Efficacy of third-party chimeric antigen receptor modified peripheral blood natural killer cells for adoptive cell therapy of B-cell precursor acute lymphoblastic leukemia.

We developed an innovative and efficient, feeder-free culture method to genetically modify and expand peripheral blood-derived NK cells with high proliferative capacity, while preserving the responsiveness of their native activating receptors. Activated peripheral blood NK cells were efficiently transduced by a retroviral vector, carrying a second-generation CAR targeting CD19. CAR expression was demonstrated across the different NK-cell subsets. CAR.CD19-NK cells display higher antileukemic activity toward CD19+ cell lines and primary blasts obtained from patients with B-cell precursor ALL compared with unmodified NK cells. In vivo animal model data showed that the antileukemia activity of CAR.CD19-NK cell is superimposable to that of CAR-T cells, with a lower xenograft toxicity profile. These data support the feasibility of generating feeder-free expanded, genetically modified peripheral blood NK cells for effective "off-the-shelf" immuno-gene-therapy, while their innate alloreactivity can be safely harnessed to potentiate allogeneic cell therapy.

An optically coupled system for quantitative monitoring of MRI gradient currents induced into endocardial leads.

The time-varying gradient fields generated during Magnetic Resonance Imaging (MRI) procedures have the potential to induce electrical current on implanted endocardial leads. Whether this current can result in undesired cardiac stimulation is unknown. This paper presents an optically coupled system with the potential to quantitatively measure the currents induced by the gradient fields into endocardial leads during MRI procedures. Our system is based on a microcontroller that works as analog-to-digital (A/D) converter and sends the current signal acquired from the lead to an optical high-speed light-emitting-diode transmitter. Plastic fiber guides the light outside the MRI chamber, to a photodiode receiver and then to an acquisition board connected to a PC. The preliminary characterization of the performances of the system is also presented.

Tumores del sistema nervioso central de localización selar y supraselar. Alteraciones neuro-oftalmológicas y de la función endocrina al diagnóstico / Sellar and suprasellar central nervous system tumors. Neuro-ophthalmological and endocrine function impairment at diagnosis

Los tumores (Tu) del SNC constituyen la segunda enfermedad oncológica en edad pediátrica, con una incidencia referida aproximada que oscila entre el 10 y 15%. En 309 pacientes con tumores selares y supraselares, seguidos durante 15 años, se evaluó en función de los distintos oncotipos tumorales, síntomas iniciales y alteraciones endocrinológicas previas al inicio del tratamiento. De ellos, 227 pacientes presentaron el tumor a edad prepuberal. Los oncotipos tumorales más frecuentes fueron craneofaringioma (CRA), glioma (GLIA) y tumor de células germinales (GERM). También, se encontró una mayor incidencia de presentación en varones. En edad puberal (n:92), el oncotipo tumoral más frecuente fue adenoma hipofisario (ADENO), seguido de GLIA y CRA. En este ultimo oncotipo tumoral, y, a diferencia del grupo prepuberal, su incidencia fue significativamente mayor en niñas. Aproximadamente 90% de los pacientes tuvieron anormalidades neuro-oftalmológicas (hipertensión craneal, dolores de cabeza, vómitos y pérdida progresiva de la visión) como uno de los signos y/o síntomas iniciales. Alteraciones clínicas endocrinológicas como baja talla, velocidad de crecimiento anormal, diabetes insípida y alteraciones del tempo puberal son frecuentes en estos pacientes y están habitualmente asociadas con las alteraciones clínico-neuro-oftalmológicas como las ya mencionadas. No obstante, la mayoría de los tumores del SNC localizados en la línea media suelen ser diagnosticados por manifestaciones neuro-oftalmológicas. Los resultados del estudio muestran alteración de la función endócrina al diagnóstico del Tu. Se concluye que en todo paciente con crecimiento lento o baja talla, así como también signos clínicos que orienten a un diagnóstico de pubertad precoz y/o retardada, el pediatra debe incluir dentro de los diagnósticos diferenciales, el diagnóstico del tumor selar o supraselar. La morbilidad aumenta frecuentemente luego de la cirugía (AU)

During the last 15 years, 309 patients with tumors of the sellar and suprasellar areas of CNS were followed in our Hospital (Endocrine Service). Tumor oncotype, initial symptoms and endocrine disturbances before any treatment was started are presented. In 227 patients, the tumor was diagnosed at prepubertal age. In this group, the most frequent tumoral oncotypes were craniopharyngioma (CRA), glial tumors (GLIA) and germ cells tumors (GERM). The incidence was higher in boys. At pubertal age (n:92), the most frequent tumoral oncotype was pituitary adenoma (ADENO), followed by GLIA and CRA. In the latter, and different from the prepubertal group, the incidence was significantly higher in girls. Approximately 90% of patients had neuro-ophtalmological abnormalities (cranial hypertension, headaches, vomits, and progressive loss of vision) as one of the initial signs and/or symptoms. Clinical endocrine disorders, such as short stature, low growth velocity, diabetes insipidus, and alterations in pubertal "tempo" are frequent in these patients and are often associated with the neuro-ophtalmological abnormalities mentioned above. This clinical symptomatology has to alert the medical team to discard the presence of a CNS tumor at the sellar and/or suprasellar level. We conclude that tumors of the SNC localized in the midline, have potential capacity to provoke abnormalities in endocrine function. Morbidity is often increased after surgery (AU)

Efecto de la dipirona sobre la agregación plaquetaria / Effect of dipyrone on platelet aggregation

El presente estudio se realizó con la finalidad de determinar si la Dipirona altera la agregación plaquetaria. Para ello, se seleccionaron 30 voluntarios sanos, los cuales fueron divididos en dos grupos y asignados al azar a recibir 1 g de Dipirona o 100 mg de Acido Acetilsalicílico. Se determinó la agregación plaquetaria a través del método turbidimétrico; utilizando adenosin difosfato, colágeno y adrenalina. En el grupo Dipirona, se observó una reducción estadísticamente significativa de la agregación plaquetaria a las 72 horas, frente a los 3 estímulos; que revirtió a las 24 horas. En el grupo Acido Acetilsalicílico, disminuyó significativamente la agregación plaquetaria a las 24 horas. Al comparar ambos grupos no hubo diferencia significativa en la muestra basal pero si a las 24 horas. Podemos concluir que la Dipirona, al igual que el resto de los antiinflamatorios no esteroideos, inhibe la agregación plaquetaria de forma reversible, al contrario del Acido Acetilsalicílico.

Peptide mimotopes as prototypic templates of broad-spectrum surrogates of carbohydrate antigens.

Peptide mimetics of carbohydrate antigens can function as templates to exploit immune mechanisms to augment vaccine design strategies as they are T cell dependent antigens. In this study we evaluate a peptide mimetic (peptide 105) of the Pneumococcal capsular polysaccharide type 14 (Pn14) as a model antigen to explore differences in antigenicity and immunogenicity of peptide mimotopes. The multiple antigenic peptide (MAP) form, by ELISA, competes with native Pn14 in a concentration-dependent manner for binding to an anti-Pn14 monoclonal antibody. It was observed that peptide priming with a conjugated form (105-BSA) and boosting with Pn14 produced higher levels of Pn14-reactive IgG1, IgG2a, IgG2b and IgG3 than priming and boosting with Pn14. This serum also displayed reactivity with multiple serotypes, as assessed by ELISA. However, when compared with serum from humans immunized with the 23-valent pneumococcal vaccines, mimetic-induced mouse serum did not display a significant ability to mediate opsonophagocytic killing of pneumococci. These results suggest the feasibility of designing mimotopes to render effective humoral responses not only to a single serotype of Streptococcus pneumoniae, but to multiple serotypes at once. Such peptides would simplify currently available vaccine approaches, yet highlights the requirement of more extensive polymerization to fully emulate native antigen.

Cambios en la composición lipídica de las membranas mitocondriales de corazón de ratas inducidos por la ingesta de aceite de pescado y su relación con la actividad mitocondrial / Changes in the lipid composition of the mitochondrial membrane of the heart rat induced by fish oil intake and its relation with mitochondrial activity

Los cambios en la composición lipídica del corazón inducidos por la dieta han sido implicados en las alteraciones de la función cardíaca. El consumo de aceite de pescado, por otra parte, ha sido relacionado con la baja incidencia de enfermedades cardiovasculares. La actividad de las mitocondrias aisladas de corazones insuficientes es menor que la observada en mitocondrias de corazones normales. En este trabajo, la administración de aceite de pescado en la dieta disminuyó el contenido de C18:2n-6 y C20:4n-6 y aumentó el de C22:6n-3 en las mitocondrias de corazón. La ingesta de aceite de pescado incrementó las velocidades de respiración en estados 3 y 4 (VR3 y VR4) sin modificar la eficiencia de fosforilación ni el índice de control respiratorio. Se discute acerca de que el posible efecto protector de los ácidos grasos poli-insaturados contra los radicales libres sea ejercido a través del aumento de VR3 y VR4

Measurement of MHC/peptide interactions by gel filtration.

This unit describes a technique for the direct and quantitative measurement of the capacity of peptide ligands to bind Class I and Class II MHC molecules. The binding of a peptide of interest to MHC is assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to MHC molecules. The establishment of an MHC/peptide binding assay, and its subsequent use in determining the MHC binding capacities of peptide ligands, requires sufficient stocks of purified MHC and both labeled and unlabeled peptides. Accordingly, this unit includes protocols for the purification of Class I and Class II MHC molecules by affinity chromatography, and for the radiolabeling of peptides using the chloramine T method. A support protocol describes alterations in the basic protocol that are necessary when performing direct binding assays, which are required for (1) selecting appropriate high-affinity, assay-specific, radiolabeled ligands and (2) determining the amount of MHC necessary to yield assays with the highest sensitivity. After a 2-day incubation, the bound and unbound radiolabeled species are separated, and their relative amounts are determined. Two methods for separation by size-exclusion gel-filtration chromatography are described, as is data analysis.

Linear PADRE T helper epitope and carbohydrate B cell epitope conjugates induce specific high titer IgG antibody responses.

Linear carbohydrate-peptide constructs based on the 13 amino acid nonnatural pan DR epitope (PADRE) and carbohydrate B cell epitopes are demonstrated to be potent immunogens. These data support our belief that PADRE should be considered as an alternative to more complex carriers for use in prophylaxis and therapeutic vaccines. Two model carbohydrate-PADRE glycoconjugates were used to demonstrate that PADRE could effectively provide T cell help for carbohydrate-specific Ab responses. Conjugates of PADRE covalently linked to the human milk oligosaccharide, lacto-N-fucopentose II or a dodecasaccharide derived from Salmonella typhimurium O-Ag induced high titer IgG Ab responses in mice, which were comparable to glycoconjugates employing human serum albumin (HSA) as the carrier protein. Different adjuvants, in combination with PADRE conjugates, allowed for the modulation of the isotype profile with alum supporting an IgG1 profile; QS-21 an IgG2a, 2b profile, while an alum/QS-21 mixture generated a balanced IgG1/IgG2b isotype profile. As defined by binding to synthetic glycoconjugates, dodecasaccharide-specific Abs exhibited fine specificity similar to protective polyclonal Ab responses previously reported for dodecasaccharide-protein conjugates. The same Abs bound to intact S. typhimurium cells, suggesting that biologically relevant specificities were produced. The affinity of the dodecasaccharide-specific Abs was further shown to be comparable to that of a well-characterized, high affinity monoclonal anti-carbohydrate Ab recognizing the same epitope.

Pan DR binding sequence provides T-cell help for induction of protective antibodies against Plasmodium yoelii sporozoites.

Pan-DR epitope (PADRE) peptides have demonstrated the capacity to deliver help for antibody responses in vivo. They were also found, fortuitously, to be able to provide significant helper T-cell activity in vivo. This suggested that linear constructs, containing the PADRE epitope, might be as efficient at generating an immune response as large multivalent antigens. Plasmodium falciparum and P. yoelii PADRE constructs were capable of inducing a high titre IgG antibody response that recognized intact sporozoites. We now report that these antibodies can inhibit sporozoite invasion of hepatocytes in vitro and that mice immunized with the PyCSP-PADRE linear construct were protected when challenged with P. yoelii sporozoites.

Utilization of MHC class I transgenic mice for development of minigene DNA vaccines encoding multiple HLA-restricted CTL epitopes.

We engineered a multiepitope DNA minigene encoding nine dominant HLA-A2.1- and A11-restricted epitopes from the polymerase, envelope, and core proteins of hepatitis B virus and HIV, together with the PADRE (pan-DR epitope) universal Th cell epitope and an endoplasmic reticulum-translocating signal sequence. Immunization of HLA transgenic mice with this construct resulted in: 1) simultaneous CTL induction against all nine CTL epitopes despite their varying MHC binding affinities; 2) CTL responses that were equivalent in magnitude to those induced against a lipopeptide known be immunogenic in humans; 3) induction of memory CTLs up to 4 mo after a single DNA injection; 4) higher epitope-specific CTL responses than immunization with DNA encoding whole protein; and 5) a correlation between the immunogenicity of DNA-encoded epitopes in vivo and the in vitro responses of specific CTL lines against minigene DNA-transfected target cells. Examination of potential variables in minigene construct design revealed that removal of the PADRE Th cell epitope or the signal sequence, and changing the position of selected epitopes, affected the magnitude and frequency of CTL responses. Our results demonstrate the simultaneous induction of broad CTL responses in vivo against multiple dominant HLA-restricted epitopes using a minigene DNA vaccine and underline the utility of HLA transgenic mice in development and optimization of vaccine constructs for human use.

Recognition of a novel naturally processed, A2 restricted, HCV-NS4 epitope triggers IFN-gamma release in absence of detectable cytopathicity.

Using short term CTL lines derived from HLA A2/Kb transgenic mice and IFN-gamma release assays we demonstrate that the NS4.1769 epitope, is generated from natural processing of the NS4 antigen, and presented in the context of the A2/Kb molecules. Interestingly, T cell recognition of the naturally processed form of the NS4. 1769 epitope was associated with significant IFN-gamma release, but no direct cytolytic activity. Epitopes of this phenotype might be of interest, in terms of therapy of chronic HCV infection by associating the benefit of localized lymphokine release with low or absent direct cytopathicity.

Characterization of the peptide binding motif of a rhesus MHC class I molecule (Mamu-A*01) that binds an immunodominant CTL epitope from simian immunodeficiency virus.

The majority of immunogenic CTL epitopes bind to MHC class I molecules with high affinity. However, peptides longer or shorter than the optimal epitope rarely bind with high affinity. Therefore, identification of optimal CTL epitopes from pathogens may ultimately be critical for inducing strong CTL responses and developing epitope-based vaccines. The SIV-infected rhesus macaque is an excellent animal model for HIV infection of humans. Although a number of CTL epitopes have been mapped in SIV-infected rhesus macaques, the optimal epitopes have not been well defined, and their anchor residues are unknown. We have now defined the optimal SIV gag CTL epitope restricted by the rhesus MHC class I molecule Mamu-A*01 and defined a general peptide binding motif for this molecule that is characterized by a dominant position 3 anchor (proline). We used peptide elution and sequencing, peptide binding assays, and bulk and clonal CTL assays to demonstrate that the optimal Mamu-A*01-restricted SIV gag CTL epitope was CTPYDINQM(181-189). Mamu-A*01 is unique in that it is found at a high frequency in rhesus macaques, and all SIV-infected Mamu-A*01-positive rhesus macaques studied to date develop an immunodominant gag-specific CTL response restricted by this molecule. Identification of the optimal SIV gag CTL epitope will be critical for a variety of studies designed to induce CD8+ CTL responses specific for SIV in the rhesus macaque.

Several common HLA-DR types share largely overlapping peptide binding repertoires.

The peptide binding specificities of HLA-DRB1*0401, DRB1*0101, and DRB1*0701 have been analyzed by the use of large collections of synthetic peptides corresponding to naturally occurring sequences. The results demonstrated that nearly all peptides binding to these DR molecules bear a motif characterized by a large aromatic or hydrophobic residue in position 1 (Y, F, W, L, I, V, M) and a small, noncharged residue in position 6 (S, T, C, A, P, V, I, L, M). In addition, allele-specific secondary effects and secondary anchors were defined, and these parameters were utilized to derive allele-specific motifs and algorithms. By the combined use of such algorithms, peptides capable of degenerate DRB1*0101, DRB1*0401, and DRB1*0701 binding were identified. Additional experiments utilizing a panel of quantitative assays specific for nine additional common DR molecules identified a large set of DR molecules, which includes at least the DRB1*0101, DRB1*0401, DRB1*0701, DRB5*0101, DRB1*1501, DRB1*0901, and DRB1*1302 allelic products, characterized by overlapping peptide-binding repertoires. These results have implications for understanding the molecular interactions involved in peptide-DR binding, as well as the genetic and structural basis of MHC polymorphism. These results also have potential practical implications for the development of epitope-based prophylactic and therapeutic vaccines.

Potent immunogenic short linear peptide constructs composed of B cell epitopes and Pan DR T helper epitopes (PADRE) for antibody responses in vivo.

Induction of humoral immune responses against protein antigen requires that two independent signals be delivered to B cells. It is currently assumed that simple monovalent synthetic peptides would not be effective immunogens for antibody responses because they would not be anticipated to effectively generate the necessary signals unless conjugated to a complex carrier system. In this study, the immunogenicity of short linear peptide constructs comprising Plasmodium vivax B cell epitopes (PVB) and non-natural Pan-DR T helper cell epitopes (PADRE) was assessed in mice and compared to other types of antigen constructs. The 33-residue PADRE-PVB linear constructs were highly immunogenic and induced responses comparable to those obtained with the multiple antigen peptides (MAP) constructs, both in terms of absolute titers and quality of antibody responses. The anti-PVB antibody responses were of long duration, composed mostly of IgG and reactive with intact sporozoites. The PADRE-PVB constructs were immunogenic when formulated in adjuvants such as Alum and Montanide ISA 51 underlining the relevance of these findings for vaccine development.

Two distinct HLA-A*0101-specific submotifs illustrate alternative peptide binding modes.

Previous studies have defined two different peptide binding motifs specific for HLA-A*0101. These motifs are characterized by the presence of tyrosine (Y) at the C-termini of 9-mer and 10-mer peptides, and either a small polar or hydrophobic (S, T, M) residue in position 2, or a negatively charged (D or E) residue in position 3. In this study, the structural requirements for peptide binding to A*0101 have been further analyzed by examining the binding capacity of large sets of peptides corresponding to naturally occurring sequences which bore one or the other of these two A*0101-specific motifs. By correlating the presence of specific residue types at each position along the peptide sequence with increased (or decreased) binding affinity, the prominent influence of secondary anchor residues was revealed. In most cases, the two anchors in positions 2 and 3 appear to act synergistically. With the exception of the DE3 submotif in 9-mer peptides, a positive role for aromatic residues in position 1 and the center of the peptide (positions 4 or 5 of 9- or 10-mer peptides, respectively), and proline at C-3, were also consistently detected. However, secondary anchor residues also appear to differ significantly between the two different submotifs, demonstrating that A*0101 can utilize alternative modes in binding its peptide ligands. According to these analyses, specific refined submotifs were also established, and their merit verified by independent sets of potential A*0101 binding peptides. Besides providing useful insight into the nature of the interaction of the A*0101 allele with its peptide ligands, such refined motifs should also facilitate accurate prediction of potential A*0101-restricted peptide epitopes.

The HLA-A*0207 peptide binding repertoire is limited to a subset of the A*0201 repertoire.

Quantitative A*0207 peptide binding assays have been developed utilizing HLA transfected cells and affinity purified molecules. By using a panel of single substitution analog peptides, it was demonstrated that A*0207 binds peptides with main anchor specificity at position 2 and the C-terminus similar to A*0201. Previous data indicating that A*0207 (but not A*0201) also requires the presence of D or P in position 3 of its peptide ligands was confirmed by the analysis of additional single substituted analogs. Finally, by analyzing the A*0201 and A*0207 binding capacities of panels of unrelated synthetic peptides, it was found that 8/15 (53.3%) A*0201 binders with D or P in position 3 bound A*0207, while only 5/72 (6.9%) A*0201 binders without D or P in position 3 also bound A*0207. Together, these data indicate that although A*0207 may be included amongst A2 supertype alleles, its peptide binding repertoire is largely limited to a subset of that bound by A*0201.

Soy allergy is not common in atopic children: a multicenter study.

The aim of the present study was to evaluate the prevalence of soy allergy (positive skin test and positive challenge test) in a large cohort of atopic children, many of them soy fed early in life for several months. In order to investigate the prevalence of soy allergy, two groups of children were enrolled into the study. The first group comprised a cohort of 505 children with personal history suggestive of food allergy. The second group included 243 children born of atopic parents, who had been soy protein formula fed for the first six months of life for the prevention of cow's milk allergy and who had been prospectively followed up, from birth to 5 years. As regards the prevalence of soy allergy in the cohort of children suffering from allergic disease: 31/505 children (6%) had positive skin prick test to soy, however only six of the 31 children with positive skin prick test to soy had positive challenge test to soy. With regard to the prevalence of soy allergy in the children who had been soy protein formula fed in the first six months of life (second group): 14/243 children (6%) had positive skin prick test to soy, but the double blind placebo control oral food challenge to soy was positive in only one of these 14 children. In conclusion documented soy allergy is not common in atopic children.

Specificity and degeneracy in peptide binding to HLA-B7-like class I molecules.

The HLA-B7-like binding supertype includes several different HLA-B molecules. Herein, the primary and secondary anchor specificities of the five most common HLA-B7-like molecules (B*0702, B*3501, B51, B*5301, and B*5401) were defined by the use of molecular binding assays, analogue peptides, and large sets of peptides corresponding to naturally occurring sequences. All five B7-like molecules analyzed preferentially bound 9-mers, with a stringent requirement for proline in position 2, while a variety of hydrophobic or aromatic residues were well tolerated at the C-terminal anchor position. Although most peptides bound in an allele-specific fashion, approximately 20% of the binders identified were degenerate and bound at least three of the five B7-like molecules analyzed with affinities of 500 nM or less. It was also noted that, in general, peptides that bind with high affinity to any given one B7-like molecule were also most frequently capable of degenerate binding. Prominent roles for secondary anchors in positions 1 and 3 were observed for most B7-like molecules, and secondary anchor motifs were utilized to derive an HLA-B7-like supermotif. The validity of this B7-like supermotif was tested by a blind prediction set. Finally, the B7-like supermotif was utilized to derive a general strategy for rationally engineering peptide analogues of naturally occurring sequences with greatly increased binding affinity and degeneracy. Such engineered supermotif binding peptides may be of significant utility in the development of peptide-based vaccines against chronic viral diseases and cancer.

Definition of an HLA-A3-like supermotif demonstrates the overlapping peptide-binding repertoires of common HLA molecules.

An HLA-A3-like supertype (minimally comprised of products from the HLA class I alleles A3, A11, A31, A*3301, and A*6801) has been defined on the basis of (a) structural similarities in the antigen-binding groove, (b) shared main anchor peptide-binding motifs, (c) the identification of peptides cross-reacting with most or all of these molecules, and (d) the definition of an A3-like supermotif that efficiently predicts highly cross-reactive peptides. Detailed secondary anchor maps for A3, A11, A31, A*3301, and A*6801 are also described. The biologic relevance of the A3-like supertype is indicated by the fact that high frequencies of the A3-like supertype alleles are conserved in all major ethnic groups. Because A3-like supertype alleles are found in most major HLA evolutionary lineages, possibly a reflection of common ancestry, the A3-like supermotif might in fact represent a primeval human HLA class I peptide-binding specificity. It is also possible that these phenomena might be related to optimal exploitation of the peptide specificity by human TAP molecules. The grouping of HLA alleles into supertypes on the basis of their overlapping peptide-binding repertoires represents an alternative to serologic or phylogenetic classification.