Capturing change in clonal composition amongst single mouse germinal centers

Understanding cellular processes occurring in vivo on time scales of days to weeks requires repeatedly interrogating the same tissue without perturbing homeostasis. We describe a novel setup for longitudinal intravital imaging of murine peripheral lymph nodes (LNs). The formation and evolution of single germinal centers (GCs) was visualized over days to weeks. Naïve B cells encounter antigen and form primary foci, which subsequently seed GCs. These experience widely varying rates of homogenizing selection, even within closely confined spatial proximity. The fluidity of GCs is greater than previously observed with large shifts in clonality over short time scales; and loss of GCs is a rare, observable event. The observation of contemporaneous, congruent shifts in clonal composition between GCs within the same animal suggests inter-GC trafficking of memory B cells. This tool refines approaches to resolving immune dynamics in peripheral LNs with high temporospatial resolution and minimal perturbation of homeostasis.

Earthquake weather and other tentative correspondences

The term ‘earthquake weather’ has had a fluctuating history being understood as both an observable event and a recurring fiction. The starting point for the term is usually associated with Aristotle who is said to have promoted the idea that winds trapped in underground caves caused earthquakes. Nineteenth century reports on the occurrence of earthquakes in Aotearoa New Zealand frequently link seismic activity with carefully detailed weather conditions. The nature of earthquake weather is variable with claims of calm expectancy, hot sultry weather or storms. Earthquake clouds currently have had some technical backing even as scientific reports largely deny associations between weather and earthquakes. The desire for such an association between the two events is of interest to this paper. Elizabeth Diller writing about the Blur project pointed out that, “Contemporary culture is addicted to weather information. … Our cultural anxiety about the weather can be attributed to its unpredictability.”[1] While weather is easily imagined as affective atmosphere we seek to illuminate the obdurate material atmosphere associated with earthquakes in an attempt to navigate the indeterminate atmospheric divide between stability and movement. Peggy Kamuf, writing on approaches to the work of Hélène Cixous, models a potential attitude for such a material writing project which might be to acknowledge “A matter of some gravity: that which pulls towards the earth, the lowest level, below ground even, the weight and volume with which heavier substances displace air or water. We say a matter is grave to remind ourselves to ponder it, to weigh it carefully, to exercise acute ethical vigilance … If the matter is grave, then, by definition it should not be taken lightly.”[2] However, Kamuf suggests a further angle of approach, which holds as imperative that one’s subject not be crushed by the weight of approach but, rather, be permitted to “escape the force of our gravity.”[3] From such an angle, “The most responsible and serious approach advances carefully and slowly, but above all lightly - which may mean obliquely, or imperceptibly, or even not at all”.[4] Here, as Hiddleson argues, Cixous’ works, “participate in the genre of theory, but allow the object of their theorising to shift and mutate, in their enjoyment of poetic associations, parallels and allusions.”[5] Like Kamuf’s dilemma of the love note, in which one does not wish to crush one’s lover under the weight of one’s own desires, Victorian and Edwardian correspondents of earthquakes and usual weather offer, time and again, paths of escape. Unexpected weathers and seismic activities are delicately laid together, but with a marked silence about the nature of their relationship. In this paper we explore the relationship offered by the correspondants through a tentative poetics of atmosphere consisting of text and accompanying images. [1] Elizabeth Diller, “Blur/Babble”, Anything, 2001, 132-139. [2] Peggy Kamuf, “To give Place: Semi-approaches to Hélène Cixous”, Yale French Studies, no. 87, 1995, 68. [3] Peggy Kamuf, Book of Addresses. California: Stanford University Press. 2005, 114). [4] Peggy Kamuf, Book of Addresses. California: Stanford University Press. 2005, 114). [5] Hiddleston, Jane. 2010. “In or out? The Dislocations of Helene Cixous” pp. 47-76. Poststructuralism and Postcoloniality: The Anxiety of Theory. (2010:51)

Risk-Informed Decision Making for High-Consequence Outcomes

The issues faced during the operation of high-consequence facilities or systems require a blended quantitative and qualitative risk-informed approach to decision-making. For example, in the case of the operation of nuclear power plants or manned space missions, a risk-informed metric one might consider is the probability of loss of the plant or failure of the mission, where this probability is balanced by the consequences of the event. However, as we will discuss, one should focus on the observable event itself (e.g., the actual loss or failure) rather than the probability of the event. We will describe the general approach of making probabilistic decisions, including the focus on predictive distributions for outcomes such as a complex system failure.

P-selectin glycoprotein ligand-1 supports rolling on E- and P-selectin in vivo

Selectin-dependent rolling is the earliest observable event in the recruitment of leukocytes to inflamed tissues. Several glycoproteins decorated with sialic acid, fucose, and/or sulfate have been shown to bind the selectins. The best-characterized selectin ligand is P-selectin glycoprotein-1 (PSGL-1) that supports P-selectin– dependent rolling in vitro and in vivo. In vitro studies have suggested that PSGL-1 may also be a ligand for E- and L-selectins. To study the in vivo function of PSGL-1, without the influence of other leukocyte proteins, the authors observed the interaction of PSGL-1–coated microspheres in mouse venules stimulated to express P- and/or E-selectin. Microspheres coated with functional recombinant PSGL-1 rolled in surgically stimulated and tumor necrosis factor alpha (TNFα)-stimulated mouse venules. P-selectin deficiency or inhibition abolished microsphere rolling in surgically and TNFα-stimulated venules, whereas E-selectin deficiency or inhibition increased microsphere rolling velocity in TNFα-stimulated venules. The results suggest that P-selectin–PSGL-1 interaction alone is sufficient to mediate rolling in vivo and that E-selectin–PSGL-1 interaction supports slow rolling.

P-selectin glycoprotein ligand-1 supports rolling on E- and P-selectin in vivo

Selectin-dependent rolling is the earliest observable event in the recruitment of leukocytes to inflamed tissues. Several glycoproteins decorated with sialic acid, fucose, and/or sulfate have been shown to bind the selectins. The best-characterized selectin ligand is P-selectin glycoprotein-1 (PSGL-1) that supports P-selectin– dependent rolling in vitro and in vivo. In vitro studies have suggested that PSGL-1 may also be a ligand for E- and L-selectins. To study the in vivo function of PSGL-1, without the influence of other leukocyte proteins, the authors observed the interaction of PSGL-1–coated microspheres in mouse venules stimulated to express P- and/or E-selectin. Microspheres coated with functional recombinant PSGL-1 rolled in surgically stimulated and tumor necrosis factor alpha (TNFα)-stimulated mouse venules. P-selectin deficiency or inhibition abolished microsphere rolling in surgically and TNFα-stimulated venules, whereas E-selectin deficiency or inhibition increased microsphere rolling velocity in TNFα-stimulated venules. The results suggest that P-selectin–PSGL-1 interaction alone is sufficient to mediate rolling in vivo and that E-selectin–PSGL-1 interaction supports slow rolling.

The passage of Ca2+ and fluorescent markers between the sperm and egg after fusion in the mouse

Mouse sperm-egg fusion was examined using two-photon and confocal microscopy. A delay of several minutes occurred between the first observable event of fusion (which was the diffusion of Ca2+-sensitive dyes from egg into sperm) and any change in egg cytoplasmic Ca2+. When indo-1 dextran was used to obtain ratiometric two-photon images, there was no detectable local increase in egg cytoplasmic Ca2+ near the site of sperm fusion. However, the sperm underwent a Ca2+ transient which appeared to be coincident with the egg cytoplasm Ca2+ transient, which suggested that there was a high permeability pathway for Ca2+ between egg and sperm. To exclude this pathway from providing trigger Ca2+ for the egg transient, we reduced bathing [Ca2+] to approx. 18 microM and 13nM (with EGTA). In these conditions the first egg Ca2+ transient was not prevented, which makes an obligatory role for extracellular Ca2+ in the initiation of the egg Ca2+ transient unlikely. Both FITC-albumin (70 kDa) and 10 kDa dextran-linked Ca2+ indicators were able to diffuse into the sperm from the egg. In addition, phycoerythrin (240 kDa) rapidly diffused into the sperm shortly after fusion (but before any changes in Ca2+ occurred). This suggests that the ‘pore(s)’ that form during sperm-egg fusion must be at least 8 nm in diameter. These data are compatible with the idea that a diffusible sperm protein could trigger the observed changes in intracellular Ca2+ in the egg, but do not exclude the possibility that other second messengers are generated during sperm-egg fusion.

Sialyl Lewisx (sLex) and an sLexMimetic, CGP69669A, Disrupt E-Selectin–Dependent Leukocyte Rolling In Vivo

Leukocyte rolling is the earliest observable event in their recruitment from the circulation to inflamed tissue. This rolling is mediated largely by interaction between the selectin family of adhesion molecules and their glycosylated ligands. Although the nature of these ligands and their interaction with the selectins is not fully understood, it is accepted that expression of fucosylated sialylated glycans such as sialyl Lewisx (sLex) is required for function. Despite findings that sLex inhibits binding of leukocytes to E-selectin in vitro, and has beneficial effects in inflammatory disease models, inhibition of E-selectin–dependent leukocyte rolling in vivo has not been described. Functional overlap between the selectins has been noted and reduction of rolling by E-selectin antibodies only occurs if P-selectin is absent or blocked. We demonstrate that leukocyte rolling velocity in tumor necrosis factor alpha (TNFα)-stimulated mouse cremaster is increased following treatment with either sLex or the sLex-mimetic CGP69669A and that rolling is dramatically reduced if CGP69669A is applied in the presence of anti–P-selectin antibody. These effects are characteristic of E-selectin antagonism. In contrast, surgically stimulated (L- or P-selectin–dependent) rolling is unaffected by either sLex or CGP69669A. Our data demonstrate that CGP69669A is an effective and selective antagonist of E-selectin in vivo.

Sialyl Lewisx (sLex) and an sLexMimetic, CGP69669A, Disrupt E-Selectin–Dependent Leukocyte Rolling In Vivo

Leukocyte rolling is the earliest observable event in their recruitment from the circulation to inflamed tissue. This rolling is mediated largely by interaction between the selectin family of adhesion molecules and their glycosylated ligands. Although the nature of these ligands and their interaction with the selectins is not fully understood, it is accepted that expression of fucosylated sialylated glycans such as sialyl Lewisx (sLex) is required for function. Despite findings that sLex inhibits binding of leukocytes to E-selectin in vitro, and has beneficial effects in inflammatory disease models, inhibition of E-selectin–dependent leukocyte rolling in vivo has not been described. Functional overlap between the selectins has been noted and reduction of rolling by E-selectin antibodies only occurs if P-selectin is absent or blocked. We demonstrate that leukocyte rolling velocity in tumor necrosis factor alpha (TNFα)-stimulated mouse cremaster is increased following treatment with either sLex or the sLex-mimetic CGP69669A and that rolling is dramatically reduced if CGP69669A is applied in the presence of anti–P-selectin antibody. These effects are characteristic of E-selectin antagonism. In contrast, surgically stimulated (L- or P-selectin–dependent) rolling is unaffected by either sLex or CGP69669A. Our data demonstrate that CGP69669A is an effective and selective antagonist of E-selectin in vivo.