A diverse Ediacara assemblage survived under low-oxygen conditions.

The Ediacaran biota were soft-bodied organisms, many with enigmatic phylogenetic placement and ecology, living in marine environments between 574 and 539 million years ago. Some studies hypothesize a metazoan affinity and aerobic metabolism for these taxa, whereas others propose a fundamentally separate taxonomic grouping and a reliance on chemoautotrophy. To distinguish between these hypotheses and test the redox-sensitivity of Ediacaran organisms, here we present a high-resolution local and global redox dataset from carbonates that contain in situ Ediacaran fossils from Siberia. Cerium anomalies are consistently >1, indicating that local environments, where a diverse Ediacaran assemblage is preserved in situ as nodules and carbonaceous compressions, were pervasively anoxic. Additionally, δ238U values match other terminal Ediacaran sections, indicating widespread marine euxinia. These data suggest that some Ediacaran biotas were tolerant of at least intermittent anoxia, and thus had the capacity for a facultatively anaerobic lifestyle. Alternatively, these soft-bodied Ediacara organisms may have colonized the seafloor during brief oxygenation events not recorded by redox proxy data. Broad temporal correlations between carbon, sulfur, and uranium isotopes further highlight the dynamic redox landscape of Ediacaran-Cambrian evolutionary events.

Cervical Spinal Cord Stimulator Lead Migration and Transection Adjacent to the Foramen Magnum: A Case Report.

BACKGROUND: Spinal cord stimulation (SCS) is a relatively safe therapy for the treatment of pain but has the potential for several complications, including lead migration and breakage. While instances of lead breakage and electrode shearing have been described, there are no reported cases of stimulator lead transection and migration to the foramen magnum. AIMS: We describe the case of a 53-year-old woman who reported that her cervical spinal cord stimulator was no longer functioning after a traumatic fall. CASE: Fluoroscopy of the neck revealed that one of the MRI conditional leads had migrated cephalad, and the distal aspect appeared to be transected. This was confirmed by computerized tomography, which showed a transected portion of the lead in the epidural space, just inferior to the posterior aspect of the foramen magnum. An SCS device revision was performed to replace the lead, but the distal transected tip was left in place in the epidural space adjacent to the foramen magnum to avoid complications of retrieval. DISCUSSION/CONCLUSION: Given the location of the transected portion of the lead, we recommended avoiding MRI imaging. In addition, we advised the patient that a repeat x-ray may be necessary if she has increased neck pain or any other concerning symptoms. In this report, we discuss the known complications with SCS, as well as management of a retained lead fragment.

Spinal Cord Stimulator Electrode Dislodging into the Ligamentum Flavum: A Case Report.

To date, no case studies specifically reporting an electrode dislodging from its lead wire have been reported. Here we describe a case involving an electrode shearing from the spinal cord stimulator lead, and lodging into the ligamentum flavum during implantation. In this case, an experienced board certified interventional pain management specialist was performing the implantation procedure of a magnetic resonance imaging (MRI) compatible spinal cord stimulator with MR conditional leads. After successful placement of the first lead, the epidural space was accessed via a T11/12 interlaminar approach using loss of resistance technique. When the lead would not advance past the tip of the needle, it was removed in order to reposition the needle slightly. Upon removal of the lead, it was discovered that the first electrode was no longer attached to the wire. Subsequent fluoroscopic imaging revealed that the electrode had lodged within the ligamentum flavum. Upon discussion with the medical director of the device company, it was agreed upon that the electrode should be left in place. The decision was made to proceed with only one lead in place and the remainder of the procedure was completed uneventfully. The patient followed up two weeks later in clinic, and no adverse effect related to the dislodged electrode was reported. The indications and common complications associated with spinal cord stimulation are discussed, followed by factors to consider to help guide decision making in the event of a retained foreign body during a procedure.

Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells.

Adoptive cellular therapy using chimeric antigen receptor (CAR) T cell therapies have produced significant objective responses in patients with CD19+ hematological malignancies, including durable complete responses. Although the majority of clinical trials to date have used autologous patient cells as the starting material to generate CAR T cells, this strategy poses significant manufacturing challenges and, for some patients, may not be feasible because of their advanced disease state or difficulty with manufacturing suitable numbers of CAR T cells. Alternatively, T cells from a healthy donor can be used to produce an allogeneic CAR T therapy, provided the cells are rendered incapable of eliciting graft versus host disease (GvHD). One approach to the production of these cells is gene editing to eliminate expression of the endogenous T cell receptor (TCR). Here we report a streamlined strategy for generating allogeneic CAR T cells by targeting the insertion of a CAR transgene directly into the native TCR locus using an engineered homing endonuclease and an AAV donor template. We demonstrate that anti-CD19 CAR T cells produced in this manner do not express the endogenous TCR, exhibit potent effector functions in vitro, and mediate clearance of CD19+ tumors in an in vivo mouse model.

Anti-coreceptor therapy drives selective T cell egress by suppressing inflammation-dependent chemotactic cues.

There continues to be a need for immunotherapies to treat type 1 diabetes in the clinic. We previously reported that nondepleting anti-CD4 and -CD8 Ab treatment effectively reverses diabetes in new-onset NOD mice. A key feature of the induction of remission is the egress of the majority of islet-resident T cells. How this occurs is undefined. Herein, the effects of coreceptor therapy on islet T cell retention were investigated. Bivalent Ab binding to CD4 and CD8 blocked TCR signaling and T cell cytokine production, while indirectly downregulating islet chemokine expression. These processes were required for T cell retention, as ectopic IFN-γ or CXCL10 inhibited Ab-mediated T cell purging. Importantly, treatment of humanized mice with nondepleting anti-human CD4 and CD8 Ab similarly reduced tissue-infiltrating human CD4+ and CD8+ T cells. These findings demonstrate that Ab binding of CD4 and CD8 interrupts a feed-forward circuit by suppressing T cell-produced cytokines needed for expression of chemotactic cues, leading to rapid T cell egress from the islets. Coreceptor therapy therefore offers a robust approach to suppress T cell-mediated pathology by purging T cells in an inflammation-dependent manner.

Microparticles bearing encephalitogenic peptides induce T-cell tolerance and ameliorate experimental autoimmune encephalomyelitis.

Aberrant T-cell activation underlies many autoimmune disorders, yet most attempts to induce T-cell tolerance have failed. Building on previous strategies for tolerance induction that exploited natural mechanisms for clearing apoptotic debris, we show that antigen-decorated microparticles (500-nm diameter) induce long-term T-cell tolerance in mice with relapsing experimental autoimmune encephalomyelitis. Specifically, intravenous infusion of either polystyrene or biodegradable poly(lactide-co-glycolide) microparticles bearing encephalitogenic peptides prevents the onset and modifies the course of the disease. These beneficial effects require microparticle uptake by marginal zone macrophages expressing the scavenger receptor MARCO and are mediated in part by the activity of regulatory T cells, abortive T-cell activation and T-cell anergy. Together these data highlight the potential for using microparticles to target natural apoptotic clearance pathways to inactivate pathogenic T cells and halt the disease process in autoimmunity.

Long-term remission of diabetes in NOD mice is induced by nondepleting anti-CD4 and anti-CD8 antibodies.

Residual ß-cells found at the time of clinical onset of type 1 diabetes are sufficient to control hyperglycemia if rescued from ongoing autoimmune destruction. The challenge, however, is to develop an immunotherapy that not only selectively suppresses the diabetogenic response and efficiently reverses diabetes, but also establishes long-term ß-cell-specific tolerance to maintain remission. In the current study, we show that a short course of nondepleting antibodies (Abs) specific for the CD4 and CD8 coreceptors rapidly reversed clinical disease in recent-onset diabetic NOD mice. Once established, remission was maintained indefinitely and immunity to foreign antigens unimpaired. Induction of remission involved selective T-cell purging of the pancreas and draining pancreatic lymph nodes and upregulation of transforming growth factor (TGF)-ß1 by pancreas-resident antigen-presenting cells. Neutralization of TGF-ß blocked the induction of remission. In contrast, maintenance of remission was associated with tissue-specific immunoregulatory T cells. These findings demonstrate that the use of nondepleting Ab specific for CD4 and CD8 is a robust approach to establish long-term ß-cell-specific T-cell tolerance at the onset of clinical diabetes.

Tolerance induced by apoptotic antigen-coupled leukocytes is induced by PD-L1+ and IL-10-producing splenic macrophages and maintained by T regulatory cells.

Ag-specific tolerance is a highly desired therapy for immune-mediated diseases. Intravenous infusion of protein/peptide Ags linked to syngeneic splenic leukocytes with ethylene carbodiimide (Ag-coupled splenocytes [Ag-SP]) has been demonstrated to be a highly efficient method for inducing peripheral, Ag-specific T cell tolerance for treatment of autoimmune disease. However, little is understood about the mechanisms underlying this therapy. In this study, we show that apoptotic Ag-SP accumulate in the splenic marginal zone, where their uptake by F4/80(+) macrophages induces production of IL-10, which upregulates the expression of the immunomodulatory costimulatory molecule PD-L1 that is essential for Ag-SP tolerance induction. Ag-SP infusion also induces T regulatory cells that are dispensable for tolerance induction but required for long-term tolerance maintenance. Collectively, these results indicate that Ag-SP tolerance recapitulates how tolerance is normally maintained in the hematopoietic compartment and highlight the interplay between the innate and adaptive immune systems in the induction of Ag-SP tolerance. To our knowledge, we show for the first time that tolerance results from the synergistic effects of two distinct mechanisms, PD-L1-dependent T cell-intrinsic unresponsiveness and the activation of T regulatory cells. These findings are particularly relevant as this tolerance protocol is currently being tested in a Phase I/IIa clinical trial in new-onset relapsing-remitting multiple sclerosis.

Ethylenecarbodiimide-treated splenocytes carrying male CD4 epitopes confer histocompatibility Y chromosome antigen transplant protection by inhibiting CD154 upregulation.

In humans and certain strains of laboratory mice, male tissue is recognized as nonself and destroyed by the female immune system via recognition of histocompatibility Y chromosome Ag (Hya). Male tissue destruction is thought to be accomplished by CTLs in a helper-dependent manner. We show that graft protection induced with the immunodominant Hya-encoded CD4 epitope (Dby) attached to female splenic leukocytes (Dby-SPs) with the chemical cross-linker ethylenecarbodiimide significantly, and often indefinitely, prolongs the survival of male skin graft transplants in an Ag-specific manner. In contrast, treatments with the Hya CD8 epitopes (Uty-/Smcy-SPs) failed to prolong graft survival. Dby-SP-tolerized CD4(+) T cells fail to proliferate, secrete IFN-gamma, or effectively prime a CD8 response in recipients of male grafts. Ag-coupled splenocyte treatment is associated with defective CD40-CD40L interactions as demonstrated by the observation that CD4 cells from treated animals exhibit a defect in CD40L upregulation following in vitro Ag challenge. Furthermore, treatment with an agonistic anti-CD40 Ab at the time of transplantation abrogates protection from graft rejection. Interestingly, anti-CD40 treatment completely restores the function of Dby-specific CD4 cells but not Uty- or Smcy-specific CD8 cells.

TCRbeta enhancer activation in early and late lymphoid progenitors.

An earlier report from our laboratory indicates that the activation of the T cell receptor (TCR) beta enhancer (Ebeta) is not always an indicator of T lineage potential in bone marrow-resident pre-lymphocytes. In order to more precisely investigate the consequences of Ebeta activation in lymphopoiesis, a genetic reporter animal, in which the expression of green fluorescent protein (GFP) is controlled by Ebeta, was used to examine two well-defined lymphopotent populations. Adoptive transfer experiments suggest that primitive lymphoid precursor populations (specifically, hematopoietic stem cells) consist of two discrete-populations discernible by Ebeta-GFP activation, although the two populations display no overt differences in lineage potential. In contrast, subsets of more differentiated pre-lymphocytes (specifically, common lymphoid progenitors), while also discernible by Ebeta-GFP activation, display different capacities for reconstituting lymphoid compartments. Interestingly, late lymphoid progenitors containing inactive Ebeta elements generated both T and B cells in vivo, in accord with the original description of this population; however, progenitors containing active Ebeta elements displayed an unexpected bias toward the B lineage. Our findings suggest that Ebeta activation is an indicator of B lineage specification in late, but not early lymphoid precursors.

TCRbeta enhancer activation occurs in some but not all cells with T cell lineage developmental potential.

Previous studies have shown that murine bone marrow contains a fraction of CD3(-)/B220(-)/Thy1(lo) cells that have pre T cell activity following adoptive transfer and produce sterile transcripts of the T cell receptor beta chain gene. The relationship between progenitors and TCRbeta transcription has not been examined. Transgenic mice were generated that express green fluorescent protein under the control of the TCRbeta enhancer (Ebeta). Phenotypic analysis of the founders revealed faithful expression of GFP in populations that express TCRbeta transcripts. Examination of the bone marrow showed two populations, CD3(-)/B220(-)/Thy1(-) and CD3(-)/B220(-)/Thy1(lo), which were GFP(+). Both populations were analyzed for their developmental potential following intrathymic transfer into recipient mice. Surprisingly, the GFP(+)/CD3(-)/B220(-)/Thy1(lo) cells failed to reconstitute; however, the GFP(+)/CD3(-)/B220(-)/Thy1(-) cells exhibited thymic repopulation. These data demonstrate that Ebeta is active pre-thymically; however, pre-thymic transcription of the TCRbeta chain gene is neither required for T cell development, nor is it limited to pre T cells.