Cloning and characterization of the mouse homologue of the human dendritic cell maturation marker CD208/DC-LAMP

DC-LAMP, a member of the lysosomal-associated membrane protein (LAMP) family, is specifically expressed by human dendritic cells (DC) upon activation and therefore serves as marker of human DC maturation. DC-LAMP is detected first in activated human DC within MHC class II molecules-containing compartments just before the translocation of MHC class II-peptide complexes to the cell surface, suggesting a possible involvement in this process. The present study describes the cloning and characterization of mouse DC-LAMP, whose predicted protein sequence is over 50% identical to the human counterpart. The mouse DC-LAMP gene spans over 25 kb and shares syntenic chromosomal localization (16B2-B4 and 3q26) and conserved organization with the human DC-LAMP gene. Analysis of mouse DC-LAMP mRNA and protein revealed the expression in lung peripheral cells, but also its unexpected absence from mouse lymphoid organs and from mouse DC activated either in vitro or in vivo. In conclusion, mouse DC-LAMP is not a marker of mature mouse DC and this observation raises new questions regarding the role of human DC-LAMP in human DC.

High concentrations of dexamethasone suppresses the proliferation but not the differentiation of further maturation of human osteoblast precursor in vitro: relevance to glucocorticoid-induced osteoporosis

Objective. The use of glucocorticoids (GCs) in the treatment of RA is a frequent cause of bone loss. In vitro, however, this same class of steroids has been shown to promote the recruitment and/or maturation of primitive osteogenic precursors present in the colony forming unit-fibroblastic (CFU-F) fraction of human bone and marrow. In an effort to reconcile these conflicting observations, we investigated the effects of the synthetic GC dexamethasone (Dx) on parameters of growth and osteogenic differentiation in cultures of bone marrow stromal cells derived from a large cohort of adult human donors (n=30). Methods. Marrow suspensions were cultured in the absence and presence of Dx at concentrations between 10 pm and 1 µm. After 28 days we determined the number and diameter of colonies formed, the total number of cells, the surface expression of receptors for selected growth factors and extracellular matrix proteins and, based on the expression of the developmental markers alkaline phosphatase (AP) and the antigen recognized by the STRO-1 monoclonal antibody, the proportion of cells undergoing osteogenic differentiation and their extent of maturation. Results. At a physiologically equivalent concentration, Dx had no effect on the adhesion of CFU-F or on their subsequent proliferation, but did promote their osteogenic differentiation and further maturation. These effects were independent of changes in the expression of the receptors for fibroblast growth factors, insulin-like growth factor 1, nerve growth factor, platelet-derived growth factors and parathyroid hormone/parathyroid hormone-related protein, but were associated with changes in the number of cells expressing the 2 and 4, but not ß1, integrin subunits. At supraphysiological concentrations, the effects of Dx on the osteogenic recruitment and maturation of CFU-F and their progeny were maintained but at the expense of a decrease in cell number. Conclusions. A decrease in the proliferation of osteogenic precursors, but not in their differentiation or maturation, is likely to be a key factor in the genesis of GC-induced bone loss.

Protein expression profiling during chick retinal maturation: a proteomics-based approach

Background: The underlying pathways that drive retinal neurogenesis and synaptogenesis are still relatively poorly understood. Protein expression analysis can provide direct insight into these complex developmental processes. The aim of this study was therefore to employ proteomic analysis to study the developing chick retina throughout embryonic (E) development commencing at day 12 through 13, 17, 19 and post-hatch (P) 1 and 33 days.

Results: 2D proteomic and mass spectrometric analysis detected an average of 1514 spots per gel with 15 spots demonstrating either modulation or constitutive expression identified via MS. Proteins identified included alpha and beta-tubulin, alpha enolase, B-creatine kinase, gamma-actin, platelet-activating factor (PAF), PREDICTED: similar to TGF-beta interacting protein 1, capping protein (actin filament muscle Z line), nucleophosmin 1 (NPM1), dimethylarginine dimethylaminohydrolase, triosphoaphate isomerase, DJ1, stathmin, fatty acid binding protein 7 (FABP7/B-FABP), beta-synuclein and enhancer of rudimentary homologue.

Conclusion: This study builds upon previous proteomic investigations of retinal development and represents the addition of a unique data set to those previously reported. Based on reported bioactivity some of the identified proteins are most likely to be important to normal retinal development in the chick. Continued analysis of the dynamic protein populations present at the early stages and throughout retinal development will increase our understanding of the molecular events underpinning retinogenesis.

Intracellular survival of Burkholderia cenocepacia in macrophages is associated with a delay in the maturation of bacteria-containing vacuoles

Strains of the Burkholderia cepacia complex (Bcc) are opportunistic bacteria that can cause life-threatening infections in patients with cystic fibrosis and chronic granulomatous disease. Previous work has shown that Bcc isolates can persist in membrane-bound vacuoles within amoeba and macrophages without bacterial replication, but the detailed mechanism of bacterial persistence is unknown. In this study, we have investigated the survival of the Burkholderia cenocepacia strain J2315 within RAW264.7 murine macrophages. Strain J2315 is a prototypic isolate of the widespread and transmissible ET12 clone. Unlike heat-inactivated bacteria, which reach lysosomes shortly after internalization, vacuoles containing live B. cenocepacia J2315 accumulate the late endosome/lysosome marker LAMP-1 and start fusing with lysosomal compartments only after 6 h post internalization. Using fluorescent fluid-phase probes, we also demonstrated that B. cenocepacia-containing vacuoles continued to interact with newly formed endosomes, and maintained a luminal pH of 6.4 +/- 0.12. In contrast, vacuoles containing heat-inactivated bacteria had an average pH of 4.8 +/- 0.03 and rapidly merged with lysosomes. Additional experiments using concanamycin A, a specific inhibitor of the vacuolar H+-ATPase, revealed that vacuoles containing live bacteria did not exclude the H+-ATPase. This mode of bacterial survival did not require type III secretion, as no differences were found between wild type and a type III secretion mutant strain. Collectively, our results suggest that intracellular B. cenocepacia cause a delay in the maturation of the phagosome, which may contribute to facilitate bacterial escape from the microbicidal activities of the host cell.

Cognitive maturation in preterm and term born adolescents

Background: Adolescence is a critical period of brain structural reorganisation and maturation of cognitive abilities. This relatively late developmental reorganisation may be altered in individuals who were born preterm.

Methods: We carried out longitudinal neuropsychological testing in 94 very preterm individuals (VPT; before 33 weeks' gestation) and 44 term born individuals at mean ages of 15.3 years ( adolescence) and 19.5 years (young adulthood).

Results: Full scale, verbal and performance IQ and phonological verbal fluency were significantly lower in the VPT group than the term group at both ages. Repeated measures ANOVA showed only one group by time point interaction for semantic verbal fluency (F= 10.25; df = 107; p = 0.002). Paired- sample t tests showed that semantic verbal fluency increased significantly in the term group over adolescence (t = -5.10; df = 42; p < 0.001), but did not increase in the VPT group (t = 0.141; df = 69; p = 0.889). For verbal IQ, there was a significant interaction between time point and sex (F = 4.48; df = 1; p = 0.036) with paired- sample t tests showing that verbal IQ decreased in males between adolescence and adulthood (t = 3.35; df = 71; p = 0.001), but did not change significantly in females (t = 0.20; df = 52; p = 0.845).

Conclusion: Decrements of intellectual functioning in VPT individuals persist into adulthood. Additionally, there is a deficit in the adolescent maturation of semantic verbal fluency in individuals born VPT.

Slower postnatal growth is associated with delayed cerebral cortical maturation in preterm newborns

Slower postnatal growth is an important predictor of adverse neurodevelopmental outcomes in infants born preterm. However, the relationship between postnatal growth and cortical development remains largely unknown. Therefore, we examined the association between neonatal growth and diffusion tensor imaging measures of microstructural cortical development in infants born very preterm. Participants were 95 neonates born between 24 and 32 weeks gestational age studied twice with diffusion tensor imaging: scan 1 at a median of 32.1 weeks (interquartile range, 30.4 to 33.6) and scan 2 at a median of 40.3 weeks (interquartile range, 38.7 to 42.7). Fractional anisotropy and eigenvalues were recorded from 15 anatomically defined cortical regions. Weight, head circumference, and length were recorded at birth and at the time of each scan. Growth between scans was examined in relation to diffusion tensor imaging measures at scans 1 and 2, accounting for gestational age, birth weight, sex, postmenstrual age, known brain injury (white matter injury, intraventricular hemorrhage, and cerebellar hemorrhage), and neonatal illness (patent ductus arteriosus, days intubated, infection, and necrotizing enterocolitis). Impaired weight, length, and head growth were associated with delayed microstructural development of the cortical gray matter (fractional anisotropy: P <0.001), but not white matter (fractional anisotropy: P = 0.529), after accounting for prenatal growth, neonatal illness, and brain injury. Avoiding growth impairment during neonatal care may allow cortical development to proceed optimally and, ultimately, may provide an opportunity to reduce neurological disabilities related to preterm birth.

The spirit of palliative practice:A qualitative inquiry into the spiritual journey of palliative care physicians

Objective: Much is known about the important role of spirituality in the delivery of multidimensional care for patients at the end of life. Establishing a strong physician-patient relationship in a palliative context requires physicians to have the self-awareness essential to establishing shared meaning and relationships with their patients. However, little is known about this phenomenon and therefore, this study seeks a greater understanding of physician spirituality and how caring for the terminally ill influences this inner aspect. Method: A qualitative descriptive study was used involving face-to-face interviews with six practicing palliative care physicians. Results: Conceptualized as a separate entity from religion, spirituality was described by participants as a notion relating to meaning, personal discovery, self-reflection, support, connectedness, and guidance. Spirituality and the delivery of care for the terminally ill appeared to be interrelated in a dynamic relationship where a physician's spiritual growth occurred as a result of patient interaction and that spiritual growth, in turn, was essential for providing compassionate care for the palliative patient. Spirituality also served as an influential force for physicians to engage in self-care practices. Significance of results: With spirituality as a pervasive force not only in the lives of palliative care patients, but also in those of healthcare providers, it may prove to be beneficial to use this information to guide future practice in training and education for palliative physicians in both the spiritual care of patients and in practitioner self care. Copyright © Cambridge University Press 2010.