In vitro maturation of B cells in chronic lymphocytic leukemia. I. Synergistic action of phorbol ester and interleukin 2 in the induction of Tac antigen expression and interleukin 2 responsiveness in leukemic B cells

Recent evidence indicates that interleukin 2 (IL 2), formerly thought to serve as growth factor exclusively for activated T cells, is directly involved in human B cell differentiation. We have investigated the role of IL 2 and IL 2 receptors (as defined by monoclonal anti-Tac antibody) in the phorbol ester-induced in vitro maturation of leukemic B cells from patients with chronic lymphocytic leukemia (CLL). Peripheral blood lymphocytes from B cells from CLL patients with high (greater than 10(5)/microliters) white blood cell counts were depleted of residual T lymphocytes and low-density cells (primarily macrophages) by consecutive steps of E rosetting, complement-mediated lysis of OKT3+ and OKT4+ cells, and Percoll density gradient centrifugation. No OKT3+ T cells were detectable in these cell populations before or after culture. When incubated for 3 days with phorbol ester plus recombinant human IL 2 (rIL 2), 12 to 57% of highly purified B cells from four of five tested patients expressed Tac antigen. Both phorbol ester and rIL 2 were required for maximal Tac antigen expression. Functional studies revealed that phorbol ester-activated (but not resting) CLL B cells responded to rIL 2 with [3H]thymidine incorporation and with enhanced secretion of IgM. Tac+ B cells were isolated in two cases on a fluorescence-activated cell sorter. In one patient, stimulation of Tac+ B cells with rIL 2 resulted in enhanced [3H]thymidine incorporation but no change in IgM secretion, as compared with Tac- B cells; in the second patient, stimulation of Tac+ B cells with rIL 2 did not result in [3H]thymidine uptake, but did result in significant IgM secretion. These findings indicate that certain leukemic B lymphocytes can be induced to express IL 2 receptors and respond to IL 2. The use of resting clonal B cell populations arrested at distinct stages of differentiation may help to better define the stage(s) at which IL 2 acts directly on B cells to induce proliferation and/or terminal differentiation.     

The expression of a viral microRNA is regulated by clustering to allow optimal B cell transformation

The Epstein-Barr virus (EBV) transforms B cells by expressing latent proteins and the BHRF1 microRNA cluster. MiR-BHRF1–3, its most transforming member, belongs to the recently identified group of weakly expressed microRNAs. We show here that miR-BHRF1–3 displays an unusually low propensity to form a stem–loop structure, an effect potentiated by miR-BHRF1–3''s proximity to the BHRF1 polyA site. Cloning miR-BHRF1–2 or a cellular microRNA, but not a ribozyme, 5′ of miR-BHRF1–3 markedly enhanced its expression. However, a virus carrying mutated miR-BHRF1–2 seed regions expressed miR-BHRF1–3 at normal levels and was fully transforming. Therefore, miR-BHRF1–2''s role during transformation is independent of its seed regions, revealing a new microRNA function. Increasing the distance between miR-BHRF1–2 and miR-BHRF1–3 in EBV enhanced miR-BHRF1–3''s expression but decreased its transforming potential. Thus, the expression of some microRNAs must be restricted to a narrow range, as achieved by placing miR-BHRF1–3 under the control of miR-BHRF1–2.     

Isolation by distance explains genetic structure of Buggy Creek virus,a bird-associated arbovirus

Many of the arthropod-borne viruses (arboviruses) show extensive genetic variability and are widely distributed over large geographic areas. Understanding how virus genetic structure varies in space may yield insight into how these pathogens are adapted to and dispersed by different hosts or vectors, the relative importance of mutation, drift, or selection in generating genetic variability, and where and when epidemics or epizootics are most likely to occur. However, because most arboviruses tend to be sampled opportunistically and often cannot be isolated in large numbers at a given locale, surprisingly little is known about their spatial genetic structure on the local scale at which host/vector/virus interactions typically occur. Here, we examine fine-scale spatial structure of two sympatric lineages of Buggy Creek virus (BCRV, Togaviridae), an alphavirus transmitted by the ectoparasitic swallow bug (Oeciacus vicarius) to colonially nesting cliff swallows (Petrochelidon pyrrhonota) and invasive house sparrows (Passer domesticus) in North America. Data from 377 BCRV isolates at cliff swallow colony sites in western Nebraska showed that both virus lineages were geographically structured. Most haplotypes were detected at a single colony or were shared among nearby colonies, and pair-wise genetic distance increased significantly with geographic distance between colony sites. Genetic structure of both lineages is consistent with isolation by distance. Sites with the most genetically distinct BCRV isolates were occupied by large numbers of house sparrows, suggesting that concentrations of invasive sparrows may represent foci for evolutionary change in BCRV. Our results show that bird-associated arboviruses can show genetic substructure over short geographic distances.     

Temperature effects on anaerobic fermentation of domestic refuse

Anaerobic fermentation of organic solid waste can provide a significant source of fuel gas (methane). Application of this process requires a better understanding of the kinetics of the biological system. The literature is replete with kinetic studies of this process as applied to waste solids from water pollution control systems. Much of this work has been conducted in the mesophilic temperature range. Increased temperatures yield higher reaction rates that will improve the economics of the process. The rate limiting step in the fermentation of refuse is the hydrolysis of the complex organic solids, in particular cellulose. Cellulose is a major component of the refuse. A laboratory study employing domestic refuse has shown the effect of temperature on the rate of methane fermentation. The optimum mesophilic temperature was found to be 42°C, while the optimum thermophilic temperature was at least 60°C. No data was obtained beyond the 60°C temperature. Reaction rate constants are presented for anaerobic fermentation of domestic refuse. Because of the characteristics of the substrate it^?was not possible to obtain the necessary measurements for evaluation of constants in the Monod model. An overall system constant was developed.     

LPS targets host guanylate‐binding proteins to the bacterial outer membrane for non‐canonical inflammasome activation

Pathogenic and commensal Gram‐negative bacteria produce and release outer membrane vesicles (OMVs), which present several surface antigens and play an important role for bacterial pathogenesis. OMVs also modulate the host immune system, which makes them attractive as vaccine candidates. At the cellular level, OMVs are internalized by macrophages and deliver lipopolysaccharide (LPS) into the host cytosol, thus activating the caspase‐11 non‐canonical inflammasome. Here, we show that OMV‐induced inflammasome activation requires TLR4‐TRIF signaling, the production of type I interferons, and the action of guanylate‐binding proteins (GBPs), both in macrophages and in vivo. Mechanistically, we find that isoprenylated GBPs associate with the surface of OMVs or with transfected LPS, indicating that the key factor that determines GBP recruitment to the Gram‐negative bacterial outer membranes is LPS itself. Our findings provide new insights into the mechanism by which GBPs target foreign surfaces and reveal a novel function for GBPs in controlling the intracellular detection of LPS derived from extracellular bacteria in the form of OMVs, thus extending their function as a hub between cell‐autonomous immunity and innate immunity.