Toxicity Testing of Pristine and Aged Silver Nanoparticles in Real Wastewaters Using Bioluminescent Pseudomonas putida.

Impact of aging on nanoparticle toxicity in real matrices is scarcely investigated due to a lack of suitable methodologies. Herein, the toxicity of pristine and aged silver nanoparticles (Ag NPs) to a bioluminescent Pseudomonas putida bioreporter was measured in spiked crude and final wastewater samples (CWs and FWs, respectively) collected from four wastewater treatment plants (WWTPs). Results showed lower toxicity of pristine Ag NPs in CWs than in FWs. The effect of the matrix on the eventual Ag NP toxicity was related to multiple physico-chemical parameters (biological oxygen demand (BOD), chemical oxygen demand (COD), total suspended solids (TSS) pH, ammonia, sulfide and chloride) based on a multivariate analysis. However, no collection site effect was concluded. Aged Ag NPs (up to eight weeks) were found less toxic than pristine Ag NPs in CWs; evident increased aggregation and decreased dissolution were associated with aging. However, Ag NPs exhibited consistent toxicity in FWs despite aging; comparable results were obtained in artificial wastewater (AW) simulating effluent. The study demonstrates the potency of performing nanoparticle acute toxicity testing in real and complex matrices such as wastewaters using relevant bacterial bioreporters.

The poxvirus encoded ubiquitin ligase, p28, is regulated by proteasomal degradation and autoubiquitination.

Virus manipulation of the ubiquitin-proteasome system has become increasingly apparent. Ubiquitin is a 76 amino acid protein that is post-translationally conjugated to target proteins, while poly-ubiquitination subsequently leads to degradation via the 26S proteasome. Target specificity is determined by a large family of ubiquitin ligases. Poxviruses encode p28, a highly conserved ubiquitin ligase expressed in a wide range of poxviruses (J. Virol. 79:597). Here we investigate the relationship between p28 and ubiquitination. Confocal microscopy indicated that orthologs of p28 co-localized with ubiquitin at the virus factory. Flow cytometry assays further demonstrated that p28 was regulated by proteasomal degradation. Moreover, when the ubiquitin ligase activity of p28 was disrupted by mutating the RING domain conjugated ubiquitin still localized to the viral factories, indicating that an unknown ubiquitin ligase(s) was responsible for regulating p28. Our observations indicate that p28 is a ubiquitin ligase that is regulated by ubiquitination and proteasomal degradation.

Hematopoietic stem cell responsiveness to exogenous signals is limited by caspase-3.

Limited responsiveness to inflammatory cytokines is a feature of adult hematopoietic stem cells and contributes to the relative quiescence and durability of the stem cell population in vivo. Here we report that the executioner Caspase, Caspase-3, unexpectedly participates in that process. Mice deficient in Caspase-3 had increased numbers of immunophenotypic long-term repopulating stem cells in association with multiple functional changes, most prominently cell cycling. Though these changes were cell autonomous, they reflected altered activation by exogenous signals. Caspase-3(-/-) cells exhibited cell type-specific changes in phosphorylated members of the Ras-Raf-MEK-ERK pathway in response to specific cytokines, while notably, members of other pathways, such as pSTAT3, pSTAT5, pAKT, pp38 MAPK, pSmad2, and pSmad3, were unaffected. Caspase-3 contributes to stem cell quiescence, dampening specific signaling events and thereby cell responsiveness to microenvironmental stimuli.

Heparan sulfate and heparin enhance ERK phosphorylation and mediate preBCR-dependent events during B lymphopoiesis.

As B lineage cells develop, they interact with cells, proteins, and extracellular matrix components of the surrounding microenvironment. In vitro, one critical checkpoint for developing cells occurs as they lose responsiveness to IL-7. These cells require contact with either stromal cells or other B lineage cells to mature. Our results demonstrate that heparan sulfate and heparin are able to promote this transition when added exogenously to the culture system or when heparan sulfate-bearing cell lines are cocultured with primary B cell progenitors. Addition of heparan sulfate or heparin to LPS-stimulated cultures of primary B cell progenitors resulted in more IgM secreted compared with untreated cultures. Heparan sulfate has been reported to be a ligand for the pre-B cell receptor (preBCR). Extending this observation, we found that treatment of preBCR+ cells with heparan sulfate before anti-micro stimulation leads to increased phosphorylation of ERK1/2. Consequently, preBCR+ cells proliferate more in the presence of IL-7 and heparan sulfate, whereas preBCR- cells are unaffected, suggesting that in these experiments, heparan sulfate is not directly affecting IL-7 activity. Heparin treatment of cultures induces many of the same biological effects as treatment with heparan sulfate, including elevated pERK levels in preBCR+ cells. However, heparin reduces the proliferation of cells expressing only the preBCR (opposed to both the preBCR and BCR) possibly due to internalization of the preBCR. Heparan sulfates are present on stromal cells and B lineage cells present in hemopoietic tissues and may provide stimulation to preB cells testing the signaling capacity of the preBCR.

Tachykinins in the immune system.

Until recently, the mammalian tachykinins included substance P, neurokinin A and neurokinin B. Following the discovery of the fourth member of this family, hemokinin 1, a diverse group of novel tachykinins and tachykinin gene-related peptides have been identified in mammals. These newly identified members are preferentially expressed in peripheral tissues. Currently, the impact of these new tachykinin peptides on the immune system remains unclear. Some data imply an important role for hemokinin 1 in the generation of lymphocytes. Tachykinins are traditionally viewed as neuropeptides with well-defined functions as neurotransmitters. Many studies however, indicate that they may also be produced by non-neuronal cells, and exert profound influence on inflammatory responses by affecting multiple aspects of immune cell function. It is of great importance to determine whether the new tachykinin peptides have similar effects. A more detailed understanding of the interactions between tachykinins and immune cells may provide the basis for the development of new therapies for inflammatory and immune-mediated diseases.

IL-7: a key regulator of B lymphopoiesis.

Interleukin-7 plays important roles in the B cell developmental pathway including events leading to commitment, survival, proliferation, and maturation. Because of its central role in adult murine B lymphopoiesis, IL-7 is frequently used to generate B cell progenitors in vitro. We have shown that differentiation of IL-7-responsive cells in these cultures is influenced by CD45, pre-B cell receptor, and other downstream signaling molecules. A common, but often overlooked aspect of IL-7 containing cultures is the routine maturation of cells to the sIgM(+) stage. The production of B cells in IL-7 containing cultures is balanced by cell death, since such cells fail to survive for long without additional stimulation.

IL-7 does not prevent pro-B/pre-B cell maturation to the immature/sIgM(+) stage.

IL-7 plays many fundamental roles during murine B lineage development. One reported function is to maintain progenitors in a developmentally immature state by preventing differentiation to the surface IgM (sIgM)(+) stage. Withdrawal of IL-7 from cultures has been shown to lead to increases in mature traits such as RAG expression, IgL rearrangements and expression of sIgM. These observations have been interpreted as an inductive event promoting the differentiation of progenitor cells. In contrast to this, we reproducibly observe sIgM(+) cells that have differentiated in cultures containing IL-7. We find that sIgM(+) cells arise as a normal consequence when B lineage cells are cultured in the presence of IL-7. However, these cells are short-lived and are quickly replaced by newly emerging sIgM(+) cells that differentiate from sIgM(-) progenitors. Withdrawal of IL-7 from these cultures only prevents the survival and proliferation of CD2(-)sIgM(-) pro-B cells but does not change the number of cells that differentiate to the sIgM(+) stage. This changes the ratio of sIgM(-):sIgM(+) cells and results only in an apparent maturation of the culture as a whole. Therefore withdrawal of IL-7 from these cultures acts as a selection event, not an induction event, for populations that are normally present.

CD45-deficient mice accumulate Pro-B cells both in vivo and in vitro.

Efficient generation of mature B lineage cells requires the participation of the BCR, the pre-BCR, accessory coreceptors, and growth factor receptors. Together these receptors integrate cell intrinsic signals with regulatory pathways initiated by surrounding cells and structures. CD45 is a receptor tyrosine phosphatase expressed at high levels on all hemopoietic cells, and has been shown to modulate many signaling cascades in both positive and negative manners. In the absence of B220, the B lineage isoform of CD45, differentiation to the mature B cell stage is incomplete. We demonstrate that CD45-deficient mice also accumulate pro-B cells in the bone marrow. In vitro differentiation is altered in that B lineage populations exhibit prolonged survival in the presence of high concentrations of IL-7. Cell lines derived from CD45-deficient animals experience prolonged JAK/STAT activation in response to IL-7 stimulation, and constitutively elevated levels of phosphorylated src kinases. Aberrant IL-7Ralpha expression is observed in vivo, and may be responsible for the skewed development present in CD45(-/-) animals. Demonstrating that CD45-deficient pro-B cells are affected by the absence of B220 highlights a previously unrecognized parallel in B and T lineage precursors, and emphasizes that the presence of normal numbers of peripheral B cells does not assure that the bone marrow compartment is intact.

Mechanisms of selection mediated by interleukin-7, the preBCR, and hemokinin-1 during B-cell development.

Many of the stromal-derived signals and factors that regulate B lymphopoiesis have been identified. We review recent evidence from our laboratory that shows that there are at least three phases during B-cell development when cells direct their own maturation, independent of stromal cells. Following the expression of the preB-cell receptor (preBCR), cells acquire the ability to proliferate in low levels of interleukin-7 (IL-7), which acts as a self-selecting mechanism to expand cells that have successfully expressed a preBCR in environments that are non-permissive to preBCR- cells. Second, the preBCR is required for a contact-mediated event between B-cell progenitors. Disruption at this stage prevents the further maturation of progenitors to the lipopolysaccharide (LPS)-responsive stage. Finally, the transition from IL-7 receptor to mature antigen receptor-based signaling is enhanced by a novel member of the tachykinin family, hemokinin-1. This series of maturation, survival, and differentiation signals is generated by B-lineage cells as they progress through developmental checkpoints on the way to becoming functionally mature cells.