Direct intracardiac injection of umbilical cord-derived stromal cells and umbilical cord blood-derived endothelial cells for the treatment of ischemic cardiomyopathy

The development of new therapeutic strategies is necessary to reduce the worldwide social and economic impact of cardiovascular disease, which produces high rates of morbidity and mortality. A therapeutic option that has emerged in the last decade is cell therapy. The aim of this study was to compare the effect of transplanting human umbilical cord-derived stromal cells (UCSCs), human umbilical cord blood-derived endothelial cells (UCBECs) or a combination of these two cell types for the treatment of ischemic cardiomyopathy (IC) in a Wistar rat model. IC was induced by left coronary artery ligation, and baseline echocardiography was performed seven days later. Animals with a left ventricular ejection fraction (LVEF) of ≤40% were selected for the study. On the ninth day after IC was induced, the animals were randomized into the following experimental groups: UCSCs, UCBECs, UCSCs plus UCBECs, or vehicle (control). Thirty days after treatment, an echocardiographic analysis was performed, followed by euthanasia. The animals in all of the cell therapy groups, regardless of the cell type transplanted, had less collagen deposition in their heart tissue and demonstrated a significant improvement in myocardial function after IC. Furthermore, there was a trend of increasing numbers of blood vessels in the infarcted area. The median value of LVEF increased by 7.19% to 11.77%, whereas the control group decreased by 0.24%. These results suggest that UCSCs and UCBECs are promising cells for cellular cardiomyoplasty and can be an effective therapy for improving cardiac function following IC.     

High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Brief antigenic stimulation generates effector CD8 T cells with low cytotoxic activity and high IL-2 production

It is currently believed that a brief antigenic stimulation is sufficient to induce CD8 T cells to complete their differentiation program, become effector T cells, and subsequently generate memory. Because this concept was derived from studies in which only a single effector function was analyzed (either IFN-gamma production or target cell lysis), we wondered whether monitoring for multiple effector functions might reveal novel characteristics of effector CD8 T cells elicited by brief or prolonged Ag exposure. Using an in vitro system to generate effector T cells and an in vivo adoptive transfer model to track donor CD8 T cells, we found that the differentiation programs acquired by CD8 T cells after brief or prolonged antigenic stimulation were different. Although the frequencies of IFN-gamma and TNF-alpha producers were comparable for both effector CD8 T cell populations, there were major differences in cytotoxic potential and IL-2 production. Whereas prolonged (>24 h) Ag exposure stimulated effector CD8 T cells with high cytotoxic activity and low IL-2 production, brief (<24 h) stimulation generated effector CD8 T cells with low cytotoxic activity and high IL-2 production. The latter effector T cells rapidly converted into central memory-like CD8 T cells, exhibited long-term survival in adoptively transferred hosts, and gave robust recall responses upon Ag challenge. These data suggest that not all functions of effector CD8 T cells are equally inherited after brief or prolonged antigenic stimulation.     

Mapping essential domains of Mycobacterium smegmatis WhmD: insights into WhiB structure and function

A growing body of evidence suggests that the WhiB-like proteins exclusive to the GC-rich actinomycete genera play significant roles in pathogenesis and cell division. Each of these proteins contains four invariant cysteine residues and a conserved helix-turn-helix motif. whmD, the Mycobacterium smegmatis homologue of Streptomyces coelicolor whiB, is essential in M. smegmatis, and the conditionally complemented mutant M. smegmatis 628-53 undergoes filamentation under nonpermissive conditions. To identify residues critical to WhmD function, we developed a cotransformation-based assay to screen for alleles that complement the filamentation phenotype of M. smegmatis 628-53 following inducer withdrawal. Mycobacterium tuberculosis whiB2 and S. coelicolor whiB complemented the defect in M. smegmatis 628-53, indicating that these genes are true functional orthologues of whmD. Deletion analysis suggested that the N-terminal 67 and C-terminal 12 amino acid residues are dispensable for activity. Site-directed mutagenesis indicated that three of the four conserved cysteine residues (C90, C93, and C99) and a conserved aspartate (D71) are essential. Mutations in a predicted loop glycine (G111) and an unstructured leucine (L116) were poorly tolerated. The region essential for WhmD activity encompasses 6 of the 10 residues conserved in all seven M. tuberculosis WhiBs, as well as in most members of the WhiB family identified thus far. WhmD structure was found to be sensitive to the presence of a reducing agent, suggesting that the cysteine residues are involved in coordinating a metal ion. Iron-specific staining strongly suggested that WhmD contains a bound iron atom. With this information, we have now begun to comprehend the functional significance of the conserved sequence and structural elements in this novel family of proteins.     

Characterization of the low molecular weight human serum proteome

Serum potentially carries an archive of important histological information whose determination could serve to improve early disease detection. The analysis of serum, however, is analytically challenging due to the high dynamic concentration range of constituent protein/peptide species, necessitating extensive fractionation prior to mass spectrometric analyses. The low molecular weight (LMW) serum proteome is that protein/peptide fraction from which high molecular weight proteins, such as albumin, immunoglobulins, transferrin, and lipoproteins, have been removed. This LMW fraction is made up of several classes of physiologically important proteins such as cytokines, chemokines, peptide hormones, as well as proteolytic fragments of larger proteins. Centrifugal ultrafiltration of serum was used to remove the large constituent proteins resulting in the enrichment of the LMW proteins/peptides. Because albumin is known to bind and transport small molecules and peptides within the circulatory system, the centrifugal ultrafiltration was conducted under solvent conditions effecting the disruption of protein-protein interactions. The LMW serum proteome sample was digested with trypsin, fractionated by strong cation exchange chromatography, and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. Analysis of the tandem mass spectra resulted in the identification of over 340 human serum proteins; however, not a single peptide from serum albumin was observed. The large number of proteins identified demonstrates the efficacy of this method for the removal of large abundant proteins and the enrichment of the LMW serum proteome.     

Most nuclear systemic autoantigens are extremely disordered proteins: implications for the etiology of systemic autoimmunity

Patients with systemic autoimmune diseases usually produce high levels of antibodies to self-antigens (autoantigens). The repertoire of common autoantigens is remarkably limited, yet no readily understandable shared thread links these apparently diverse proteins. Using computer prediction algorithms, we have found that most nuclear systemic autoantigens are predicted to contain long regions of extreme structural disorder. Such disordered regions would generally make poor B cell epitopes and are predicted to be under-represented as potential T cell epitopes. Consideration of the potential role of protein disorder may give novel insights into the possible role of molecular mimicry in the pathogenesis of autoimmunity. The recognition of extreme autoantigen protein disorder has led us to an explicit model of epitope spreading that explains many of the paradoxical aspects of autoimmunity – in particular, the difficulty in identifying autoantigen-specific helper T cells that might collaborate with the B cells activated in systemic autoimmunity. The model also explains the experimentally observed breakdown of major histocompatibility complex (MHC) class specificity in peptides associated with the MHC II proteins of activated autoimmune B cells, and sheds light on the selection of particular T cell epitopes in autoimmunity. Finally, the model helps to rationalize the relative rarity of clinically significant autoimmunity despite the prevalence of low specificity/low avidity autoantibodies in normal individuals.