Sticholysin II shows similar immunostimulatory properties to LLO stimulating dendritic cells and MHC-I restricted T cell responses of heterologous antigen.

Induction of CD8+ T cell responses against tumor cells and intracellular pathogens is an important goal of modern vaccinology. One approach of translational interest is the use of liposomes encapsulating pore-forming proteins (PFPs), such as Listeriolysin O (LLO), which has shown efficacy at priming strong and sustained CD8+ T cell responses. Recently, we have demonstrated that Sticholysin II (StII), a PFP from the sea anemone Stichodactyla helianthus, co-encapsulated into liposomes with ovalbumin (OVA) was able to stimulate, antigen presenting cells, antigen-specific CD8+ T cells and anti-tumor activity in mice. In the present study, we aimed to compare StII and LLO in terms of their abilities to stimulate dendritic cells and to induce major histocompatibility complex (MHC) class I restricted T cell responses against OVA. Interestingly, StII exhibited similar abilities to LLO in vitro of inducing dendritic cells maturation, as measured by increased expression of CD40, CD80, CD86 and MHC-class II molecules, and of stimulating OVA cross-presentation to a CD8+ T cell line. Remarkably, using an ex vivo Enzyme-Linked ImmunoSpot Assay (ELISPOT) to monitor gamma interferon (INF-γ) producing effector memory CD8+ T cells, liposomal formulations containing either StII or LLO induced comparable frequencies of OVA-specific INF-γ producing CD8+ T cells in mice that were sustained in time. However, StII-containing liposomes stimulated antigen-specific memory CD8+ T cells with a higher potential to secrete IFN-γ than liposomes encapsulating LLO. This StII immunostimulatory property further supports its use for the rational design of T cell vaccines against cancers and intracellular pathogens. In summary, this study indicates that StII has immunostimulatory properties similar to LLO, despite being evolutionarily distant PFPs.

Preclinical modeling of EGFR-specific antibody resistance: oncogenic and immune-associated escape mechanisms.

To define the molecular basis of secondary resistance to epidermal growth factor receptor (EGFR)-specific antibodies is crucial to increase clinical benefit in patients. The limited access to posttreatment tumor samples constitutes the major barrier to conduct these studies, representing preclinical experimentation as a useful alternative. Anti-EGFR antibody-based therapy has been reported to mediate tumor regression by interrupting oncogenic signals and, more recently, by inducing antitumor immunological responses. However, resistance models have been focused only on tumor escape associated with EGFR blockade, whereas studies describing immune-associated escape mechanisms have not been reported thus far. To address this idea, we modeled resistance induction in D122 metastasis-bearing C57BL/6 mice treated with 7A7 (an anti-murine EGFR antibody). Similarly to patients receiving EGFR-specific antibodies, 7A7 resistance promotion represents an important drawback to successful therapy. Characterization of primary cultures derived from metastasis in 7A7-treated mice revealed a high frequency of tumor variants resistant to in vivo and in vitro antibody treatment. We showed, for the first time, the convergence of alterations in oncogenic and immunological pathways in 7A7-resistant variants. To identify key molecules behind resistance, seven 7A7-resistant variants were screened. HER3 overexpression and PTEN deficiency leading to hyperactivation of protumoral downstream signaling were found in these variants as a consequence of 7A7-mediated EGFR inhibition. Concomitantly, we found a high percentage of resistant variants carrying abnormalities in the constitutive and/or interferon gamma (IFN-γ)-inducible major histocompatibility complex I (MHC-I) expression. A significant decrease in mRNA levels for MHC-I heavy chains, ß2-microglogulin and antigen processing machinery genes as well as transcriptional alterations in IFN-γ pathway components were identified as the main mechanisms underlying MHC-I expression defects in 7A7-resistant variants. Notably, these defects have not been previously associated with EGFR-specific antibody resistance, providing novel immunological escape mechanisms. This study has strong implications for the development of new combination strategies to overcome anti-EGFR antibodies refractoriness.

Experimental and theoretical vibrational study of methylene bis(thiocyanate), CH2(SCN)2. A comparison with thiocyanogen, (SCN)2.

The infrared and Raman spectra of methylene bis(thiocyanate), CH2(SCN)2, were obtained. The observed bands were assigned to the different normal modes of vibration using the results of a DFT calculation of the molecular vibrational properties. These results and the experimental data were used to define a Scaled Quantum Mechanics force field for the molecule. A similar treatment was applied to the thiocyanogen molecule, (SCN)2, for which the experimental frequencies were already reported in the literature. The sets of internal force constants for both molecules show very similar values.

Cytofluorimetric evaluation of N-glycolylated GM3 ganglioside expression on murine leukocytes.

Gangliosides are considered relevant components of lipid rafts at the plasma membrane. Antigen encounter, immunological synapse assembly and signal transduction modify lipid raft composition and distribution on immune system cells. On the contrary of other gangliosides, differential expression of the N-glycolylated variant of GM3 (NGcGM3) on murine leukocytes has received limited attention. In particular, whether cell activation modulates the expression of NGcGM3 on lymphoid and myeloid cells is still unexplored. Availability of the NGcGM3 specific 14F7 MAb allows us to characterize by cytofluorimetric assays the presence of this molecule on resting and activated immune system cells. On T cells, preferential expression of NGcGM3 was detected on CD4(+) single positive thymocytes, peripheral CD4(+) lymphocytes and natural occurring regulatory T cells. In comparison with peritoneal B1 cells, reduced expression of NGcGM3 was observed in peritoneal B2 and splenic B cell subpopulations. Of note, activation of CD4(+) and NK 1.1(+) cells abrogated NGcGM3 expression while LPS-maturated DC increased the ganglioside level at the plasma membrane. Modifications on the presence of NGcGM3 mediated by cell activation did not influence the expression of the N-acetylated variant of GM3 (NAcGM3). In addition to extend previous descriptions of NGcGM3 expression on immunity cell subpopulations, this work highlights the opposite effect of cellular activation over NGcGM3 levels on lymphoid and myeloid cellular series. Obtained results complement the evaluation of a tumor-specific, non-human sialic acid containing ganglioside that has been considered an attractive target for cancer immunotherapy.

Innate-immunity cytokines induced by very small size proteoliposomes, a Neisseria-derived immunological adjuvant.

Neisserial outer membrane proteins have been combined with monosialoganglioside GM3 to form very small size proteoliposomes (VSSP), a nanoparticulated formulation used as a cancer vaccine for the treatment of cancer patients with GM3-positive tumours. VSSP were shown to elicit anti-GM3 and anti-tumour immune responses. VSSP have also been shown to be an efficient adjuvant for tumour-cell and peptide-antigen vaccines in mice. In vitro studies showed that VSSP promote maturation of both murine and human dendritic cells, suggesting that VSSP could be used as efficient adjuvants. In order to study further the capacity of VSSP to elicit innate immune responses, human peripheral blood mononuclear cells and monocytes derived thereof were assessed for in vitro secretion of interleukin (IL)-10, IL-6, IL-12 and interferon (IFN)-gamma. VSSP most prominently induced the secretion of IL-6. IL-10 was secreted at a lower level. IL-12 p40 (but no p70) was also detected. IFN-gamma response was observed in 56% of the tested samples. Cytokine secretion was not related to lipopolysaccharide (LPS) content and involved Toll-like receptor 2 (TLR2)-mediated signal transduction. VSSP also induced DC maturation and a cytokine secretion pattern (high IL-6/low IL-10) which differs from that induced by LPS. The observed proinflammatory cytokine secretion pattern and the capacity of VSSP to drive DC maturation are examined in the light of the properties of other bacterial derivatives currently being user for immunotherapy purposes. Our results suggest that VSSP could be tested in clinical settings where T helper 1-type immune responses would be beneficial.

Role of receptor interaction in the mode of action of insecticidal Cry and Cyt toxins produced by Bacillus thuringiensis.

Cry toxins from Bacillus thuringiensis are used for insect control. Their primary action is to lyse midgut epithelial cells. In this review we will summarize recent findings on the Cry toxin-receptor interaction and the role of receptor recognition in their mode of action. Cry toxins interact sequentially with multiple receptors. In lepidopteran insects, Cry1A monomeric toxins interact with the first receptor and this interaction triggers oligomerization of the toxins. The oligomer then interacts with second receptor inducing insertion into membrane microdomains and larval death. In the case of mosquitocidal toxins, Cry and Cyt toxins play a part. These toxins have a synergistic effect and Cyt1Aa overcomes Cry toxin resistance. Recently, it was proposed that Cyt1Aa synergizes or suppresses resistance to Cry toxins by functioning as a membrane-bound receptor for Cry toxin.

Vibrational spectra of the trifluoromethylsulfinate anion and scaled quantum mechanical force fields for CF3SO2- and CF3SeO2-.

The infrared and Raman spectra of KCF(3)SO(2) were obtained and the observed spectral features assigned to the expected normal modes of vibration. Besides, the vibrational properties of the CF(3)SO(2)(-) and CF(3)SeO(2)(-) related anions were studied by means of density functional theory (DFT) techniques. After obtaining the optimized geometrical parameters and conformations, the vibrational wavenumbers and the associated force constants were calculated. The original force fields in cartesian coordinates were transformed to local symmetry coordinates and subsequently scaled to reproduce the experimental wavenumbers. Some trends observed in the force constants of the studied species and of the related CF(3)SO(3)(-) anion could be explained by the differences in geometrical parameters.

Therapeutic cancer vaccines: at midway between immunology and pharmacology.

The pursuit of active specific immunotherapy of cancer re-emerged vigorously in the 90s. More than 50 vaccines are currently under clinical testing, and more than 400 clinical trials have been conducted. This wave of enthusiasm is rooted in fundamental immunology, as new paradigms, such as the dominant tolerance through T-regulatory cells and the instructive role of the innate immune system on the adaptive immune system, opened the possibility that an efficient cancer vaccination could be achieved even without the need of cancer neoantigens, provided that antigen presentation could be increased, and that regulatory circuits could be controlled. However, recent failures in some large trials have brought disappointment and have highlighted the differences between experiments in young, healthy mice with small transplanted tumours, and clinical testing in aged, ill patients with advanced spontaneous tumours, driving the attention to issues such as tumour editing, tumour-induced immunosuppression, and immunosenescence. The molecular basis of these phenomena is only partially known. Additionally, the inherent complexity of the immune system as a network of multiple interactions and redundant control loops among a huge diversity of components sets another barrier to the translation of in vitro reductionist knowledge into rationally designed clinical trials. All this calls for a new therapeutic paradigm in cancer vaccines, moving beyond the analogy with the classic drug-target approach, and targeting the immune system regulation as a whole, and its interaction with the tumour, in all its complexity. Early mathematical modelling of cancer immunotherapy has suggested how to go about it. This re-evaluation of the cancer vaccine landscape, suggests that future successful cancer immunotherapy will be combined immunotherapy, will be exquisitely schedule-dependent and will need new experimental models allowing for the exploration of the mechanisms of resistance and tumour escape, such as tumour editing and tumour induced immunosuppression, in the context of the physiology of the immune system of the elderly. These shifts will put cancer vaccines closer to pharmacology than to conventional preventive vaccinology, or at least at the midway. A change in the design and the ultimate goals of the clinical trials will also be needed, identifying long term stabilization of the disease and quality of life as main endpoints, again closer to the clinical management of most chronic noncommunicable diseases.

A scaled quantum mechanical force field for the sulfuryl halides II. The SO(2)XF (X=Cl, Br) halides.

Force fields and vibrational wavenumbers were calculated for the molecules SO(2)XF (X=Cl, Br) using DFT techniques. The previously available experimental data and assignments for SO(2)ClF and SO(2)BrF were partially confirmed by the theoretical results. These data were subsequently used in the definition of scaled quantum mechanics force fields for such molecules. A comparison of the obtained force constants is made with results previously published for the SO(2)X(2) molecules.

Experimental and theoretical vibrational study of 2,2,2-trifluoroethyl trifluoromethanesulfonate, CF3SO2OCH2CF3.

The infrared spectra of 2,2,2-trifluoroethyl trifluoromethanesulfonate (CF3SO2OCH2CF3) were obtained in the gaseous, liquid and solid states as well as the Raman spectrum of the liquid. Quantum chemistry calculations using the density functional theory were used to predict the most stable geometry and conformation of the studied molecule. Subsequently, the harmonic vibrational frequencies and force field were calculated. An assignment of the observed spectral features made after comparison with the related molecules and with the predicted frequencies was used as the basis of a scaling of the original force field in order to reproduce as well as possible the experimental frequencies. With this purpose a set of scale factors was calculated by a least square procedure, leading to a final root mean square deviation (RMSD) of 9.7 cm(-1).

A scaled quantum mechanical force field for the sulfuryl halides. I. The symmetric halides SO2X2 (X=F, Cl, Br).

Force fields and vibrational wavenumbers were calculated for the molecules SO2X2 (X=F, Cl, Br) using DFT techniques. The previously available experimental data and assignments for SO2F2 and SO2Cl2 were compared with the theoretical results and revised, and new low temperature infrared and Raman data were obtained for SO2Cl2. These data were subsequently used in the definition of scaled quantum mechanics force fields for such molecules. Adjusted wavenumbers were also predicted for the still unknown SO2Br2. A comparison is made with results published for the VO2X2- anions.

Evaluación del diferencial leucocitario realizado por el coulter STKS y el coulter MAXM / Evaluation of differential leukocyte performance by coulter STKS and coulter MAXM

Para evaluar la exactitud del recuento diferencial leucocitario realizado por los autoanalizadores Coulter STKS y Coulter MAXM se emplearon un total de 184 muestras, de una población aparentemente sana de la Escuela de Bioanálisis de la UCV. El diferencial manual de referencia se realizó según los procedimientos descritos en el protocolo H20-A. Con envoltura binomial encontramos que para: neutrófilos sobresale 12 por ciento de los limites, linfocitos 47 por ciento cae por debajo del límite inferior, monocitos 74 por ciento cae por encima del limite del 95 por ciento, eosinófilos 5 por ciento, basófilos 48,5 por ciento. Los errores sistemáticos para neutrófilos, linfocitos y eosinófilos fueron menores que los errores permitidos: para monocitos y basófilos los errores sistematicos fueron mayores que los permitidos. La correlación para todos los subtipos celulares fue buena excepto para monocitos y basófilos. La exactitud del contaje diferencial leucocitario realizado por el Coulter STKS y el coulter MAXM resultó ser satisfactoria para la mayoría de los subtipos celulares

Toxicity of a GM3 cancer vaccine in Macaca fascicularis monkey: a 12-month study.

GM3 is a ganglioside that has been biochemically identified as dominating the cell surface of several human tumours, but is also found on human normal cells at much lower density. Since GM3 is widely distributed in essentially all types of animal cells, there is a conflict with the concepts of tumour-associated antigen, immunogen, and toxicity. We have designed a GM3-based cancer vaccine for the treatment of human breast and melanoma tumours. Prior to the Phase I clinical trial, we carried out a 12-month dose repeated toxicity study in five male Macaca fascicularis monkeys. Four male monkeys were treated with placebo in a similar way. During the study, no differences were observed between control and treated monkeys related to daily clinical observations (other than local damage) including rectal temperature, blood pressure, respiratory and cardiac rates, weight gain, biochemical and hematological parameters (with the exception of transitory pathological changes), and anti-DNA and anti-nuclear antibodies, although treated monkeys consistently developed both IgM- and IgG-specific anti-GM3 antibodies. Sixty per cent of treated monkeys developed moderate local reactions at the injection site, which disappeared without sequels. We concluded that this GM3 cancer vaccine overcame in monkeys the natural tolerance to GM3 ganglioside evidenced by a strong immune response, while the local reactions elicited-were transitory without apparent important systemic toxicity effects.

Preparation of deacetyl-, lyso-, and deacetyl-lyso-GM(3) by selective alkaline hydrolysis of GM3 ganglioside.

Three methods (using GM3 quantities ranging from a few milligrams to grams) have been developed to prepare, in high yield, the three derivatives of ganglioside GM3 [alpha-Neu5Ac-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-ceramide]: deacetyl-GM3 [alpha-Neu-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-ceramide], lyso-GM3 [alpha-Neu5Ac-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-sphingosine], and deacetyl-lyso-GM3 [alpha-Neu-(2-3)-beta-Gal-(1-4)-beta-Glc-(1-1)-sphingosine]. This is the first report of the preparation of lyso-GM3 by a one-pot reaction. We can now define the optimal conditions for the different preparations. Preparation of deacetyl-GM3: alkaline reagent, 2 M KOH in water; GM3 concentration, 33 mg/ml; reaction temperature, 90 degrees C; reaction time, 3.5 h; nitrogen atmosphere. Preparation of deacetyl-lyso-GM3: alkaline reagent, 8 M KOH in water; GM3 concentration, 10 mg/ml; reaction temperature, 90 degrees C; reaction time, 18 h; nitrogen atmosphere. Preparation of lyso-GM(3): alkaline reagent, 1 M sodium tert-butoxide in methanol; GM3 concentration, 10 mg/ml; reaction temperature, 80 degrees C; reaction time, 18 h; anhydrous conditions. The percentage yield of deacetyl-GM3 was 70;-75%, that of deacetyl-lyso-GM3 100%, and of lyso-GM3 36;-40%.Deacetyl-GM3, deacetyl-lyso-GM3, and lyso-GM3 were purified by column chromatography, and chemical structures were confirmed by electron spray-mass spectrometry.

A purified GM3 ganglioside conjugated vaccine induces specific, adjuvant-dependent and non-transient antitumour activity against B16 mouse melanoma in vitro and in vivo.

The presence of substantial amounts of GM3 ganglioside on human melanomas and other tumours, together with its peculiar biological properties, makes this glycolipid a unique target for cancer immunotherapy. B16 mouse melanoma expresses GM3 and constitutes an appropriate model for the development of novel GM3-based vaccines. Recently, we hydrophobically incorporated purified GM3 into the outer membrane protein complex from Neisseria meningitidis to form very small size proteoliposomes (GM3/VSSP). We have examined the antitumour properties of GM3/VSSP vaccine and compared it with GM3 incorporated in very low density serum lipoproteins (GM3/VLDL). Immunization with four doses of GM3/VSSP vaccine (120 microg of ganglioside) plus Freund's adjuvant or Montanide ISA 51 significantly increased the overall survival of mice inoculated in the subcutis with 103 B16-F1 cells, whereas the GM3/VLDL immunogen was ineffective. The non-transient character of tumour protection was confirmed in animals surviving the first challenge and re-inoculated with 5 x 103 cells. GM3/VSSP vaccine also reduced the subcutaneous growth of highly aggressive B16-F10 cells. The importance of ganglioside structure in the tumour-protective effect of GM3/VSSP vaccine was confirmed using GM3 containing N-glycolylneuraminic acid, a ganglioside absent in melanoma cells. Immunostaining and enzyme-linked immunosorbent assay (ELISA) experiments showed a high specificity of immune sera against GM3 and the presence of all four IgG subclasses, with a preponderance of IgG2b and IgG3. In addition, a strong anti-B16 complement-mediated cytotoxicity was induced by vaccination with GM3/VSSP. The present data indicate the molecular specificity of GM3/VSSP vaccine as well as the adjuvant-dependent and non-transient character of tumour protection in the B16 mouse model. These findings suggest that an appropriate GM3 vaccine may be capable of inducing prolonged tumour protection in melanoma patients.

Characterization of a recombinant monoclonal antibody by mass spectrometry combined with liquid chromatography.

In this report, we present the characterization of a humanized monoclonal antibody specific for the human epidermal growth factor receptor (hEGFR). Direct analysis by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) of peptide mixtures and chromatographically isolated fractions allowed identification of 94.0% and 85.4% of the amino acid sequence of light and heavy chains, respectively. Microheterogeneity sources were identified in light and heavy chains and a previously unreported posttranslational modification for immunoglobulins was found. One N-glycosylation site was identified in the heavy chain with non-sialylated bianntenary fucosylated structures. This study is one of the first to assess the potential of MALDI-MS in combination with more conventional protein chemistry techniques for the characterization of monoclonal antibodies.

Infrared and Raman spectra and theoretical study of methyl trifluoromethyl sulfone, CF3SO2CH3.

The infrared and Raman spectra were obtained for liquid CF3SO2CH3, as well as the infrared spectrum of the gaseous substance. The molecular geometry was optimized by means of the Hartree-Fock (HF), second order electron correlation (MP2) and density functional theory (DFT) procedures of quantum chemistry, resulting in a structure with Cs symmetry. The wavenumbers corresponding to the normal modes of vibration were calculated using the DFT (B3LYP/6-31G**) approximation and their agreement with the measured values improved after scaling of the associated force field. An assignment of bands is proposed on the basis of such calculations and the comparison with related molecules.

A mouse IgG1 monoclonal antibody specific for N-glycolyl GM3 ganglioside recognized breast and melanoma tumors.

14F7 murine monoclonal antibody (MAb) is an IgG1 immunoglobulin that is generated by immunizing Balb/c mice with GM3(NeuGc) ganglioside hydrophobically conjugated with human very-low-density lipoproteins and in the presence of Freund's adjuvants. 14F7 MAb binds specifically to GM3(NeuGc), whereas neither N-glycolyl or N-acetyl gangliosides, nor a sulfated glycolipid, are recognized as assessed by enzyme-linked immunosorbent assay or immunostaining on thin layer chromatograms. Immunohistochemical studies in fresh tumor tissues showed that 14F7 MAb strongly recognized in antigen expressed in human breast and melanoma tumors.

Experimental and theoretical vibrational study of silyl trifluoromethanesulfonate, CF3SO2OSiH3.

Infrared and Raman spectra were obtained for liquid silyl trifluoromethanesulfonate, a silylating agent of limited stability. The molecular geometry was optimized by means of density functional theory and Möller-Plesset second order perturbation theory methods, using different basis sets. The optimized structure presents a gauche conformation, similar to that adopted by methyl trifluoromethanesulfonate, which was determined experimentally a short time ago. The wavenumbers for the normal modes of vibration and the corresponding force constants were also calculated, facilitating the interpretation of the vibrational data. The harmonic force constants given by theory were scaled to reproduce adequately the experimental wavenumbers.

Enhancement of the immune response to poorly immunogenic gangliosides after incorporation into very small size proteoliposomes (VSSP).

Certain gangliosides are tumor-associated antigens that constitute potential targets for cancer immunotherapy. A major drawback in the design of ganglioside-based cancer vaccines, however, is the poor immunogenicity of these glycolipids. Here we report the immunological and physicochemical properties of very small size proteoliposomes (VSSP) obtained by using anionic detergents to incorporate gangliosides into the outer membrane protein complex (OMPC) of N. meningitidis. VSSP of three different gangliosides, GM3, NGcGM3 and GD3, were tested. These gangliosides differ in level of expression in normal tissues and in immunogenicity in different animal species. We show that the immunization with VSSP in an oil adjuvant consistently induced both IgM and IgG anti-ganglioside antibodies. In the mouse, the anti-ganglioside IgG fraction was not restricted to the typical T-independent isotype IgG3. Unexpectedly, significant levels of the T-dependent IgG1, IgG2a and particularly IgG2b were also found. VSSP-mediated enhancement of the immunogenicity was not restricted to the relatively immunogenic ganglioside GD3, satisfactory immune responses against highly tolerated GM3 and NGcGM3 were also obtained. Similar results were achieved in chickens and monkeys. No reactogenicity was observed even when self-gangliosides were used for immunization. VSSP overcame natural tolerance to gangliosides in an adjuvant dependent fashion.