεRI(+) cells without inducing T(H)17 cells or autoantibodies. CONCLUSIONS The codelivery of an allergen with a modified adenine as a conjugate inducing modulatory cytokines from innate cells redirects in vitro and in vivo pathogenic TH2 responses without eliciting harmful effects.

hERG1 positivity and Glut-1 negativity identifies high-risk TNM stage I and II colorectal cancer patients, regardless of adjuvant chemotherapy.

Background The identification of early-stage colorectal cancer (CRC) with high risk of progression is one major clinical challenge, mainly due to lack of validated biomarkers. The aims of the present study were to analyze the prognostic impact of three molecular markers belonging to the ion channels and transporters family: the ether-à-go-go-related gene 1 (hERG1) and the calcium-activated KCa3.1 potassium channels, as well as the glucose transporter 1 (Glut-1); and to define the impact of adjuvant chemotherapy in conjunction with the abovementioned biomarkers, in a cohort of radically resected stage I–III CRC patients. Patients and methods The expressions of hERG1, KCa3.1, and Glut-1 were tested by immunohistochemistry on 162 surgical samples of nonmetastatic, stage I–III CRC patients. The median follow-up was 32 months. The association between biological markers, clinicopathological features, and survival outcomes was investigated by evaluating both disease-free survival and overall survival. Results Although no prognostic valence emerged for KCa3.1, evidence of a negative impact of hERG1 expression on survival outcomes was provided. On the contrary, Glut-1 expression had a positive impact. According to the results of the multivariate analysis, patients were stratified in four risk groups, based on TNM stage and hERG1/Glut-1 expression. After adjusting for adjuvant therapy, stage I and II, Glut-1-negative, and hERG1-positive patients showed the worst survival experience. Conclusion This study strongly indicates that the combination of hERG1 positivity and Glut-1 negativity behaves as a prognostic biomarker in radically resected CRC patients. This combination identifies a group of stage I and II CRC patients with a bad prognosis, even worse than that of stage III patients, regardless of adjuvant therapy accomplishment.

Treatment with 8-OH-modified adenine (TLR7 ligand)-allergen conjugates decreases T helper type 2-oriented murine airway inflammation

A strategy to improve allergen‐specific immunotherapy is to employ new adjuvants stably linked to allergens. The study is addressed to evaluate the in vivo and in vitro effects of allergens [natural Dermatophagoides pteronyssinus 2 (nDer p 2) and ovalbumin (OVA)] chemically bound to an 8‐OH‐modified adenine. Humoral and cellular responses were analysed in allergen‐sensitized and challenged mice by using conjugates (Conj) in a therapeutic setting. The in vitro activity of the conjugates on cytokine production induced by bone marrow dendritic cells and the co‐culture system was also investigated. The nDer p 2‐Conj treatment in nDer p 2‐primed and challenged BALB/c mice reduced the numbers of eosinophils in bronchoalveolar lavage fluid and lung, airway allergen‐driven interleukin‐13 (IL‐13) production in lung mononuclear cells and IgE, in comparison with nDer p 2‐treated mice. The increase of IgG2a paralleled that of interferon‐γ (IFN‐γ) and IL‐10 in allergen‐stimulated spleen cells. Similar effects were elicited by treatment with OVA‐Conj in an OVA‐driven BALB/c model. The nDer p 2‐Conj or OVA‐Conj redirected memory T helper type 2 cells towards the production of IL‐10 and IFN‐γ also in C57BL/6 mice and when subcutaneously administered. Interleukin‐10, IL‐12 and IL‐27 were produced in vitro by Conj‐stimulated bone marrow dendritic cells, whereas IL‐10 and IFN‐γ were up‐regulated in co‐cultures of CD11c+ and CD4+ T cells from Conj‐treated mice stimulated with allergen. Cytofluorometric analysis indicated that the Conj expanded IFN‐γ‐ and IL‐10‐ producing memory T cells. The Conj effects on IL‐10−/− and IL‐12−/− mice confirmed the role of IL‐10 and IFN‐γ in inducing a protective and balanced redirection the T helper type 2‐mediated airway inflammation.