Terminal deoxynucleotidyltransferase is required for the establishment of private virus-specific CD8+ TCR repertoires and facilitates optimal CTL responses

Virus-immune CD8(+) TCR repertoires specific for particular peptide-MHC class I complexes may be substantially shared between (public), or unique to, individuals (private). Because public TCRs can show reduced TdT-mediated N-region additions, we analyzed how TdT shapes the heavily public (to D(b)NP(366)) and essentially private (to D(b)PA(224)) CTL repertoires generated following influenza A virus infection of C57BL/6 (B6, H2(b)) mice. The D(b)NP(366)-specific CTL response was virtually clonal in TdT(-/-) B6 animals, with one of the three public clonotypes prominent in the wild-type (wt) response consistently dominating the TdT(-/-) set. Furthermore, this massive narrowing of TCR selection for D(b)NP(366) reduced the magnitude of D(b)NP(366)-specific CTL response in the virus-infected lung. Conversely, the D(b)PA(224)-specific responses remained comparable in both magnitude and TCR diversity within individual TdT(-/-) and wt mice. However, the extent of TCR diversity across the total population was significantly reduced, with the consequence that the normally private wt D(b)PA(224)-specific repertoire was now substantially public across the TdT(-/-) mouse population. The key finding is thus that the role of TdT in ensuring enhanced diversity and the selection of private TCR repertoires promotes optimal CD8(+) T cell immunity, both within individuals and across the species as a whole.     

Yeast Est2p affects telomere length by influencing association of Rap1p with telomeric chromatin

In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.     

Herpesviral inclusion body disease in owls and falcons is caused by the pigeon herpesvirus (columbid herpesvirus 1)

A herpesviral disease of Rock Pigeons (Columba livia), called "inclusion body disease" or "inclusion body hepatitis," was first described in the 1940s. The disease involves hepatic and splenic necrosis with associated intranuclear inclusion bodies and occurs primarily in young squabs. A similar herpesviral disease occurs in falcons and owls. Serologic and restriction endonuclease digestion studies indicate that herpesviruses from pigeons, falcons, and owls are very closely related and that most reported cases of disease in falcons and owls involve prior documented or possible ingestion of pigeons. These findings led to the hypothesis that an endemic herpesvirus of pigeons may be causing disease in falcons and owls. In order to test this hypothesis, we sequenced a fragment of the herpesviral DNA polymerase gene from naturally infected owls, falcons, and pigeons with inclusion body disease collected between 1991 and 2006. Sequences from all three sources were almost identical, and we therefore propose that the usual agent of inclusion body hepatitis in owls and falcons is columbid herpesvirus 1.     

Bacterial communities of Bartonella-positive fleas: diversity and community assembly patterns

We investigated the bacterial communities of nine Bartonella-positive fleas (n = 6 Oropsylla hirsuta fleas and n = 3 Oropsylla montana fleas), using universal primers, clone libraries, and DNA sequencing. DNA sequences were used to classify bacteria detected in a phylogenetic context, to explore community assembly patterns within individual fleas, and to survey diversity patterns in dominant lineages.     

Detection of phencyclidine in human oral fluid using solid-phase extraction and liquid chromatography with tandem mass spectrometric detection

An analytical procedure for the determination of phencyclidine in oral fluid has been developed and validated using liquid chromatography with tandem mass spectral detection, following initial screening with enzyme linked immunosorbent assay. The oral fluid samples were collected using the Quantisal device, and any drugs present were quantified using mixed mode solid-phase extraction followed by mass spectrometric detection in positive atmospheric pressure chemical ionization mode. For confirmation, two transitions were monitored and one ratio determined, which had to be within 20% of that of the known calibration standard. The monitoring of the qualifying transition and requirement for its presence within a specific ratio to the primary ion has the potential of limiting the sensitivity of the assay, however, the additional confidence in the final result as well as forensic defensibility were considered to be of greater importance. The limit of quantitation was 5ng/mL; the intra-day precision of the assay (n=5) was 3.04%; inter-day precision 3.35% (n=5) at a concentration of 10ng/mL. The accuracy was determined at four concentrations (5, 10, 20 and 40ng/mL) within the linear range of the assay. The percentage recovery of phencyclidine from the oral fluid collection pad was 81.7% (n=6). The methods were applied to both proficiency specimens and to samples obtained during research studies in the USA.     

Cord blood ASP is predicted by maternal lipids and correlates with fetal birth weight

Background: The acylation stimulating protein (ASP) is a potent lipogenic adipokine that correlates with postprandial triglyceride (TG) clearance and is linked to the pathophysiology of obesity and related disorders. Objective: To investigate ASP levels in cord blood and its relation to maternal and cord blood lipid parameters and fetal birth weight. Methods and Procedures: Thirty nondiabetic pregnant women, their newborns, and thirty‐three nonpregnant controls were included in this study. Fasting maternal and cord blood ASP, TGs, nonesterified fatty acids (NEFAs), cholesterol, glucose levels, in addition to maternal BMI and fetal birth weight were measured. Results: No significant difference was found between cord blood ASP (16.3 ± 0.96 nmol/l) and ASP levels in the adult controls (15.7 ± 1.0 nmol/l). Cord blood ASP, however, was lower than maternal plasma ASP levels (25.4 ± 1.6 nmol/l, P < 0.001). Yet, lipid levels in cord blood, particularly TGs were markedly decreased compared to control and maternal TG levels (threefold and 7.4‐fold, P < 0.001 respectively). Maternal TGs significantly correlated with fetal birth weight (r = 0.54, P = 0.002). Multiple regression analysis showed that maternal TGs (β = 0.57, P = 0.01) and NEFAs (β = 0.43, P = 0.024) predicted 45% variation in cord blood ASP levels, independent of all measured maternal and cord blood parameters. Cord blood ASP showed a positive correlation with fetal birth weight (r = 0.524, P = 0.037) in neonates above average fetal birth weight of the studied population. Discussion: This is the first study investigating ASP in cord blood. We suggest that maternal hypertriglyceridemia is associated with increased fetal ASP production, thus enhancing fetal fat storage independent of maternal glucose variations in nondiabetic women.     

Age-specific differences in oncogenic pathway deregulation seen in human breast tumors

Purpose

To define the biology driving the aggressive nature of breast cancer arising in young women.

Experimental Design

Among 784 patients with early stage breast cancer, using prospectively-defined, age-specific cohorts (young ≤45 years; older ≥65 years), 411 eligible patients (n = 200≤45 years; n = 211≥65 years) with clinically-annotated Affymetrix microarray data were identified. GSEA, signatures of oncogenic pathway deregulation and predictors of chemotherapy sensitivity were evaluated within the two age-defined cohorts.

Results

In comparing deregulation of oncogenic pathways between age groups, a higher probability of PI3K (p = 0.006) and Myc (p = 0.03) pathway deregulation was observed in breast tumors arising in younger women. When evaluating unique patterns of pathway deregulation, a low probability of Src and E2F deregulation in tumors of younger women, concurrent with a higher probability of PI3K, Myc, and β-catenin, conferred a worse prognosis (HR = 4.15). In contrast, a higher probability of Src and E2F pathway activation in tumors of older women, with concurrent low probability of PI3K, Myc and β-catenin deregulation, was associated with poorer outcome (HR = 2.7). In multivariate analyses, genomic clusters of pathway deregulation illustrate prognostic value.

Conclusion

Results demonstrate that breast cancer arising in young women represents a distinct biologic entity characterized by unique patterns of deregulated signaling pathways that are prognostic, independent of currently available clinico-pathologic variables. These results should enable refinement of targeted treatment strategies in this clinically challenging situation.     

Identification of the endostyle as a stem cell niche in a colonial chordate

Stem cell populations exist in "niches" that hold them and regulate their fate decisions. Identification and characterization of these niches is essential for understanding stem cell maintenance and tissue regeneration. Here we report on the identification of a novel stem cell niche in Botryllus schlosseri, a colonial urochordate with high stem cell-mediated developmental activities. Using in vivo cell labeling, engraftment, confocal microscopy, and time-lapse imaging, we have identified cells with stemness capabilities in the anterior ventral region of the Botryllus' endostyle. These cells proliferate and migrate to regenerating organs in developing buds and buds of chimeric partners but do not contribute to the germ line. When cells are transplanted from the endostyle region, they contribute to tissue development and induce long-term chimerism in allogeneic tissues. In contrast, cells from other Botryllus' regions do not show comparable stemness capabilities. Cumulatively, these results define the Botryllus' endostyle region as an adult somatic stem cell niche.     

Mechanical unloading impairs keratinocyte migration and angiogenesis during cutaneous wound healing.

Although initially thought to improve an individual's ability to heal, mechanical unloading promoted by extended periods of bed rest has emerged as a contributing factor to delayed or aberrant tissue repair. Using a rat hindlimb unloading (HLU) model of hypogravity, we mimicked some aspects of physical inactivity by removing weight-bearing loads from the hindlimbs and producing a systemic cephalic fluid shift. This model simulates bed rest in that the animal undergoes physiological adaptations, resulting in a reduction in exercise capability, increased frequency of orthostatic intolerance, and a reduction in plasma volume. To investigate whether changes associated with prior prolonged bed rest correlate with impaired cutaneous wound healing, we examined wound closure, angiogenesis, and collagen content in day 2 to day 21 wounds from rats exposed to HLU 2 wk before excisional wounding. Wound closure was delayed in day 2 wounds from HLU rats compared with ambulatory controls. Although the levels of proangiogenic growth factors, fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor (VEGF) were similar between the two groups, wound vascularity was significantly reduced in day 7 wounds from HLU animals. To further examine this disparity, total collagen content was assessed but found to be similar between the two groups. Taken together, these results suggest that keratinocyte and endothelial cell function may be impaired during the wound healing process under periods of prolonged inactivity or bed rest.