α1G T-type calcium channel determines the angiogenic potential of pulmonary microvascular endothelial cells.

Pulmonary microvascular endothelial cells (PMVECs) display a rapid angioproliferative phenotype, essential for maintaining homeostasis in steady-state and promoting vascular repair after injury. Although it has long been established that endothelial cytosolic Ca2+ ([Ca2+]i) transients are required for proliferation and angiogenesis, mechanisms underlying such regulation and the transmembrane channels mediating the relevant [Ca2+]i transients remain incompletely understood. In the present study, the functional role of the microvascular endothelial site-specific α1G T-type Ca2+ channel in angiogenesis was examined. PMVECs intrinsically possess an in vitro angiogenic "network formation" capacity. Depleting extracellular Ca2+ abolishes network formation, whereas blockade of vascular endothelial growth factor receptor or nitric oxide synthase has little or no effect, suggesting that the network formation is a [Ca2+]i-dependent process. Blockade of the T-type Ca2+ channel or silencing of α1G, the only voltage-gated Ca2+ channel subtype expressed in PMVECs, disrupts network formation. In contrast, blockade of canonical transient receptor potential (TRP) isoform 4 or TRP vanilloid 4, two other Ca2+ permeable channels expressed in PMVECs, has no effect on network formation. T-type Ca2+ channel blockade also reduces proliferation, cell-matrix adhesion, and migration, three major components of angiogenesis in PMVECs. An in vivo study demonstrated that the mice lacking α1G exhibited a profoundly impaired postinjury cell proliferation in the lungs following lipopolysaccharide challenge. Mechanistically, T-type Ca2+ channel blockade reduces Akt phosphorylation in a dose-dependent manner. Blockade of Akt or its upstream activator, phosphatidylinositol-3-kinase (PI3K), also impairs network formation. Altogether, these findings suggest a novel functional role for the α1G T-type Ca2+ channel to promote the cell's angiogenic potential via a PI3K-Akt signaling pathway.

Basophil Activation-Dependent Autoantibody and Interleukin-17 Production Exacerbate Systemic Lupus Erythematosus.

OBJECTIVE: Autoantibody and inflammatory cytokines play crucial roles in the development of systemic lupus erythematosus (SLE); however, the regulation of their production warrants further investigation. This study aimed to investigate the role of basophil activation in the development of SLE based on studies in patients with SLE and spontaneous lupus-prone MRL-lpr/lpr mice. METHODS: The phenotypes of peripheral basophils and the production of autoantibody and interleukin (IL)-17 in patients with SLE were determined by flow cytometry and enzyme-linked immunosorbent assay, and also their correlations were investigated by statistical analysis. Thereafter, the effect of basophils on autoantibody production by B cells and Th17 differentiation in SLE were evaluated in vitro. Finally, the effect of basophil depletion on the development of autoimmune disorders in spontaneous lupus-prone MRL-lpr/lpr mice was examined. RESULTS: The decreased numbers and an increased activation of peripheral basophils were found to be correlated with increased autoantibody production and disease activity in patients with SLE. Correspondingly, in vitro coculture studies showed that basophils obtained from patients with SLE promoted autoantibody production by SLE B cells and promoted Th17 differentiation from SLE naïve CD4+ T cells. The decrease of peripheral basophils in patients with SLE might be due to their migration to lymph nodes post their activation mediated by (autoreactive) IgE as supported by their increased CD62L and CCR7 expressions and accumulation in the lymph nodes of MRL-lpr/lpr mice. Furthermore, an increased activation of peripheral basophils was identified in MRL-lpr/lpr mice. Importantly, basophil-depleted MRL-lpr/lpr mice exhibited an extended life span, improved renal function, and lower serum levels of autoantibodies and IL-17, while basophil-adoptive-transferred mice exhibited the opposite results. CONCLUSION: These finding suggest that basophil activation-dependent autoantibody and IL-17 production may constitute a critical pathogenic mechanism in SLE.

Factors That Influence a Mother's Willingness to Preserve Umbilical Cord Blood: A Survey of 5120 Chinese Mothers.

BACKGROUND: Umbilical Cord blood (UCB), which contains a substantive number of stem cells, could be widely used in transplants to treat a variety of oncologic, genetic, hematologic, and immunodeficiency disorders. However, only a small portion of mothers preserve or donate their UCB in China. The limited availability of UCB has hampered stem cell research and therapy nowadays. To date, no systemic investigations regarding factors that influence a mother's willingness to preserve UCB have been performed in China. In the current study, we are trying to determine those factors which will provide useful information for national health policy development and will raise awareness of the importance of UCB preservation. METHODS: During 2011 to 2013, 5120 mothers with the average age of 26.1±8.4 years were included in this study. Those mothers participated in a standardized survey. The information gathered consisted of delivery time, occupation, level of education, knowledge of preservation of UCB, willingness to store UCB, and related concerns. The results have been analyzed with SPSS 16.0. RESULTS: The results showed that first-time mothers showed a greater willingness to preserve their UCB (73.3%) compared to those having their second (48.9%) or third child (40.3%). Mothers who were employed at Government Agencies and Organizations were more willing to preserve their UCB (87.3%) than those employed at factories (62.0%), and those who were unemployed (27.3%). Mothers holding master's or college degrees were more willing to preserve their UCB (72.5% and 71.1%, respectively) than mothers with high school diplomas (48.7%) or those who only went to preliminary school or middle school (40.7%). The two strongest factors that influenced an unwillingness to preserve UCB were the high cost and concerns regarding the safety of the preservation. CONCLUSIONS: The results showed that mothers with higher education or those having better occupations are more likely to preserve their UCB in China. These mothers have related knowledge and understand the importance of the preservation and they could more readily afford the relatively high cost. The government, clinicians and UCB banks should combine efforts to take measures, such as increasing public knowledge of the importance of UCB preservation and decreasing the high cost for its storage will most likely increase the frequency of UCB preservation which will further benefit stem cell research and therapy.

Contributions of CTCF and DNA methyltransferases DNMT1 and DNMT3B to Epstein-Barr virus restricted latency.

Establishment of persistent Epstein-Barr virus (EBV) infection requires transition from a program of full viral latency gene expression (latency III) to one that is highly restricted (latency I and 0) within memory B lymphocytes. It is well established that DNA methylation plays a critical role in EBV gene silencing, and recently the chromatin boundary protein CTCF has been implicated as a pivotal regulator of latency via its binding to several loci within the EBV genome. One notable site is upstream of the common EBNA gene promoter Cp, at which CTCF may act as an enhancer-blocking factor to initiate and maintain silencing of EBNA gene transcription. It was previously suggested that increased expression of CTCF may underlie its potential to promote restricted latency, and here we also noted elevated levels of DNA methyltransferase 1 (DNMT1) and DNMT3B associated with latency I. Within B-cell lines that maintain latency I, however, stable knockdown of CTCF, DNMT1, or DNMT3B or of DNMT1 and DNMT3B in combination did not result in activation of latency III protein expression or EBNA gene transcription, nor did knockdown of DNMTs significantly alter CpG methylation within Cp. Thus, differential expression of CTCF and DNMT1 and -3B is not critical for maintenance of restricted latency. Finally, mutant EBV lacking the Cp CTCF binding site exhibited sustained Cp activity relative to wild-type EBV in a recently developed B-cell superinfection model but ultimately was able to transition to latency I, suggesting that CTCF contributes to but is not necessarily essential for the establishment of restricted latency.

trans-Repression of protein expression dependent on the Epstein-Barr virus promoter Wp during latency.

An ordered silencing of Epstein-Barr virus (EBV) latency gene transcription is critical for establishment of persistent infection within B lymphocytes, yet the mechanisms responsible and the role that the virus itself may play are unclear. Here we describe two B-cell superinfection models with which to address these problems. In the first, Burkitt lymphoma (BL) cells that maintain latency I, when superinfected, initially supported transcription from the common EBNA promoters Wp and Cp (latency III) but ultimately transitioned to latency I (Cp/Wp silent), an essential requirement for establishment of EBV latency in vivo. We used this model to test whether the early lytic-cycle gene BHLF1, implicated in silencing of the Cp/Wp locus, is required to establish latency I. Upon superinfection with EBV deleted for the BHLF1 locus, however, we have demonstrated that BHLF1 is not essential for this aspect of EBV latency. In the second model, BL cells that maintain Wp-restricted latency, a variant program in which Cp is silent but Wp remains active, sustained the latency III program of transcription from the superinfecting-virus genomes, failing to transition to latency I. Importantly, there was substantial reduction in Wp-mediated protein expression from endogenous EBV genomes, in the absence of Cp reactivation, that could occur independent of a parallel decrease in mRNA. Thus, our data provide evidence of a novel, potentially posttranscriptional mechanism for trans-repression of Wp-dependent gene expression. We suggest that this may ensure against overexpression of the EBV nuclear antigens (EBNAs) prior to the transcriptional repression of Wp in cis that occurs upon activation of Cp.