[Abstract unavailable

Epigenetic genome modifications are thought to be important for specifying the lineage and developmental stage of cells within a multicellular organism. Here, we show that the epigenetic profile of pluripotent embryonic stem cells (ES) is distinct from that of embryonic carcinoma cells, haematopoietic stem cells (HSC) and their differentiated progeny. Silent, lineage-specific genes replicated earlier in pluripotent cells than in tissue-specific stem cells or differentiated cells and had unexpectedly high levels of acetylated H3K9 and methylated H3K4. Unusually, in ES cells these markers of open chromatin were also combined with H3K27 trimethylation at some non-expressed genes. Thus, pluripotency of ES cells is characterized by a specific epigenetic profile where lineage-specific genes may be accessible but, if so, carry repressive H3K27 trimethylation modifications. H3K27 methylation is functionally important for preventing expression of these genes in ES cells as premature expression occurs in embryonic ectoderm development (Eed)-deficient ES cells. Our data suggest that lineage-specific genes are primed for expression in ES cells but are held in check by opposing chromatin modifications.

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Regulatory T cells have been clearly implicated in the control of disease in murine models of autoimmunity. The paucity of data regarding the role of these lymphocytes in human autoimmune disease has prompted us to examine their function in patients with rheumatoid arthritis (RA). Regulatory (CD4+CD25+) T cells isolated from patients with active RA displayed an anergic phenotype upon stimulation with anti-CD3 and anti-CD28 antibodies, and suppressed the proliferation of effector T cells in vitro. However, they were unable to suppress proinflammatory cytokine secretion from activated T cells and monocytes, or to convey a suppressive phenotype to effector CD4+CD25− T cells. Treatment with antitumor necrosis factor α (TNFα; Infliximab) restored the capacity of regulatory T cells to inhibit cytokine production and to convey a suppressive phenotype to “conventional” T cells. Furthermore, anti-TNFα treatment led to a significant rise in the number of peripheral blood regulatory T cells in RA patients responding to this treatment, which correlated with a reduction in C reactive protein. These data are the first to demonstrate that regulatory T cells are functionally compromised in RA, and indicate that modulation of regulatory T cells by anti-TNFα therapy may be a further mechanism by which this disease is ameliorated.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

Protective humoral memory forms in secondary lymphoid organs where B cells undergo affinity maturation and differentiation into memory or plasma cells. Here, we provide a comprehensive roadmap of human B cell maturation with single-cell transcriptomics matched with bulk and single-cell antibody repertoires to define gene expression, antibody repertoires, and clonal sharing of B cell states at single-cell resolution, including memory B cell heterogeneity that reflects diverse functional and signaling states. We reconstruct gene expression dynamics during B cell activation to reveal a pre–germinal center state primed to undergo class switch recombination and dissect how antibody class–dependent gene expression in germinal center and memory B cells is linked with a distinct transcriptional wiring with potential to influence their fate and function. Our analyses reveal the dynamic cellular states that shape human B cell–mediated immunity and highlight how antibody isotype may play a role during their antibody-based selection.

Intrahepatic cholestasis of pregnancy (ICP) is a liver disease of pregnancy with serious consequences for the mother and fetus. Two pedigrees have been reported with ICP in the mothers of children with a subtype of autosomal recessive progressive familial intrahepatic cholestasis (PFIC) with raised serum gamma-glutamyl transpeptidase (gamma-GT). Affected children have homozygous mutations in the MDR3 gene (also called ABCB4 ), and heterozygous mothers have ICP. More frequently, however, ICP occurs in women with no known family history of PFIC and the genetic basis of this disorder is unknown. We investigated eight women with ICP and raised serum gamma-GT, but with no known family history of PFIC. DNA sequence analysis revealed a C to A transversion in codon 546 in exon 14 of MDR3 in one patient, which results in the missense substitution of the wild-type alanine with an aspartic acid. We performed functional studies of this mutation introduced into MDR1, a closely related homologue of MDR3. Fluorescence activated cell sorting (FACS) and western analysis indicated that this missense mutation causes disruption of protein trafficking with a subsequent lack of functional protein at the cell surface. The demonstration of a heterozygous missense mutation in the MDR3 gene in a patient with ICP with no known family history of PFIC, analysed by functional studies, is a novel finding. This shows that MDR3 mutations are responsible for the additional phenotype of ICP in a subgroup of women with raised gamma-GT.

Summary. Experiments were performed to determine whether a modern flow cytometer could be used to study bacterial populations in suspension, with particular reference to their morphological characteristics and their responses to antibiotics. The FACScan, a commercial benchtop flow cytometer fitted with an air-cooled laser, designed primarily for the study of eukaryotic peripheral blood mononuclear cells, yielded reproducible data relating to bacterial shape and internal architecture. It was sensitive enough to detect changes in bacterial morphology on entry into the growth cycle and after exposure to antibiotics. Antibioticinduced morphological changes affecting subpopulations of bacteria were sufficiently specific to allow differentiation between antibiotics with different cell-wall enzyme targets. Simultaneously, the effect of such antibiotics on the integrity of the outer cell membrane of Escherichia coli was assessed by measurement of the association of the nucleic acid-binding dye propidium iodide with the bacteria. These experiments demonstrated complex patterns of probable cell-wall leakage, related to the modes of action of the antibiotics. The FACScan is a useful and sensitive tool for the study of the morphology and physiology of bacterial populations in suspension, and is especially applicable to the study of antibiotic action.