Correlation of effector function with phenotype and cell division after in vitro differentiation of naive MART-1-specific CD8+ T cells.

Adoptive transfer of antigen-specific CD8+ T cells may represent an effective strategy for immunotherapy of tumors such as melanoma, but is limited by the number and functionality of in vitro expanded T cells. Here, we document that although ELAGIGILTV-specific CD8+ T cells from different donors initially possessed a naive phenotype, after antigen-induced in vitro expansion two distinct phenotypes correlating with cell proliferation rate emerged in the different donors. Those cultures achieving fewer cumulative population doublings (CPDs) were cytotoxic and displayed a CD45RA+CCR7- phenotype. In contrast, cultures reaching higher CPDs were non-cytotoxic T cells with a CD45RA-CCR7- phenotype. Thus, the generation of larger numbers of ELAGIGILTV-specific CD8+ T cells correlates negatively with the acquisition of a CD45RA+CCR7- phenotype and cytotoxic capacity. A better understanding of the differentiation pathways of cytotoxic T cells to obtain optimally efficient cells for adoptive transfer will allow the development of new immunotherapy protocols.

TIPSS procedure in the treatment of a single patient after recent heart transplantation because of refractory ascites due to cardiac cirrhosis.

We present the case of a female patient with arrhythmogenic dysplasia of the right ventricle who evolved to refractory heart failure, ascites, and peripheral edema. As a result, heart transplantation was performed. Subsequently, refractory ascites impaired the patient's respiratory function, resulting in prolonged mechanical ventilation. She was successfully treated with transjugular intrahepatic portosystemic shunt (TIPSS) placement, which allowed satisfactory weaning of ventilatory support.

Peptide rearrangement during quadrupole ion trap fragmentation: added complexity to MS/MS spectra.

The emergence of proteomics has placed great interest in the understanding of the mechanisms of MS/MS fragmentation of peptides under low-energy collision-induced dissociation. In this work, we describe the presence of anomalous fragments, which correspond to neutral loss elimination of internal amino acids from ions of the b series in quadrupole ion trap MS/MS spectra from naturally occurring peptides. Internal amino acid elimination occurred preferentially with aliphatic amino acids. The phenomenon was more apparent when doubly charged precursors were fragmented and was inhibited when peptides were N-acetylated at the N-terminus. Fragmentation of isomeric peptides where some internal amino acids were relocated in N-terminal position produced MSn spectra indistinguishable from those of the original peptides, indicating that some b ions underwent a structural rearrangement process. Formation of anomalous fragments required a minimum activation time. Our data are consistent with a nucleophile attack of the N-terminal nitrogen over the electrophilic carbonyl carbon at one peptide bond, forming a cyclic b ion intermediate that, by reopening at preferential sites, exposes internal amino acids to the C-terminal side.

Direct visualization by confocal fluorescent microscopy of the permeation of myristoylated peptides through the cell membrane.

To study the permeability through the cellular membrane of synthetic peptides containing an hydrophobic moiety, we used a 13-mer myristoylated peptide labeled with a N-terminal fluorescent probe. After 2 h of incubation, the subcellular distribution was analyzed in intact chromaffin cells by confocal fluorescent microscopy. Our results demonstrate that myristoylated peptides diffuse into intact cells, showing an heterogeneous distribution, but they do not reach the cellular nucleous, at least during the time range used.