MicroRNAs regulate dendritic cell differentiation and function.

MicroRNAs (miRNAs) are an important class of cellular regulators that modulate gene expression and thereby influence cell fate and function. In the immune system, miRNAs act at checkpoints during hematopoietic development and cell subset differentiation, they modulate effector cell function, and they are implicated in the maintenance of homeostasis. Dendritic cells (DCs), the professional APCs involved in the coordination of adaptive immune responses, are also regulated by miRNAs. Some DC-relevant miRNAs, including miR-155 and miR-146a, are shared with other immune cells, whereas others have been newly identified. In this review, we summarize the current understanding of where miRNAs are active during DC development from myeloid precursors and differentiation into specialized subsets, and which miRNAs play roles in DC function.

B cells do not take up bacterial DNA: an essential role for antigen in exposure of DNA to toll-like receptor-9.

Murine dendritic cells (DC) and macrophages respond to bacterial CpG DNA through toll-like receptor 9 (TLR9). Although it is frequently assumed that bacterial DNA is a direct stimulus for B cells, published work does not reliably show responses of purified B cells. Here we show that purified splenic B cells did not respond to Escherichia coli DNA with induction of CD86, despite readily responding to single-stranded (ss) phosphodiester CpG oligodeoxynucleotides (ODN). This was due to a combination of weak responses to both long and double-stranded (ds) DNA. B-cell DNA uptake was greatly reduced with increasing DNA length. This contrasts with macrophages where DNA uptake and subsequent responses were enhanced with increasing DNA length. However, when DNA was physically linked to hen egg lysozyme (HEL), HEL-specific B cells showed efficient uptake of DNA, and limited proliferation in response to the HEL-DNA complex. We propose that, in the absence of other signals, B cells have poor uptake and responses to long dsDNA to prevent polyclonal activation. Conversely, when DNA is physically linked to a B-cell receptor (BCR) ligand, its uptake is increased, allowing TLR9-dependent B-cell activation in an antigen-specific manner. We could not generate fragments of E. coli DNA by limited DNaseI digestion that could mimic the stimulatory effect of ss CpG ODN on naïve B cells. We suggest that the frequently studied polyclonal B-cell responses to CpG ODN are relevant to therapeutic applications of phosphorothioate-modified CpG-containing ODN, but not to natural responses to foreign or host dsDNA.

Elevated Mcl-1 perturbs lymphopoiesis, promotes transformation of hematopoietic stem/progenitor cells, and enhances drug resistance.

Diverse human cancers with poor prognosis, including many lymphoid and myeloid malignancies, exhibit high levels of Mcl-1. To explore the impact of Mcl-1 overexpression on the hematopoietic compartment, we have generated vavP-Mcl-1 transgenic mice. Their lymphoid and myeloid cells displayed increased resistance to a variety of cytotoxic agents. Myelopoiesis was relatively normal, but lymphopoiesis was clearly perturbed, with excess mature B and T cells accumulating. Rather than the follicular lymphomas typical of vavP-BCL-2 mice, aging vavP-Mcl-1 mice were primarily susceptible to lymphomas having the phenotype of a stem/progenitor cell (11 of 30 tumors) or pre-B cell (12 of 30 tumors). Mcl-1 overexpression dramatically accelerated Myc-driven lymphomagenesis. Most vavP-Mcl-1/ Eµ-Myc mice died around birth, and transplantation of blood from bitransgenic E18 embryos into unirradiated mice resulted in stem/progenitor cell tumors. Furthermore, lethally irradiated mice transplanted with E13 fetal liver cells from Mcl-1/Myc bitransgenic mice uniformly died of stem/progenitor cell tumors. When treated in vivo with cyclophosphamide, tumors coexpressing Mcl-1 and Myc transgenes were significantly more resistant than conventional Eµ-Myc lymphomas. Collectively, these results demonstrate that Mcl-1 overexpression renders hematopoietic cells refractory to many cytotoxic insults, perturbs lymphopoiesis and promotes malignant transformation of hematopoietic stem and progenitor cells.

High-affinity B cell receptor ligation by cognate antigen induces cytokine-independent isotype switching.

The selection of an appropriate Ig isotype is critical for an effective immune response against pathogens. Isotype regulation is sensitive to external signals, particularly cytokines secreted by Th cells. For example, IL-4 induces isotype switching to IgG1 via a STAT6-dependent signaling pathway. In this study, we show that BCR ligation also induces IgG1 switching in mouse B cells. The extent of switch induction by Ag is affinity-dependent, and high-affinity Ag binding leads to IgG1 switching levels comparable to those induced by saturating IL-4. However, the Ag-induced IgG1 switch does not require additional cytokine signals and occurs in a STAT6-independent manner. Thus, BCR ligation represents a novel pathway for direct isotype switching leading to IgG1 secretion.

Strategies for implementing and sustaining therapeutic lifestyle changes as part of hypertension management in African Americans.

African Americans with high blood pressure (BP) can benefit greatly from therapeutic lifestyle changes (TLC) such as diet modification, physical activity, and weight management. However, they and their health care providers face many barriers in modifying health behaviors. A multidisciplinary panel synthesized the scientific data on TLC in African Americans for efficacy in improving BP control, barriers to behavioral change, and strategies to overcome those barriers. Therapeutic lifestyle change interventions should emphasize patient self-management, supported by providers, family, and the community. Interventions should be tailored to an individual's cultural heritage, beliefs, and behavioral norms. Simultaneously targeting multiple factors that impede BP control will maximize the likelihood of success. The panel cited limited progress with integrating the Dietary Approaches to Stop Hypertension (DASH) eating plan into the African American diet as an example of the need for more strategically developed interventions. Culturally sensitive instruments to assess impact will help guide improved provision of TLC in special populations. The challenge of improving BP control in African Americans and delivery of hypertension care requires changes at the health system and public policy levels. At the patient level, culturally sensitive interventions that apply the strategies described and optimize community involvement will advance TLC in African Americans with high BP.

Quantitative regulation of B cell division destiny by signal strength.

Differentiation to Ab secreting and isotype-switched effector cells is tightly linked to cell division and therefore the degree of proliferation strongly influences the nature of the immune response. The maximum number of divisions reached, termed the population division destiny, is stochastically distributed in the population and is an important parameter in the quantitative outcome of lymphocyte responses. In this study, we further assessed the variables that regulate B cell division destiny in vitro in response to T cell- and TLR-dependent stimuli. Both the concentration and duration of stimulation were able to regulate the average maximum number of divisions undergone for each stimulus. Notably, a maximum division destiny was reached during provision of repeated saturating stimulation, revealing that an intrinsic limit to proliferation exists even under these conditions. This limit was linked directly to division number rather than time of exposure to stimulation and operated independently of the survival regulation of the cells. These results demonstrate that a B cell population's division destiny is regulable by the stimulatory conditions up to an inherent maximum value. Division destiny is a crucial parameter in regulating the extent of B cell responses and thereby also the nature of the immune response mounted.

Determining the expected variability of immune responses using the cyton model.

During an adaptive immune response, lymphocytes proliferate for 5-20 cell divisions, then stop and die over a period of weeks. The cyton model for regulation of lymphocyte proliferation and survival was introduced by Hawkins et al. (Proc. Natl. Acad. Sci. USA 104, 5032-5037, 2007) to provide a framework for understanding this response and its regulation. The model assumes stochastic values for division and survival times for each cell in a responding population. Experimental evidence indicates that the choice of times is drawn from a skewed distribution such as the lognormal, with the fate of individual cells being potentially highly variable. For this reason we calculate the higher moments of the model so that the expected variability can be determined. To do this we formulate a new analytic framework for the cyton model by introducing a generalization to the Bellman-Harris branching process. We use this framework to introduce two distinct approaches to predicting variability in the immune response to a mitogenic signal. The first method enables explicit calculations for certain distributions and qualitatively exhibits the full range of observed immune responses. The second approach does not facilitate analytic solutions, but allows simple numerical schemes for distributions for which there is little prospect of analytic formulae. We compare the predictions derived from the second method to experimentally observed lymphocyte population sizes from in vivo and in vitro experiments. The model predictions for both data sets are remarkably accurate. The important biological conclusion is that there is limited variation around the expected value of the population size irrespective of whether the response is mediated by small numbers of cells undergoing many divisions or for many cells pursuing a small number of divisions. Therefore, we conclude the immune response is robust and predictable despite the potential for great variability in the experience of each individual cell.

Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data.

Cellular proliferation is an essential feature of the adaptive immune response. The introduction of the division tracking dye carboxyfluorescein diacetate succinimidyl ester (CFSE) has made it possible to monitor the number of cell divisions during proliferation and to examine the relationship between proliferation and differentiation. Although qualitative examination of CFSE data may be useful, substantially more information about division and death rates can be extracted from quantitative CFSE time-series experiments. Quantitative methods can reveal in detail how lymphocyte proliferation and survival are regulated and altered by signals such as those received from co-stimulatory molecules, drugs and genetic polymorphisms. In this protocol, we present a detailed method for examining time-series data using graphical and computer-based procedures available to all experimenters.

High affinity germinal center B cells are actively selected into the plasma cell compartment.

A hallmark of T cell-dependent immune responses is the progressive increase in the ability of serum antibodies to bind antigen and provide immune protection. Affinity maturation of the antibody response is thought to be connected with the preferential survival of germinal centre (GC) B cells that have acquired increased affinity for antigen via somatic hypermutation of their immunoglobulin genes. However, the mechanisms that drive affinity maturation remain obscure because of the difficulty in tracking the affinity-based selection of GC B cells and their differentiation into plasma cells. We describe a powerful new model that allows these processes to be followed as they occur in vivo. In contrast to evidence from in vitro systems, responding GC B cells do not undergo plasma cell differentiation stochastically. Rather, only GC B cells that have acquired high affinity for the immunizing antigen form plasma cells. Affinity maturation is therefore driven by a tightly controlled mechanism that ensures only antibodies with the greatest possibility of neutralizing foreign antigen are produced. Because the body can sustain only limited numbers of plasma cells, this "quality control" over plasma cell differentiation is likely critical for establishing effective humoral immunity.

A systematic review of interactive computer-assisted technology in diabetes care. Interactive information technology in diabetes care.

BACKGROUND: Excellent diabetes care and self-management depends heavily on the flow of timely, accurate information to patients and providers. Recent developments in information technology (IT) may, therefore, hold great promise. OBJECTIVE: To determine, in a systematic review, how emerging interactive IT has been used to enhance care for adults with type 2 diabetes. METHOD: Eligible studies were randomized controlled trials (RCTs) and observational studies (both before-after designs and post-intervention assessments) focused on computer-assisted interactive IT that included > or =10 adults with diabetes (> or =50% type 2) and reported in English. We searched 4 electronic databases (up to 2003) using terms for diabetes and technology, reviewed bibliographies, and handsearched Diabetes Care (January 1990 to February 2004). Two reviewers independently selected articles and worked serially on data extraction with adjudication of discrepancies by consensus. RESULTS: There were 26 studies (27 reports): internet (n=6; 3 RCTs), telephone (n=7; 4 RCTs), and computer-assisted integration of clinical information (n=13, 7 RCTs). The median (range) sample size was 165 (28 to 6,469 participants) for patients and 37 (15 to 67) for providers; the median duration was 6 (1 to 29) months. Ethnic minorities or underserved populations were described in only 8 studies. Six of 14 interventions demonstrated moderate to large significant declines in hemoglobin A1c levels compared with controls. Most studies reported overall positive results and found that IT-based interventions improved health care utilization, behaviors, attitudes, knowledge, and skills. CONCLUSIONS: There is growing evidence that emerging IT may improve diabetes care. Future research should characterize benefits in the long term (>1 year), establish methods to evaluate clinical outcomes, and determine the cost-effectiveness of using IT.

Computer and internet use among urban African Americans with type 2 diabetes.

Previous studies have identified a "digital divide" between African Americans and whites, with African Americans having substantially lower rates of Internet use. However, use of the Internet to access health information has not been sufficiently evaluated in this population. Therefore, we conducted a telephone survey to determine the prevalence of computer and Internet use among 457 African American adults with type 2 diabetes. Participants were 78% female, with a mean age of 57 +/- 11 years, and about one-third had a yearly income % $7,500. Forty percent of the participants reported having a computer at home and 46% reported knowing how to use a computer. Most participants (58%) reported that they had, at some point, used a computer, and of those, 40% reported that they used the computer to find health information. In a stratified analysis, participants with lower education levels (

Antigens of selected Acanthamoeba species detected with monoclonal antibodies.

Acanthamoeba species are ubiquitous soil and freshwater protozoa that have been associated with infections of the human brain, skin, lungs and eyes. Our aim was to develop specific antibodies to aid in rapid and specific diagnosis of clinically important isolates. Mice were variously immunised with live mixtures of Acanthamoeba castellanii strain 112 (AC112) trophozoites and cysts, or with sonicated, formalin-fixed or heat-treated trophozoites, or with a trophozoite membrane preparation. Eight hybridoma cell lines secreting monoclonal antibodies reactive with A. castellanii epitopes were generated. Seven of the new antibodies (designated AMEC1-3 and MTAC1-4) were isotyped as IgMkappa and one (MTAC5) as IgG1kappa. All of the novel antibodies bound to AC112 cysts, and MTAC4 and MTAC5 also bound to trophozoites as measured by flow cytometry on unfixed cells. Single chain antibody fragments that retained parental antibody binding characteristics were engineered from three of the hybridomas (AMEC1, MTAC3 and MTAC4). Four monoclonal antibodies (AMEC1, AMEC3, MTAC1, MTAC3) bound reliably to unfixed cysts of clinical isolates of A. castellanii (two strains) and Acanthamoeba polyphaga (two strains), belonging to Pussard-Pons morphological group II, and to Acanthamoeba lenticulata and Acanthamoeba culbertsoni, belonging to Pussard-Pons morphological group III. None of the antibodies bound to cysts or trophozoites of the environmental group I species, Acanthamoeba tubiashi. Antibodies AMEC1, MTAC3, MTAC4 and MTAC5 reacted with buffered formalin-fixed AC112 by immunohistochemistry, and also stained Acanthamoeba in sections of infected rat cornea and buffered formalin-fixed, paraffin-embedded infected human cornea. These antibodies may be useful in diagnosing pathogenic Acanthamoeba species in clinical specimens, provided that cysts are present.