CD101 surface expression discriminates potency among murine FoxP3+ regulatory T cells

CD4+CD25+FoxP3+ regulatory T cells (Treg) have been shown to be protective in animal models of autoimmunity and acute graft-vs-host disease. However, owing to the functional heterogeneity among CD4+CD25+ T cells, surface markers expressed selectively on functionally active Treg would be useful for purposes of identifying and isolating such cells. We generated a rabbit mAb against murine CD101, a transmembrane glycoprotein involved in T cell activation. Among freshly isolated T cells, CD101 was detected on 25-30% of CD4+CD25+ Treg and approximately 20% of conventional memory T cells. CD101(high) Treg displayed greater in vitro suppression of alloantigen-driven T cell proliferation as compared with CD101(low) Treg. In a model of graft-vs-host disease induced by allogeneic bone marrow transplantation in vivo bioluminescence imaging demonstrated reduced expansion of donor-derived luciferase-labeled conventional T cells in mice treated with CD101(high) Treg, compared with CD101(low) Treg. Moreover, treatment with CD101(high) Treg resulted in improved survival, reduced proinflammatory cytokine levels and reduced end organ damage. Among the CD101(high) Treg all of the in vivo suppressor activity was contained within the CD62L(high) subpopulation. We conclude that CD101 expression distinguishes murine Treg with potent suppressor activity.     

Evolutionary origins of members of a superfamily of integral membrane cytochrome c biogenesis proteins

We have analyzed the relationships of homologues of the Escherichia coli CcmC protein for probable topological features and evolutionary relationships. We present bioinformatic evidence suggesting that the integral membrane proteins CcmC (E. coli; cytochrome c biogenesis System I), CcmF (E. coli; cytochrome c biogenesis System I) and ResC (Bacillus subtilis; cytochrome c biogenesis System II) are all related. Though the molecular functions of these proteins have not been fully described, they appear to be involved in the provision of heme to c-type cytochromes, and so we have named them the putative Heme Handling Protein (HHP) family (TC #9.B.14). Members of this family exhibit 6, 8, 10, 11, 13 or 15 putative transmembrane segments (TMSs). We show that intragenic triplication of a 2 TMS element gave rise to a protein with a 6 TMS topology, exemplified by CcmC. This basic 6 TMS unit then gave rise to two distinct types of proteins with 8 TMSs, exemplified by ResC and the archaeal CcmC, and these further underwent fusional or insertional events yielding proteins with 10, 11 and 13 TMSs (ResC homologues) as well as 15 TMSs (CcmF homologues). Specific evolutionary pathways taken are proposed. This work provides the first evidence for the pathway of appearance of distantly related proteins required for post-translational maturation of c-type cytochromes in bacteria, plants, protozoans and archaea.     

Mechanisms controlling cell cycle exit upon terminal differentiation

Coordinating terminal differentiation with permanent exit from the cell cycle is crucial for proper organogenesis, yet how the cell cycle is blocked in differentiated tissues remains unclear. Important roles for retinoblastoma family proteins and Cyclin-dependent kinase inhibitors have been delineated, but in many cases it remains unclear what triggers cell cycle exit. This review focuses on describing recent advances in deciphering how terminal differentiation and exit from the cell cycle are coordinated.     

Use of amaranthus leucocarpus lectin to differentiate cervical dysplasia (CIN)

Alterations in O-glycosylation of proteins in cell surfaces can originate disorder in cellular function, as well as in cell transformation and tumoral differentiation. In this work, we investigate changes in O-glycosylation in cervical intraepithelial dysplasia (CIN) at different stages of differentiation (CIN I, CIN II, and CIN III) using lectins specific for O-glycosidically linked glycans. Twenty cases with CIN I, CIN II, and CIN III dysplasias each, and 20 normal cases were studied by lectin histochemistry and evaluated under optical microscopy. The lectins from Glycine max and Griffonia simplicifolia showed no differences in their recognition pattern among the different CIN stages and normal tissue. Dolichos Biflorus lectin recognized CIN I dysplasia. Lectin from Amaranthus leucocarpus showed increased reactivity in the presence of CIN II dysplasia, compared with CIN I and CIN III. These results suggest that subtle modifications in the O-glycosylation pattern could be considered in diagnosis or prognosis of cervical precancerous stages.     

Paradoxical effect of salbutamol in a model of acute organophosphates intoxication in guinea pigs: role of substance P release

Organophosphates induce bronchoobstruction in guinea pigs, and salbutamol only transiently reverses this effect, suggesting that it triggers additional obstructive mechanisms. To further explore this phenomenon, in vivo (barometric plethysmography) and in vitro (organ baths, including ACh and substance P concentration measurement by HPLC and immunoassay, respectively; intracellular Ca2+) measurement in single myocytes) experiments were performed. In in vivo experiments, parathion caused a progressive bronchoobstruction until a plateau was reached. Administration of salbutamol during this plateau decreased bronchoobstruction up to 22% in the first 5 min, but thereafter airway obstruction rose again as to reach the same intensity as before salbutamol. Aminophylline caused a sustained decrement (71%) of the parathion-induced bronchoobstruction. In in vitro studies, paraoxon produced a sustained contraction of tracheal rings, which was fully blocked by atropine but not by TTX, omega-conotoxin (CTX), or epithelium removal. During the paraoxon-induced contraction, salbutamol caused a temporary relaxation of approximately 50%, followed by a partial recontraction. This paradoxical recontraction was avoided by the M2- or neurokinin-1 (NK1)-receptor antagonists (methoctramine or AF-DX 116, and L-732138, respectively), accompanied by a long-lasting relaxation. Forskolin caused full relaxation of the paraoxon response. Substance P and, to a lesser extent, ACh released from tracheal rings during 60-min incubation with paraoxon or physostigmine, respectively, were significantly increased when salbutamol was administered in the second half of this period. In myocytes, paraoxon did not produce any change in the intracellular Ca2+ basal levels. Our results suggested that: 1) organophosphates caused smooth muscle contraction by accumulation of ACh released through a TTX- and CTX-resistant mechanism; 2) during such contraction, salbutamol relaxation is functionally antagonized by the stimulation of M2 receptors; and 3) after this transient salbutamol-induced relaxation, a paradoxical contraction ensues due to the subsequent release of substance P.     

Incorporation of CpG oligonucleotide ligand into protein-loaded particle vaccines promotes antigen-specific CD8 T-cell immunity

The development of multicomponent biotherapeutic carriers is an important challenge in the field of drug delivery, particularly in the area of protein-based vaccines. While the delivery of protein antigens to antigen presenting cells (APCs) is crucial for this type of vaccination, the incorporation of additional adjuvants may be just as important in order to generate more potent immune responses. This article presents the synthesis and biological evaluation of carrier particles that both deliver a protein payload to APCs and display receptor ligands for the enhancement of APC immunostimulation. Particles displaying CpG oligonucleotide ligands for Toll-like receptor 9 were synthesized. The addition of CpG DNA to the particles led to a 45-fold increase in the secretion of interleukin-12, a cytokine that aids in T-cell activation, and a significant increase in the expression of costimulatory molecules by APCs. Moreover, vaccination with particles containing both ovalbumin (OVA) and CpG DNA induced a superior OVA-specific CD8 T-cell response in vivo, as measured by increased OVA-specific CD8 T-cell proliferation, secretion of the proinflammatory cytokine IFN-gamma, and the induction of OVA-specific cytotoxicity.     

Assessment of algorithms for high throughput detection of genomic copy number variation in oligonucleotide microarray data

Background

Genomic deletions and duplications are important in the pathogenesis of diseases, such as cancer and mental retardation, and have recently been shown to occur frequently in unaffected individuals as polymorphisms. Affymetrix GeneChip whole genome sampling analysis (WGSA) combined with 100 K single nucleotide polymorphism (SNP) genotyping arrays is one of several microarray-based approaches that are now being used to detect such structural genomic changes. The popularity of this technology and its associated open source data format have resulted in the development of an increasing number of software packages for the analysis of copy number changes using these SNP arrays.

Results

We evaluated four publicly available software packages for high throughput copy number analysis using synthetic and empirical 100 K SNP array data sets, the latter obtained from 107 mental retardation (MR) patients and their unaffected parents and siblings. We evaluated the software with regards to overall suitability for high-throughput 100 K SNP array data analysis, as well as effectiveness of normalization, scaling with various reference sets and feature extraction, as well as true and false positive rates of genomic copy number variant (CNV) detection.

Conclusion

We observed considerable variation among the numbers and types of candidate CNVs detected by different analysis approaches, and found that multiple programs were needed to find all real aberrations in our test set. The frequency of false positive deletions was substantial, but could be greatly reduced by using the SNP genotype information to confirm loss of heterozygosity.