A novel C3d-containing oligomeric vaccine provides insight into the viability of testing human C3d-based vaccines in mice.

The use of C3d, the final degradation product of complement protein C3, as a "natural" adjuvant has been widely examined since the initial documentation of its immunogenicity-enhancing properties as a consequence of binding to complement receptor 2. Subsequently it was demonstrated that these effects are most evident when oligomeric, rather than when monomeric forms of C3d, are linked to various test protein antigens. In this study, we examined the feasibility of enhancing the adjuvant properties of human C3d further by utilizing C4b-binding protein (C4BP) to provide an oligomeric arrayed scaffold fused to the model antigen, tetanus toxin C fragment (TTCF). High molecular weight, C3d-containing oligomeric vaccines were successfully expressed, purified from mammalian cells and used to immunize groups of mice. Surprisingly, anti-TTCF antibody responses measured in these mice were poor. Subsequently we established by in vitro and in vivo analysis that, in the presence of mouse C3, human C3d does not interact with either mouse or even human complement receptor 2. These data confirm the requirement to develop murine versions of C3d based adjuvant compounds to test in mice or that mice would need to be developed that express both human C3 and human CR2 to allow the testing of human C3d based adjuvants in mouse in any capacity.

Real-Time Fluorescence Measurements of ROS and [Ca2+] in Ischemic / Reperfused Rat Hearts: Detectable Increases Occur only after Mitochondrial Pore Opening and Are Attenuated by Ischemic Preconditioning.

Mitochondrial permeability transition pore (mPTP) opening is critical for ischemia / reperfusion (I/R) injury and is associated with increased [Ca2+] and reactive oxygen species (ROS). Here we employ surface fluorescence to establish the temporal sequence of these events in beating perfused hearts subject to global I/R. A bespoke fluorimeter was used to synchronously monitor surface fluorescence and reflectance of Langendorff-perfused rat hearts at multiple wavelengths, with simultaneous measurements of hemodynamic function. Potential interference by motion artefacts and internal filtering was assessed and minimised. Re-oxidation of NAD(P)H and flavoproteins on reperfusion (detected using autofluorescence) was rapid (t0.5 < 15 s) and significantly slower following ischemic preconditioning (IP). This argues against superoxide production from reduced Complex 1 being a critical mediator of initial mPTP opening during early reperfusion. Furthermore, MitoPY1 (a mitochondria-targeted H2O2-sensitive fluorescent probe) and aconitase activity measurements failed to detect matrix ROS increases during early reperfusion. However, two different fluorescent cytosolic ROS probes did detect ROS increases after 2-3 min of reperfusion, which was shown to be after initiation of mPTP opening. Cyclosporin A (CsA) and IP attenuated these responses and reduced infarct size. [Ca2+]i (monitored with Indo-1) increased progressively during ischemia, but dropped rapidly within 90 s of reperfusion when total mitochondrial [Ca2+] was shown to be increased. These early changes in [Ca2+] were not attenuated by IP, but substantial [Ca2+] increases were observed after 2-3 min reperfusion and these were prevented by both IP and CsA. Our data suggest that the major increases in ROS and [Ca2+] detected later in reperfusion are secondary to mPTP opening. If earlier IP-sensitive changes occur that might trigger initial mPTP opening they are below our limit of detection. Rather, we suggest that IP may inhibit initial mPTP opening by alternative mechanisms such as prevention of hexokinase 2 dissociation from mitochondria during ischemia.

Divergent effect of mammalian PLCζ in generating Ca²âº oscillations in somatic cells compared with eggs.

Sperm PLCζ (phospholipase Cζ) is a distinct phosphoinositide-specific PLC isoform that is proposed to be the physiological trigger of egg activation and embryo development at mammalian fertilization. Recombinant PLCζ has the ability to trigger Ca²âº oscillations when expressed in eggs, but it is not known how PLCζ activity is regulated in sperm or eggs. In the present study, we have transfected CHO (Chinese-hamster ovary) cells with PLCζ fused with either YFP (yellow fluorescent protein) or luciferase and found that PLCζ-transfected cells did not display cytoplasmic Ca²âº oscillations any differently from control cells. PLCζ expression was not associated with changes in CHO cell resting Ca²âº levels, nor with a significantly changed Ca²âº response to extracellular ATP compared with control cells transfected with either YFP alone, a catalytically inactive PLCζ or luciferase alone. Sperm extracts containing PLCζ also failed to cause Ca²âº oscillations in CHO cells. Despite these findings, PLCζ-transfected CHO cell extracts exhibited high recombinant protein expression and PLC activity. Furthermore, either PLCζ-transfected CHO cells or derived cell extracts could specifically cause cytoplasmic Ca²âº oscillations when microinjected into mouse eggs. These data suggest that PLCζ-mediated Ca²âº oscillations may require specific factors that are only present within the egg cytoplasm or be inhibited by factors present only in somatic cell lines.

Redistribution of mitochondria leads to bursts of ATP production during spontaneous mouse oocyte maturation.

During mammalian oocyte maturation there are marked changes in the distribution of mitochondria that supply the majority of the cellular ATP. Such redistribution of mitochondria is critical for oocyte quality, as oocytes with a poor developmental potential display aberrant mitochondrial distribution and lower ATP levels. Here we have investigated the dynamics of mitochondrial ATP production throughout spontaneous mouse oocyte maturation, using live measurements of cytosolic and mitochondrial ATP levels. We have observed three distinct increases in cytosolic ATP levels temporally associated with discrete events of oocyte maturation. These changes in cytosolic ATP levels are mirrored by changes in mitochondrial ATP levels, suggesting that mitochondrial ATP production is stimulated during oocyte maturation. Strikingly, these changes in ATP levels correlate with the distribution of mitochondria undergoing translocation to the peri-nuclear region and aggregation into clusters. Mitochondrial clustering during oocyte maturation was concomitant with the formation of long cortical microfilaments and could be disrupted by cytochalasin B treatment. Furthermore, the ATP production bursts observed during oocyte maturation were also inhibited by cytochalasin B suggesting that mitochondrial ATP production is stimulated during oocyte maturation by microfilament-driven, sub-cellular targeting of mitochondria.

Defective B cell ontogeny and immune response in human complement receptor 2 (CR2, CD21) transgenic mice is partially recovered in the absence of C3.

Mice prematurely expressing human CR2 (hCR2) in the B cell lineage have a defective B cell ontogeny and immune response. Our recent analysis of this phenotype suggested that signaling through hCR2 and presumably mouse CD19 on the B cell surface, during bone marrow development, could result in the observed changes in B cell function in these mice. To test this hypothesis, we back crossed hCR2(high) transgenic mice onto the CD19(-/-) background. CD19(-/-)hCR2(high) mice were found to possess even fewer mature B cells than their CD19(+/+)hCR2(high) littermates, demonstrating that loss of CD19 exacerbated the effects elicited through hCR2. This data suggests that CD19 provides a survival signal during B cell development in this model. Next, we examined if the removal of the main ligand for CR2, namely C3d, through back-crossing onto the C3(-/-) background could restore normal B cell development. However, we found only minor recovery in peripheral B cell numbers and no obvious change in function. This was despite a three-fold increase in the level of hCR2 expression on B cells isolated from the spleen or bone marrow of C3(-/-)hCR2(high) mice when compared with C3 sufficient littermates. These data demonstrate that hCR2 is integrated in mouse B cell signaling and that the downstream effects of hCR2 expression during early B cell development are partially but not completely due to interaction with C3 fragments and signaling through CD19 in the bone marrow environment.

Role of phospholipase C-zeta domains in Ca2+-dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca2+ oscillations.

The sperm-specific phospholipase C-zeta (PLCzeta) elicits fertilization-like Ca2+ oscillations and activation of embryo development when microinjected into mammalian eggs (Saunders, C. M., Larman, M. G., Parrington, J., Cox, L. J., Royse, J., Blayney, L. M., Swann, K., and Lai, F. A. (2002) Development (Camb.) 129, 3533-3544; Cox, L. J., Larman, M. G., Saunders, C. M., Hashimoto, K., Swann, K., and Lai, F. A. (2002) Reproduction 124, 611-623). PLCzeta may represent the physiological stimulus for egg activation and development at mammalian fertilization. PLCzeta is the smallest known mammalian PLC isozyme, comprising two EF hand domains, a C2 domain, and the catalytic X and Y core domains. To gain insight into PLCzeta structure-function, we assessed the ability of PLCzeta and a series of domain-deletion constructs to cause phosphatidylinositol 4,5-bisphosphate hydrolysis in vitro and also to generate cytoplasmic Ca2+ changes in intact mouse eggs. PLCzeta and the closely related PLCdelta1 had similar K(m) values for phosphatidylinositol 4,5-bisphosphate, but PLCzeta was around 100 times more sensitive to Ca2+ than was PLCdelta1. Notably, specific phosphatidylinositol 4,5-bisphosphate hydrolysis activity was retained in PLCzeta constructs that had either EF hand domains or the C2 domain removed, or both. In contrast, Ca2+ sensitivity was greatly reduced when either one, or both, of the EF hand domains were absent, and the Hill coefficient was reduced upon deletion of the C2 domain. Microinjection into intact mouse eggs revealed that all domain-deletion constructs were ineffective at initiating Ca2+ oscillations. These data suggest that the exquisite Ca2+-dependent features of PLCzeta regulation are essential for it to generate inositol 1,4,5-trisphosphate and Ca2+ oscillations in intact mouse eggs.