Pandemic influenza viruses often cause severe disease in middle-aged adults without preexistent co-morbidities. The mechanism of illness associated with severe disease in this age group is not well understood1–10. Here, we demonstrate preexisting serum antibody that cross-reacts with, but does not protect against 2009 H1N1 influenza virus in middle-aged adults. Non-protective antibody is associated with immune complex(IC)-mediated disease after infection. High titers of serum antibody of low avidity for H1-2009 antigen, and low avidity pulmonary ICs against the same protein were detected in severely ill patients. Moreover, C4d deposition - a sensitive marker of complement activation mediated by ICs- was present in lung sections of fatal cases. Archived lung sections from adults with confirmed fatal influenza 1957 H2N2 infection revealed a similar mechanism of illness. These observations provide a novel biological mechanism for the unusual age distribution of severe cases during influenza pandemics.

Although the antibody-based recognition of cell-surface markers has been widely used for the identification of immune cells, overlap in the expression of markers by different cell types and the inconsistent use of antibody panels have resulted in a lack of clearly defined signatures for myeloid cell subsets. We developed a 10-fluorochrome flow cytometry panel for the identification and quantitation of myeloid cells in the lungs, including pulmonary monocytes, myeloid dendritic cells, alveolar and interstitial macrophages, and neutrophils. After the initial sorting of viable CD451 leukocytes, we detected three leukocyte subpopulations based on CD68 expression: CD68 /CD11b1 /Gr1 hi ). The validity of cellular signatures was confirmed by a morphological analysis of FACS-sorted cells, functional studies, and the depletion of specific macrophage subpopulations using liposomal clodronate. We believe our approach provides an accurate and reproducible method for the isolation, quantification, and characterization of myeloid cell subsets in the lungs, which may be useful for studying the roles of myeloid cells during various pathological processes.Keywords: interstitial macrophages; alveolar macrophages; monocytes; myeloid dendritic cells; neutrophilsThe heterogeneous population of myeloid cells in the lungs is important for maintaining homeostasis and regulating inflammation, injury, and remodeling. These cells are thought to originate from bone marrow-derived precursors, and they differentiate into mature cells with a variety of functional properties, depending on environmental cues. Multiple studies have demonstrated functional and phenotypic differences between myeloid cell subsets, including monocytes, myeloid dendritic cells, macrophages, and neutrophils. However, the specific identification and quantification of subsets of these cells in tissues are complex, and a comprehensive scheme for isolating these cells from the lungs is not readily available. To date, studies of lung myeloid cells have been limited by the inconsistent use of cellular markers and a lack of clearly defined signatures for these cells, resulting in difficulties comparing studies from different laboratories and generalizing findings across different model systems. Therefore, we undertook a study to identify and quantify myeloid cell populations comprehensively in the lungs, using flow cytometry.Based on the recent explosion of information regarding the mechanisms that underpin innate immune responses, a clear need is apparent for better strategies to identify and study myeloid cell subpopulations in the lungs. The traditional method for the identification and quantification of lung myeloid cells involves the manual counting of cells obtained from the airways by bronchoalveolar lavage (BAL). This methodology normally yields a predominance of alveolar macrophages, because these are the primary immune cells in the airspaces. Interstitial macrophages, however, comprise a substantial portion of the lung myeloid cell population (1), and may have dif...

SummaryHeterogeneity within pluripotent stem cell (PSC) populations is indicative of dynamic changes that occur when cells drift between different states. Although the role of metastability in PSCs is unclear, it appears to reflect heterogeneity in cell signaling. Using the Fucci cell-cycle indicator system, we show that elevated expression of developmental regulators in G1 is a major determinant of heterogeneity in human embryonic stem cells. Although signaling pathways remain active throughout the cell cycle, their contribution to heterogeneous gene expression is restricted to G1. Surprisingly, we identify dramatic changes in the levels of global 5-hydroxymethylcytosine, an unanticipated source of epigenetic heterogeneity that is tightly linked to cell-cycle progression and the expression of developmental regulators. When we evaluated gene expression in differentiating cells, we found that cell-cycle regulation of developmental regulators was maintained during lineage specification. Cell-cycle regulation of developmentally regulated transcription factors is therefore an inherent feature of the mechanisms underpinning differentiation.

IntroductionHematopoietic stem cells (HSCs) have the unique ability to undergo self-renewal and to differentiate into cells belonging to multiple hematopoietic lineages. 1,2 These properties allow stem cells to maintain hematopoiesis throughout the life span of an organism. The knowledge of the behavior of HSCs is limited due to their rarity, difficulty of efficient isolation, and sensitivity to manipulation. 1,2 The self-renewal capacity of several classes of stem cells is thought to be controlled by external signals and intrinsic cellular processes. [1][2][3][4] Over the last 2 decades, a variety of external stimuli (cytokines, matrix proteins) that alter HSC self-renewal have been the subject of intense investigation. Although a number of such external signals that interact with specific receptors on HSC have been identified, the signaling mechanisms that govern HSC self-renewal have eluded investigation. Intrinsic cellular mechanisms that regulate stem cell self-renewal have been explored in a variety of model systems including germline stem cells (GSCs) in several lower species. Drosophila has been a particularly useful model for studying biologic processes that are conserved in higher developmental systems. [5][6][7][8][9][10][11][12] Therefore, in an attempt to define candidate genes that are responsible for human HSC self-renewal, we have explored the expression of genes in humans that have recently been demonstrated to play a role in Drosophila GSC self-renewal.The GSCs provide a continuous source of totipotent cells for the production of gametes needed for fertilization. 8 They are similar to HSCs in their ability to not only self-renew but also to remain capable of generating large numbers of differentiated daughter cells. 8,9 The intracellular mechanisms that serve as the determinants of asymmetric-segregating cell fates of GSCs depend not only on the basic cell cycle machinery but also on a family of recently identified genes, some of which are evolutionarily conserved. 7,9 A group of somatic cells in Drosophila, termed terminal filament cells, which lie distal and immediately adjacent to the GSCs, have been shown to regulate GSC division. 8,10,11 Laser ablation of the terminal filament increases the rate of oogenesis by 40%. 12 Loss of function mutations in a gene found in the terminal filament, termed piwi, leads to a failure of stem cell maintenance 7,10 ; piwi is expressed not only in the terminal filament but also in the germline. Loss of piwi function in the germline, however, is not known to affect GSC division. The protein encoded by piwi is extraordinarily well conserved along the evolutionary tree, being found in both Caenorhabditis elegans and primates. 7 Our laboratory has attempted to determine if such genes were present in primitive hematopoietic cells and if they might play a role in HSC development. We report here the presence of a human homologue of the piwi gene, termed hiwi, in a variety of primitive hematopoietic cells. The hiwi gene represents a candidate gene that may play a ro...