Enhanced isolation of adult thymic epithelial cell subsets for multiparameter flow cytometry and gene expression analysis

The epithelial cells (TECs) are microenvironmental niche cells which support T lymphocyte development in the thymus. Most studies of TEC biology have focused on TEC at the fetal stage of development, whereas the biology of adult-stage TECs is not as well-understood. Delineating the molecular mechanisms that control adult TEC differentiation has implications for the success of T-lymphocyte based therapies for autoimmune diseases and induction of immunological tolerance to stem cell-derived tissues. Detailed analysis of adult TECs is technically challenging due to their rarity, their diminishing numbers with age, and the limited number of markers to distinguish between unique TEC subpopulations. Here, we have devised an improved isolation protocol for adult mouse TECs and combined it with six-color multiparameter flow cytometry. Using these techniques, we have identified four distinct subsets of CD45- EpCAM+ TECs in adult mice: a) UEA1(low) CDR1(low) (UC(low)); b) UEA1(high) CDR1(high)(UC(high)); c) UEA1(low) CDR1(high) MHC(high) (cTEC); and d) UEA1(high)CDR1(low) MHC(int/high) (mTEC). PCR analysis verified that these TEC subsets differentially expressed known TEC genes. TEC subsets were further analyzed using high-throughput quantitative PCR arrays to reveal novel genes that could be important for TEC subset maintenance. Intracellular staining for keratin-5 and keratin-8 can also be added, but our results suggest that keratin expression alone cannot be used to distinguish adult TEC subsets. Our enhanced isolation allows for detailed analysis of rare TEC subpopulations in the adult mouse at the cellular and molecular levels.     

Introducing seasonal influenza vaccine in low‐income countries: an adverse events following immunization survey in the Lao People's Democratic Republic

Objective

In 2012, Lao PDR introduced seasonal influenza vaccine in pregnant women, persons aged ≥50 years, persons with chronic diseases, and healthcare personnel. We assessed adverse events following immunization (AEFI).

Methods

We used a multistage randomized cluster sample design to interview vaccine recipients.

Findings

Between April and May 2012, 355 902 were vaccinated. Of 2089 persons interviewed, 261 (12·5%) reported one or more AEFI. The most commonly reported AEFIs were local reactions. No hospitalizations or deaths were reported; 16% sought medical care. Acceptance and awareness of vaccination were high.

Conclusions

Following the introduction of seasonal influenza vaccine in Lao PDR, self-reported adverse events were mild.     

Embryonic stem cell-derived hematopoietic stem cells: challenges in development, differentiation, and immunogenicity

Embryonic stem cells (ESC) can potentially be manipulated in vitro to differentiate into cells and tissues of all three germ layers. This pluripotent feature is being exploited to use ESC-derived tissues as therapies for degenerative diseases and replacement of damaged organs. Although their potential is great, the promise of ESC-derived therapies will be unfulfilled unless several challenges are overcome. For example, inefficient production of ESC-derived tissues before transplantation, inability of ESC-derived tissues to integrate well into the adult microenvironments due to developmental stage incompatibility, or active immune rejection of the ESC-derived graft are all potential challenges to successful ESC-derived therapies. One way to induce immunological tolerance to allogeneic tissues is via the establishment of mixed hematopoietic chimerism in which the host and donor cells are educated to recognize each other as "self". Proof of principle that in vitro cultured ESC-derived hematopoietic progenitors can be transplanted and induce immunological tolerance to allogeneic tissues exists in mouse models. In this review, we discuss the challenges to in vitro development of a bona fide ESC-derived hematopoietic stem cell and their differentiation fate in vivo, and provide suggestions to predict the immunogenicity of specific ESC-derived hematopoietic populations before transplantation that could be used to prevent their rejection after transplantation into an adult host.     

Absence of sclerostin adversely affects B-cell survival

Increased osteoblast activity in sclerostin-knockout (Sost(-/-)) mice results in generalized hyperostosis and bones with small bone marrow cavities resulting from hyperactive mineralizing osteoblast populations. Hematopoietic cell fate decisions are dependent on their local microenvironment, which contains osteoblast and stromal cell populations that support both hematopoietic stem cell quiescence and facilitate B-cell development. In this study, we investigated whether high bone mass environments affect B-cell development via the utilization of Sost(-/-) mice, a model of sclerosteosis. We found the bone marrow of Sost(-/-) mice to be specifically depleted of B cells because of elevated apoptosis at all B-cell developmental stages. In contrast, B-cell function in the spleen was normal. Sost expression analysis confirmed that Sost is primarily expressed in osteocytes and is not expressed in any hematopoietic lineage, which indicated that the B-cell defects in Sost(-/-) mice are non-cell autonomous, and this was confirmed by transplantation of wild-type (WT) bone marrow into lethally irradiated Sost(-/-) recipients. WT→Sost(-/-) chimeras displayed a reduction in B cells, whereas reciprocal Sost(-/-) →WT chimeras did not, supporting the idea that the Sost(-/-) bone environment cannot fully support normal B-cell development. Expression of the pre-B-cell growth stimulating factor, Cxcl12, was significantly lower in bone marrow stromal cells of Sost(-/-) mice, whereas the Wnt target genes Lef-1 and Ccnd1 remained unchanged in B cells. Taken together, these results demonstrate a novel role for Sost in the regulation of bone marrow environments that support B cells.     

A comparative study of young and mature bovine cortical bone

The mechanical properties and microstructure of young and mature bovine femur bone were investigated by optical microscopy and compression testing in the longitudinal and transverse directions for untreated, deproteinized and demineralized cases. Optical microscopy revealed that mature bone has a more established and less porous microstructure compared to young bone. Mature bone was found to be stronger in both directions for the untreated and deproteinized cases. Mature untreated bone was also found to be stiffer and less tough compared to young bone in both directions. These results are related to the increase in mineralization of mature bone and significant microstructural differences. Young bone was found to be stronger in both directions for the demineralized case, which is attributed to alterations in the collagen network with age.