TRPA1 Expression and Pathophysiology in Immune Cells.

The non-selective cation channel TRPA1 is best known as a broadly-tuned sensor expressed in nociceptive neurons, where it plays key functions in chemo-, thermo-, and mechano-sensing. However, in this review we illustrate how this channel is expressed also in cells of the immune system. TRPA1 has been detected, mainly with biochemical techniques, in eosinophils, mast cells, macrophages, dendritic cells, T cells, and B cells, but not in neutrophils. Functional measurements, in contrast, remain very scarce. No studies have been reported in basophils and NK cells. TRPA1 in immune cells has been linked to arthritis (neutrophils), anaphylaxis and atopic dermatitis (mast cells), atherosclerosis, renal injury, cardiac hypertrophy and inflammatory bowel disease (macrophages), and colitis (T cells). The contribution of TRPA1 to immunity is dual: as detector of cell stress, tissue injury, and exogenous noxious stimuli it leads to defensive responses, but in conditions of aberrant regulation it contributes to the exacerbation of inflammatory conditions. Future studies should aim at characterizing the functional properties of TRPA1 in immune cells, an essential step in understanding its roles in inflammation and its potential as therapeutic target.

Activation of Drosophila melanogaster TRPA1 Isoforms by Citronellal and Menthol.

BACKGROUND: The transient receptor potential ankyrin 1 (TRPA1) cation channels function as broadly-tuned sensors of noxious chemicals in many species. Recent studies identified four functional TRPA1 isoforms in Drosophila melanogaster (dTRPA1(A) to (D)), but their responses to non-electrophilic chemicals are yet to be fully characterized. METHODS: We determined the behavioral responses of adult flies to the mammalian TRPA1 non-electrophilic activators citronellal and menthol, and characterized the effects of these compounds on all four dTRPA1 channel isoforms using intracellular Ca2+ imaging and whole-cell patch-clamp recordings. RESULTS: Wild type flies avoided citronellal and menthol in an olfactory test and this behavior was reduced in dTrpA1 mutant flies. Both compounds activate all dTRPA1 isoforms in the heterologous expression system HEK293T, with the following sensitivity series: dTRPA1(C) = dTRPA1(D) > dTRPA1(A) ≫ dTRPA1(B) for citronellal and dTRPA1(A) > dTRPA1(D) > dTRPA1(C) > dTRPA1(B) for menthol. CONCLUSIONS: dTrpA1 was required for the normal avoidance of Drosophila melanogaster towards citronellal and menthol. All dTRPA1 isoforms are activated by both compounds, but the dTRPA1(B) is consistently the least sensitive. We discuss how these findings may guide further studies on the physiological roles and the structural bases of chemical sensitivity of TRPA1 channels.

The Citrus Flavonoid Hesperetin Has an Inadequate Anti-Arrhythmic Profile in the ΔKPQ NaV1.5 Mutant of the Long QT Type 3 Syndrome.

Type 3 long QT syndromes (LQT3) are associated with arrhythmogenic gain-of-function mutations in the cardiac voltage-gated Na+ channel (hNaV1.5). The citrus flavanone hesperetin (HSP) was previously suggested as a template molecule to develop new anti-arrhythmic drugs, as it blocks slowly-inactivating currents carried by the LQT3-associated hNaV1.5 channel mutant R1623Q. Here we investigated whether HSP also has potentially beneficial effects on another LQT3 hNaV1.5 channel variant, the ΔKPQ, which is associated to lethal ventricular arrhythmias. We used whole-cell patch-clamp to record Na+ currents (INa) in HEK293T cells transiently expressing hNaV1.5 wild type or ΔKPQ mutant channels. HSP blocked peak INa and the late INa carried by ΔKPQ mutant channels with an effective concentration of ≈300 µM. This inhibition was largely voltage-independent and tonic. HSP decreased the rate of inactivation of ΔKPQ channels and, consequently, was relatively weak in reducing the intracellular Na+ load in this mutation. We conclude that, although HSP has potential value for the treatment of the R1623Q LQT3 variant, this compound is inadequate to treat the LQT3 associated to the ΔKPQ genetic variant. Our results underscore the precision medicine rationale of better understanding the basic pathophysiological and pharmacological mechanisms to provide phenotype- genotype-directed individualization of treatment.

Obtención de una versión IgG1 de ratón del rituximab para detectar la molécula CD20 humana / Obtaining a mouse IgG1 version of rituximab to detect the human CD20 molecule

Introducción: El rituximab, anticuerpo quimérico que reconoce la molécula CD20 humana, se ha utilizado en el tratamiento de diversos trastornos linfoproliferativos de células B. Para la selección de los potenciales beneficiarios del tratamiento con rituximab se han desarrollado técnicas que, mediante el uso de anticuerpos monoclonales, detectan la presencia del CD20 en los linfocitos de estos pacientes. Objetivo: Obtener y caracterizar un anticuerpo recombinante IgG1 de ratón específico para la molécula CD20 humana, que contenga las regiones variables del anticuerpo rituximab. Métodos: Para la expresión estable del anticuerpo recombinante se empleó la transducción lentiviral de células de embrión de riñón humano (HEK293). La caracterización inmunoquímica del anticuerpo se realizó por la técnica de Western Blot y su capacidad de reconocimiento de la molécula CD20 humana se evaluó por citometría de flujo e inmunohistoquímica. Resultados: Se obtuvo el anticuerpo 1F5 que reconoce, por citometría de flujo, la molécula CD20 en líneas celulares humanas de origen linfoide, así como en células de sangre periférica de humanos sanos y pacientes con trstornos linfoproliferativos de células B. Sin embargo, la técnica de inmunohistoquímica solo permitió detectar con este anticuerpo la molécula CD20 en tejidos frescos, no así en los embebidos en parafina. Conclusiones: Este trabajo sugiere las potencialidades del uso del anticuerpo 1F5 para las mediciones de la expresión de CD20 por citometría de flujo en pacientes con leucemias B o linfomas B avanzados en fase de leucemización. Esto complementaría los estudios para la selección apropiada de pacientes para el tratamiento con el rituximab(AU)

Introduction: Rituximab, chimeric antibody specific for human CD20 molecule, has been widely used in the treatment of several B-cell linfoproliferative disorders. For the selection of patients with the greatest potential to benefit from the therapy with rituximab, a number of techniques using monoclonal antibodies have been developed to detect the CD20 molecule. Objective: To obtain and to characterize a mouse IgG1 recombinant antibody, specific for human CD20, that contains the variable regions of rituximab. Methods: The lentiviral transduction of human embryonic kidney cells (HEK293) was used for the stable expression of the recombinant antibody. The immunochemical characterization of the antibody was performed by Western Blot and the recognition of CD20 was evaluated by immunohistochemistry and flow cytometry. Results: We generated the antibody 1F5, able to recognize by flow cytometry the CD20 molecule expressed on lymphoid human cell lines, as well as peripheral blood mononuclear cells from healthy donors and patients with B-cell lymphoproliferative disorders. However, 1F5 antibody detected the CD20 molecule on fresh tissues, but not on formalin-fixed paraffin embedded tissues,by immunohistochemistry. Conclusions: This work suggests the potential use of 1F5 antibody for the measurement of CD20 expression by flow cytometry in patients with B-cell leukemias or B-cell lymphomas in phase of leukemization. This could complement the studies to ensure the appropriate selection of patients for the treatment with rituximab(AU)

Antitumor effects of the GM3(Neu5Gc) ganglioside-specific humanized antibody 14F7hT against Cmah-transfected cancer cells.

The GM3(Neu5Gc) ganglioside represents a tumor-specific antigen that is considered a promising target for cancer immunotherapy. We previously demonstrated that the humanized antibody 14F7hT, specific for this ganglioside, exhibited significant antitumor effects in preclinical hematological tumor models. As this antibody recognizes human tumor tissues from several origins, we addressed its potential effect on different tumor types. The use of cell lines for testing GM3(Neu5Gc)-targeting strategies, in particular for human malignancies, is complicated by the absence in humans of functional cytidine monophospho-N-acetyl-neuraminic acid hydroxylase (CMAH), the enzyme required for Neu5Gc sialic acid biosynthesis. Quantitative flow cytometry revealed the absence of surface GM3(Neu5Gc) in several human but also mouse cell lines, in the last case due to low expression of the enzyme. Hypoxia-induced expression of this ganglioside on human SKOV3 cells was observed upon culture in Neu5Gc-containing medium without evidence for CMAH-independent biosynthesis. However, only transfection of the mouse Cmah gene into human SKOV3 and mouse 3LL cells induced a stable expression of GM3(Neu5Gc) on the cancer cell surface, resulting in effective models to evaluate the antitumor responses by 14F7hT in vitro and in vivo. This antibody exerted antibody-dependent cell-mediated cytotoxicity (ADCC) and in vivo antitumor effects on these Cmah-transfected non-hematological tumors from both mouse and human origin. These results contribute to validate GM3(Neu5Gc) as a relevant target for cancer immunotherapy and reinforces the value of 14F7hT as a novel anti-cancer drug.

Expression and Functional Role of TRPV4 in Bone Marrow-Derived CD11c+ Cells.

The increase in cytosolic Ca2+ is essential in key effector functions of dendritic cells (DCs), including differentiation, maturation, cytokine expression, and phagocytosis. Although several Ca2+-permeable ion channels have been described in DCs, the contribution of transient receptor potential (TRP) channels remains poorly understood. Here, we investigated whether TRPV4 plays a role in the differentiation, maturation, and phagocytosis of granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced mouse bone marrow-derived cells (BMDCs). Using intracellular Ca2+ imaging experiments, we found that TRPV4 was functionally expressed in the plasma membrane of immature CD11c+ BMDCs and that its activity and expression were downregulated in CD11c+ BMDCs matured with lipopolysaccharide (LPS). Comparative analysis of the GM-CSF-stimulated cells showed that Trpv4 knockout and wild-type bone marrow cultures had a similar distribution of differentiated cells, generating a heterogenous culture population rich in CD11c+, CD11b+ cells, and low levels of F4/80+ cells. The lack of TRPV4 did not prevent the LPS-induced nuclear translocation of NF-κB, the upregulation of the proinflammatory cytokines IL-6 and IL-12, or the upregulation of the maturation markers CD40, CD80, and CD86. In contrast, TRPV4-deficient CD11c+ BMDCs exhibited a significantly reduced endocytic capacity of IgG-coated beads, but the internalization of uncoated beads in the absence of TRPV4 was not affected. Taken together, our results demonstrate that TRPV4 was dispensable in the differentiation and maturation of mouse CD11c+ BMDCs but contributed to the mechanism underlying Fc receptor-mediated phagocytosis. Overall, our results further strengthen the role of TRPV4 in immune-related processes.

Mouse TRPA1 function and membrane localization are modulated by direct interactions with cholesterol.

The cation channel TRPA1 transduces a myriad of noxious chemical stimuli into nociceptor electrical excitation and neuropeptide release, leading to pain and neurogenic inflammation. Despite emergent evidence that TRPA1 is regulated by the membrane environment, it remains unknown whether this channel localizes in membrane microdomains or whether it interacts with cholesterol. Using total internal reflection fluorescence microscopy and density gradient centrifugation we found that mouse TRPA1 localizes preferably into cholesterol-rich domains and functional experiments revealed that cholesterol depletion decreases channel sensitivity to chemical agonists. Moreover, we identified two structural motifs in transmembrane segments 2 and 4 involved in mTRPA1-cholesterol interactions that are necessary for normal agonist sensitivity and plasma membrane localization. We discuss the impact of such interactions on TRPA1 gating mechanisms, regulation by the lipid environment, and role of this channel in sensory membrane microdomains, all of which helps to understand the puzzling pharmacology and pathophysiology of this channel.

The citrus flavanone hesperetin preferentially inhibits slow-inactivating currents of a long QT syndrome type 3 syndrome Na+ channel mutation.

BACKGROUND AND PURPOSE: The citrus flavanone hesperetin has been proposed for the treatment of several human pathologies, but its cardiovascular actions remain largely unexplored. Here, we evaluated the effect of hesperetin on cardiac electrical and contractile activities, on aortic contraction, on the wild-type voltage-gated NaV 1.5 channel, and on a channel mutant (R1623Q) associated with lethal ventricular arrhythmias in the long QT syndrome type 3 (LQT3). EXPERIMENTAL APPROACH: We used cardiac surface ECG and contraction force recordings to evaluate the effects of hesperetin in rat isolated hearts and aortic rings. Whole-cell patch clamp was used to record NaV 1.5 currents (INa ) in rat ventricular cardiomyocytes and in HEK293T cells expressing hNaV 1.5 wild-type or mutant channels. KEY RESULTS: Hesperetin increased the QRS interval and heart rate and decreased the corrected QT interval and the cardiac and aortic contraction forces at concentrations equal or higher than 30 µmol·L-1 . Hesperetin blocked rat and human NaV 1.5 channels with an effective inhibitory concentration of ≈100 µmol·L-1 . This inhibition was enhanced at depolarized holding potentials and higher stimulation frequency and was reduced by the disruption of the binding site for local anaesthetics. Hesperetin increased the rate of inactivation and preferentially inhibited INa during the slow inactivation phase, these effects being more pronounced in the R1623Q mutant. CONCLUSIONS AND IMPLICATIONS: Hesperetin preferentially inhibits the slow inactivation phase of INa , more markedly in the mutant R1623Q. Hesperetin could be used as a template to develop drugs against lethal cardiac arrhythmias in LQT3.

Differential effects of lipopolysaccharide on mouse sensory TRP channels.

Acute neurogenic inflammation and pain associated to bacterial infection have been traditionally ascribed to sensitization and activation of sensory nerve afferents secondary to immune cell stimulation. However, we recently showed that lipopolysaccharides (LPS) directly activate the Transient Receptor Potential channels TRPA1 in sensory neurons and TRPV4 in airway epithelial cells. Here we investigated whether LPS activates other sensory TRP channels expressed in sensory neurons. Using intracellular Ca2+ imaging and patch-clamp we determined the effects of LPS on recombinant TRPV1, TRPV2, TRPM3 and TRPM8, heterologously expressed in HEK293T cells. We found that LPS activates TRPV1, although with lower potency than for TRPA1. Activation of TRPV1 by LPS was not affected by mutations of residues required for activation by electrophilic agents or by diacylglycerol and capsaicin. On the other hand, LPS weakly activated TRPM3, activated TRPM8 at 25 °C, but not at 35 °C, and was ineffective on TRPV2. Experiments performed in mouse dorsal root ganglion (DRG) neurons revealed that genetic ablation of Trpa1 did not abolish the responses to LPS, but remain detected in 30% of capsaicin-sensitive cells. The population of neurons responding to LPS was dramatically lower in double Trpa1/Trpv1 KO neurons. Our results show that, in addition to TRPA1, other TRP channels in sensory neurons can be targets of LPS, suggesting that they may contribute to trigger and regulate innate defenses against gram-negative bacterial infections.

TRPV4 activation triggers protective responses to bacterial lipopolysaccharides in airway epithelial cells.

Lipopolysaccharides (LPS), the major components of the wall of gram-negative bacteria, trigger powerful defensive responses in the airways via mechanisms thought to rely solely on the Toll-like receptor 4 (TLR4) immune pathway. Here we show that airway epithelial cells display an increase in intracellular Ca2+ concentration within seconds of LPS application. This response occurs in a TLR4-independent manner, via activation of the transient receptor potential vanilloid 4 cation channel (TRPV4). We found that TRPV4 mediates immediate LPS-induced increases in ciliary beat frequency and the production of bactericidal nitric oxide. Upon LPS challenge TRPV4-deficient mice display exacerbated ventilatory changes and recruitment of polymorphonuclear leukocytes into the airways. We conclude that LPS-induced activation of TRPV4 triggers signaling mechanisms that operate faster and independently from the canonical TLR4 immune pathway, leading to immediate protective responses such as direct antimicrobial action, increase in airway clearance, and the regulation of the inflammatory innate immune reaction.

Phosphocholine-Specific Antibodies Improve T-Dependent Antibody Responses against OVA Encapsulated into Phosphatidylcholine-Containing Liposomes.

Liposomes containing phosphatidylcholine have been widely used as adjuvants. Recently, we demonstrated that B-1 cells produce dipalmitoyl-phosphatidylcholine (DPPC)-specific IgM upon immunization of BALB/c mice with DPPC-liposomes encapsulating ovalbumin (OVA). Although this preparation enhanced the OVA-specific humoral response, the contribution of anti-DPPC antibodies to this effect was unclear. Here, we demonstrate that these antibodies are secreted by B-1 cells independently of the presence of OVA in the formulation. We also confirm that these antibodies are specific for phosphocholine. The anti-OVA humoral response was partially restored in B-1 cells-deficient BALB/xid mice by immunization with the liposomes opsonized with the serum total immunoglobulin (Ig) fraction containing anti-phosphocholine antibodies, generated in wild-type animals. This result could be related to the increased phagocytosis by peritoneal macrophages of the particles opsonized with the serum total Ig or IgM fractions, both containing anti-phosphocholine antibodies. In conclusion, in the present work, it has been demonstrated that phosphocholine-specific antibodies improve T-dependent antibody responses against OVA carried by DPPC-liposomes.

Gustatory-mediated avoidance of bacterial lipopolysaccharides via TRPA1 activation in Drosophila.

Detecting pathogens and mounting immune responses upon infection is crucial for animal health. However, these responses come at a high metabolic price (McKean and Lazzaro, 2011, Kominsky et al., 2010), and avoiding pathogens before infection may be advantageous. The bacterial endotoxins lipopolysaccharides (LPS) are important immune system infection cues (Abbas et al., 2014), but it remains unknown whether animals possess sensory mechanisms to detect them prior to infection. Here we show that Drosophila melanogaster display strong aversive responses to LPS and that gustatory neurons expressing Gr66a bitter receptors mediate avoidance of LPS in feeding and egg laying assays. We found the expression of the chemosensory cation channel dTRPA1 in these cells to be necessary and sufficient for LPS avoidance. Furthermore, LPS stimulates Drosophila neurons in a TRPA1-dependent manner and activates exogenous dTRPA1 channels in human cells. Our findings demonstrate that flies detect bacterial endotoxins via a gustatory pathway through TRPA1 activation as conserved molecular mechanism.

Engineered tobacco etch virus (TEV) protease active in the secretory pathway of mammalian cells.

Tobacco etch virus protease (TEVp) is a unique endopeptidase with stringent substrate specificity. TEVp has been widely used as a purified protein for in vitro applications, but also as a biological tool directly expressing it in living cells. To adapt the protease to diverse applications, several TEVp mutants with different stability and enzymatic properties have been reported. Herein we describe the development of a novel engineered TEVp mutant designed to be active in the secretory pathway. While wild type TEVp targeted to the secretory pathway of mammalian cells is synthetized as an N-glycosylated and catalytically inactive enzyme, a TEVp mutant with selected mutations at two verified N-glycosylation sites and at an exposed cysteine was highly efficient. This mutant was very active in the endoplasmic reticulum (ER) of living cells and can be used as a biotechnological tool to cleave proteins within the secretory pathway. As an immediate practical application we report the expression of a complete functional monoclonal antibody expressed from a single polypeptide, which was cleaved by our TEVp mutant into the two antibody chains and secreted as an assembled and functional molecule. In addition, we show active TEVp mutants lacking auto-cleavage activity.

Antitumor and cytotoxic properties of a humanized antibody specific for the GM3(Neu5Gc) ganglioside.

Gangliosides are sialic acid-bearing glycosphingolipids expressed on all mammalian cell membranes, and participate in several cellular processes. During malignant transformation their expression changes, both at the quantitative and qualitative levels. Of particular interest is the overexpression by tumor cells of Neu5Gc-gangliosides, which are absent, or detected in trace amounts, in human normal cells. The GM3(Neu5Gc) ganglioside in particular has been detected in many human tumors, and it is considered one of the few tumor specific antigen. We previously demonstrated that a humanized antibody specific for this molecule, named 14F7hT, retained the binding and cytotoxic properties of the mouse antibody. In this work, we confirm that 14F7hT exerts a non-apoptotic cell death mechanism in vitro and shows its potent in vivo antitumor activity on a solid mouse myeloma model. Also, we demonstrate, in contrast to the murine counterpart, the capacity of this antibody to induce antibody-dependent cell-mediated cytotoxicity using human effector cells, which increases its potential for the treatment of GM3(Neu5Gc)-expressing human tumors.

Molecular subtyping of metastatic melanoma based on cell ganglioside metabolism profiles.

BACKGROUND: In addition to alterations concerning the expression of oncogenes and onco-suppressors, melanoma is characterized by the presence of distinctive gangliosides (sialic acid carrying glycosphingolipids). Gangliosides strongly control cell surface dynamics and signaling; therefore, it could be assumed that these alterations are linked to modifications of cell behavior acquired by the tumor. On these bases, this work investigated the correlations between melanoma cell ganglioside metabolism profiles and the biological features of the tumor and the survival of patients. METHODS: Melanoma cell lines were established from surgical specimens of AJCC stage III and IV melanoma patients. Sphingolipid analysis was carried out on melanoma cell lines and melanocytes through cell metabolic labeling employing [3-3H]sphingosine and by FACS. N-glycolyl GM3 was identified employing the 14 F7 antibody. Gene expression was assayed by Real Time PCR. Cell invasiveness was assayed through a Matrigel invasion assay; cell proliferation was determined through the soft agar assay, MTT, and [3H] thymidine incorporation. Statistical analysis was performed using XLSTAT software for melanoma hierarchical clustering based on ganglioside profile, the Kaplan-Meier method, the log-rank (Mantel-Cox) test, and the Mantel-Haenszel test for survival analysis. RESULTS: Based on the ganglioside profiles, through a hierarchical clustering, we classified melanoma cells isolated from patients into three clusters: 1) cluster 1, characterized by high content of GM3, mainly in the form of N-glycolyl GM3, and GD3; 2) cluster 2, characterized by the appearance of complex gangliosides and by a low content of GM3; 3) cluster 3, which showed an intermediate phenotype between cluster 1 and cluster 3. Moreover, our data demonstrated that: a) a correlation could be traced between patients' survival and clusters based on ganglioside profiles, with cluster 1 showing the worst survival; b) the expression of several enzymes (sialidase NEU3, GM2 and GM1 synthases) involved in ganglioside metabolism was associated with patients' survival; c) melanoma clusters showed different malignant features such as growth in soft agar, invasiveness, expression of anti-apoptotic proteins. CONCLUSIONS: Ganglioside profile and metabolism is strictly interconnected with melanoma aggressiveness. Therefore, the profiling of melanoma gangliosides and enzymes involved in their metabolism could represent a useful prognostic and diagnostic tool.

Role of B-1 cells in the immune response against an antigen encapsulated into phosphatidylcholine-containing liposomes.

B-1 lymphocytes comprise a unique subset of B cells that differ phenotypically, ontogenetically and functionally from conventional B-2 cells. A frequent specificity of the antibody repertoire of peritoneal B-1 cells is phosphatidylcholine. Liposomes containing phosphatidylcholine have been studied as adjuvants and their interaction with dendritic cells and macrophages has been demonstrated. However, the role of B-1 cells in the adjuvanticity of liposomes composed of phosphatidylcholine has not been explored. In the present work, we studied the contribution of B-1 cells to the humoral response against ovalbumin (OVA) encapsulated into dipalmitoylphosphatidylcholine (DPPC) and cholesterol-containing liposomes. BALB/X-linked immunodeficient (xid) mice, which are deficient in B-1 cells, showed quantitative and qualitative differences in the anti-OVA antibody response compared with wild-type animals after immunization with these liposomes. The OVA-specific immune response was significantly increased in the BALB/xid mice when reconstituted with B-1 cells from naive BALB/c mice. Our results indicate the internalization of DPPC-containing liposomes by these cells and their migration from the peritoneal cavity to the spleen. Phosphatidylcholine significantly contributed to the immunogenicity of liposomes, as DPPC-containing liposomes more effectively stimulated the anti-OVA response compared with vesicles composed of dipalmitoylphosphatidylglycerol. In conclusion, we present evidence for a cognate interaction between B-1 cells and phosphatidylcholine liposomes, modulating the immune response to encapsulated antigens. This provides a novel targeting approach to assess the role of B-1 cells in humoral immunity.

Liposomes of phosphatidylcholine and cholesterol induce an M2-like macrophage phenotype reprogrammable to M1 pattern with the involvement of B-1 cells.

Macrophages respond to endogenous and non-self stimuli acquiring the M1 or M2 phenotypes, corresponding to classical or alternative activation, respectively. The role of B-1 cells in the regulation of macrophage polarization through the secretion of interleukin (IL)-10 has been demonstrated. However, the influence of B-1 cells on macrophage phenotype induction by an immunogen that suppress their ability to secrete IL-10 has not been explored. Here, we studied the peritoneal macrophage pattern induced by liposomes comprised of dipalmitoylphosphatidylcholine (DPPC) and cholesterol (Chol) carrying ovalbumin (OVA) (Lp DPPC/OVA), and the involvement of B-1 cells in macrophage polarization. Peritoneal cells from BALB/c, B-1 cells-deficient BALB/xid and C57BL/6 mice immunized with Lp DPPC/OVA and OVA in soluble form (PBS/OVA) were analyzed and stimulated or not in vitro with lipopolysaccharide (LPS). Peritoneal macrophages from BALB/c and C57BL/6 mice immunized with Lp DPPC/OVA showed an M2-like phenotype as evidenced by their high arginase activity without LPS stimulation. Upon stimulation, these macrophages were reprogrammable toward the M1 phenotype with the upregulation of nitric oxide (NO) and a decrease in IL-10 secretion. In addition, high IFN-γ levels were detected in the culture supernatant of peritoneal cells from BALB/c and C57BL/6 mice immunized with Lp DPPC/OVA. Nevertheless, still high levels of arginase activity and undetectable levels of IL-12 were found, indicating that the switch to a classical activation state was not complete. In the peritoneal cells from liposomes-immunized BALB/xid mice, levels of arginase activity, NO, and IL-6 were below those from wild type animals, but the last two products were restored upon adoptive transfer of B-1 cells, together with an increase in IFN-γ secretion. Summarizing, we have demonstrated that Lp DPPC/OVA induce an M2-like pattern in peritoneal macrophages reprogrammable to M1 phenotype after LPS stimulation, with the involvement of B-1 cells.

Immunological and histological evaluation of clinical samples from psoriasis patients treated with anti-CD6 itolizumab.

Psoriasis is a chronic inflammatory disease with a prevalence of approximately 2-3% in the general population. The majority of diagnosed patients have plaque psoriasis, and about 20% have moderate-to-severe disease. Itolizumab, a new monoclonal antibody specific for the CD6 molecule mainly expressed on T lymphocytes, has demonstrated to inhibit in vitro ligand-induced proliferation and pro-inflammatory cytokine production. We assessed the immunological and histopathological effect of the antibody using clinical samples taken from 26 patients with moderate-to-severe psoriasis included in a clinical trial. The precursor frequency of lymphocytes activated with anti-CD2/CD3/CD28 beads, as well as the number of interferon (IFN)-γ-secreting T cells after stimulation, were measured at different time points of the study. Serum cytokine levels and anti-idiotypic antibody response to itolizumab were also evaluated. Additionally, lymphocyte infiltration and epidermis hyperplasia were studied in five patients. A significant reduction in T cell proliferation capacity and number of IFN-γ-producing T cells was found in treated patients. Serum levels of interleukin-6, tumor necrosis factor and IFN-γ showed an overall trend toward reduction. No anti-idiotypic antibody response was detected. A significant reduction in the epidermis hyperplasia was observed in analyzed patients. These results support the relevance of the CD6 molecule as a therapeutic target for the treatment of this disease.

A shift from N-glycolyl- to N-acetyl-sialic acid in the GM3 ganglioside impairs tumor development in mouse lymphocytic leukemia cells.

Humans, in contrast to other mammals, do not synthesize N-glycolyl-neuraminic acid (Neu5Gc) due to a deletion in the gene (cmah) encoding the enzyme responsible for this conversion, the cytidine monophospho-N-acetyl-neuraminic acid hydroxylase (CMP-Neu5Ac hydroxylase). The detection of considerable amounts of Neu5Gc-sialoconjugates, in particular gangliosides, in human malignancies makes these antigens attractive targets for immunotherapy, in particular with monoclonal antibodies (mAbs). We have previously described a GM3(Neu5Gc) ganglioside-specific mAb, named 14F7, with the ability to kill tumor cells in a complement-independent manner. Silencing the cmah gene in GM3(Neu5Gc)-expressing L1210 mouse lymphocytic leukemia B cells caused the abrogation of this cytotoxic effect. We now show that cmah-silenced L1210 cells (cmah-kd) express a high level of GM3(Neu5Ac) and have an impaired ability for anchorage-independent cell growth and tumor development in vivo. No evidences of increased immunogenicity of the cmah-kd cell line were found. These results provide new evidences on the role of GM3(Neu5Gc), or Neu5Gc-sialoconjugates in general, in tumor biology. As an important tool in this study, we used the humanized version (here referred to as 7C1 mAb) of a recently described, rationally-designed mutant of 14F7 mAb that is able to bind to both GM3(Neu5Gc) and GM3(Neu5Ac). In contrast to its parental antibody, the humanized 14F7 (14F7hT) mAb, 7C1 mAb was able to kill not only GM3(Neu5Gc)-expressing L1210 wild type cells, but also GM3(Neu5Ac)-expressing cmah-kd cells, which endorses this antibody as a potential agent for cancer immunotherapy.

Idiotypes as immunogens: facing the challenge of inducing strong therapeutic immune responses against the variable region of immunoglobulins.

Idiotype (Id)-based immunotherapy has been exploited as cancer treatment option. Conceived as therapy for malignancies bearing idiotypic antigens, it has been also extended to solid tumors because of the capacity of anti-idiotypic antibodies to mimic Id-unrelated antigens. In both these two settings, efforts are being made to overcome the poor immune responsiveness often experienced when using self immunoglobulins as immunogens. Despite bearing a unique gene combination, and thus particular epitopes, it is normally difficult to stimulate the immune response against antibody variable regions. Different strategies are currently used to strengthen Id immunogenicity, such as concomitant use of immune-stimulating molecules, design of Id-containing immunogenic recombinant proteins, specific targeting of relevant immune cells, and genetic immunization. This review focuses on the role of anti-Id vaccination in cancer management and on the current developments used to foster anti-idiotypic B and T cell responses.