Quantification of antithrombin isoform proportions in plasma samples of healthy subjects, sepsis patients, and in antithrombin concentrates

Antithrombin (AT) circulates in plasma in two isoforms, AT-alpha (90-95%) and AT-beta (5-10%). AT isoform proportions were measured in plasma samples of 17 healthy subjects and 26 posttraumatic or postoperative septic patients, as well as in 4 commercially available AT concentrates. Total AT was immune-purified from plasma and concentrates. Micellar electrokinetic chromatography was used to analytically separate and quantify the isoforms. Compared with plasma samples of healthy donors, septic plasmas revealed significantly reduced AT activity (p < 0.001) and beta-isoform content (p < 0.05). AT-beta correlated inversely with urea and creatinine serum concentrations (p < 0.01), indicating a relationship between better renal function and higher beta-isoform content. beta-Isoform neither correlated with age, gender, and 28-day mortality, nor with plasma concentrations of various inflammatory and organ function parameters. The commercial AT concentrate, which is equivalent to the current WHO standard, had an AT-beta content close to that found in plasma of healthy subjects. The availability of this novel quantitative AT isoform assay allows, for the first time, a closer look at the role of AT isoforms in hemostasis and sepsis pathophysiology. Copyright (C) 2002 S. Karger AG, Basel

Synthetic Cell-Based Immunotherapies for Neurologic Diseases

The therapeutic success and widespread approval of genetically engineered T cells for a variety of hematologic malignancies spurred the development of synthetic cell-based immunotherapies for CNS lymphoma, primary brain tumors, and a growing spectrum of nononcologic disease conditions of the nervous system. Chimeric antigen receptor effector T cells bear the potential to deplete target cells with higher efficacy, better tissue penetration, and greater depth than antibody-based cell depletion therapies. In multiple sclerosis and other autoimmune disorders, engineered T-cell therapies are being designed and currently tested in clinical trials for their safety and efficacy to eliminate pathogenic B-lineage cells. Chimeric autoantibody receptor T cells expressing a disease-relevant autoantigen as cell surface domains are designed to selectively deplete autoreactive B cells. Alternative to cell depletion, synthetic antigen-specific regulatory T cells can be engineered to locally restrain inflammation, support immune tolerance, or efficiently deliver neuroprotective factors in brain diseases in which current therapeutic options are very limited. In this article, we illustrate prospects and bottlenecks for the clinical development and implementation of engineered cellular immunotherapies in neurologic diseases

PD1-Expressing T Cell Subsets Modify the Rejection Risk in Renal Transplant Patients

We tested whether multi-parameter immune phenotyping before or after renal ­transplantation can predict the risk of rejection episodes. Blood samples collected before and weekly for 3 months after transplantation were analyzed by multi-parameter flow cytometry to define 52 T cell and 13 innate lymphocyte subsets in each sample, producing more than 11,000 data points that defined the immune status of the 28 patients included in this study. Principle component analysis suggested that the patients with histologically confirmed rejection episodes segregated from those without rejection. Protein death 1 (PD-1)-expressing subpopulations of regulatory and conventional T cells had the greatest influence on the principal component segregation. We constructed a statistical tool to predict rejection using a support vector machine algorithm. The algorithm correctly identified 7 out of 9 patients with rejection, and 14 out of 17 patients without rejection. The immune profile before transplantation was most accurate in determining the risk of rejection, while changes of immune parameters after transplantation were less accurate in discriminating rejection from non-rejection. The data indicate that pretransplant immune subset analysis has the potential to identify patients at risk of developing rejection episodes, and suggests that the proportion of PD1-expressing T cell subsets may be a key indicator of rejection risk

Follicular regulatory T cells control humoral autoimmunity via NFAT2-regulated CXCR5 expression

Maturation of high-affinity B lymphocytes is precisely controlled during the germinal center reaction. This is dependent on CD4(+)CXCR5(+) follicular helper T cells (TFH) and inhibited by CD4(+)CXCR5(+)Foxp3(+) follicular regulatory T cells (TFR). Because NFAT2 was found to be highly expressed and activated in follicular T cells, we addressed its function herein. Unexpectedly, ablation of NFAT2 in T cells caused an augmented GC reaction upon immunization. Consistently, however, TFR cells were clearly reduced in the follicular T cell population due to impaired homing to B cell follicles. This was TFR-intrinsic because only in these cells NFAT2 was essential to up-regulate CXCR5. The physiological relevance for humoral (auto-)immunity was corroborated by exacerbated lupuslike disease in the presence of NFAT2-deficient TFR cells

The transcriptional coactivator Bob1 promotes the development of follicular T helper cells via Bcl6

Follicular T helper (Tfh) cells are key regulators of the germinal center reaction and long-term humoral immunity. Tfh cell differentiation requires the sustained expression of the transcriptional repressor Bcl6; however, its regulation in CD4(+) T cells is incompletely understood. Here, we report that the transcriptional coactivator Bob1, encoded by the Pou2af1 gene, promotes Bcl6 expression and Tfh cell development. We found that Bob1 together with the octamer transcription factors Oct1/Oct2 can directly bind to and transactivate the Bcl6 and Btla promoters. Mixed bone marrow chimeras revealed that Bob1 is required for the expression of normal levels of Bcl6 and BTLA, thereby controlling the pool size and composition of the Tfh compartment in a T cell-intrinsic manner. Our data indicate that T cell-expressed Bob1 is directly involved in Tfh cell differentiation and required for mounting normal T cell-dependent B-cell responses

Transitional B cell cytokines predict renal allograft outcomes

Early immunological biomarkers that predict rejection and chronic allograft loss are needed to inform preemptive therapy and improve long-term outcomes. Here, we prospectively examined the ratio of interleukin-10 (IL-10) to tumor necrosis factor–α (TNFα) produced by transitional-1 B cells (T1B) 3 months after transplantation as a predictive biomarker for clinical and subclinical renal allograft rejection and subsequent clinical course. In both Training (n = 162) and Internal Validation (n = 82) Sets, the T1B IL-10/TNFα ratio 3 months after transplantation predicted both clinical and subclinical rejection anytime in the first year. The biomarker also predicted subsequent late rejection with a lead time averaging 8 months. Among biomarker high-risk patients, 60% had early rejection, of which 48% recurred later in the first posttransplant year. Among high-risk patients without early rejection, 74% developed rejection later in the first year. In contrast, only 5% of low-risk patients had early and 5% late rejection. The biomarker also predicted rejection in an External Validation Set (n = 95) and in key patient subgroups, confirming generalizability. Biomarker high-risk patients exhibited progressively worse renal function and decreased 5-year graft survival compared to low-risk patients. Treatment of B cells with anti-TNFα in vitro augmented the IL-10/TNFα ratio, restored regulatory activity, and inhibited plasmablast differentiation. To conclude, the T1B IL-10/TNFα ratio was validated as a strong predictive biomarker of renal allograft outcomes and provides a rationale for preemptive therapeutic intervention with TNF blockade

An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells.

Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells

CD8(+) T cells retain protective functions despite sustained inhibitory receptor expression during Epstein-Barr virus infection in vivo

Epstein Barr virus (EBV) is one of the most ubiquitous human pathogens in the world, persistently infecting more than 90% of the adult human population. It drives some of the strongest human CD8+ T cell responses, which can be observed during symptomatic primary infection known as infectious mononucleosis (IM). Despite high viral loads and prolonged CD8+ T cell stimulation during IM, EBV enters latency and is under lifelong immune control in most individuals that experience this disease. We investigated whether changes in T cell function, as frequently characterized by PD-1 up-regulation, occur during IM due to the prolonged exposure to high antigen levels. We readily detected the expansion of PD-1 positive CD8+ T cells together with high frequencies of Tim-3, 2B4, and KLRG1 expression during IM and in mice with reconstituted human immune system components (huNSG mice) that had been infected with a high dose of EBV. These PD-1 positive CD8+ T cells, however, retained proliferation, cytokine production, and cytotoxic abilities. Multiple subsets of CD8+ T cells expanded during EBV infection, including PD-1+ Tim-3+ KLRG1+ cells that express CXCR5 and TCF-1 germinal center homing and memory markers, and may also contain BATF3. Moreover, blocking the PD-1 axis compromised EBV specific immune control and resulted in virus-associated lymphomagenesis. Finally, PD-1+ , Tim-3+ , and KLRG1+ CD8+ T cell expansion coincided with declining viral loads during low dose EBV infection. These findings suggest that EBV infection primes PD-1 positive CD8+ T cell populations that rely on this receptor axis for the efficient immune control of this ubiquitous human tumor virus

A bird's eye view of discard reforms: bird-borne cameras reveal seabird/fishery interactions.

notes: PMCID: PMC3590202types: Journal Article; Research Support, Non-U.S. Gov'tCopyright: © 2013 Votier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Commercial capture fisheries produce huge quantities of offal, as well as undersized and unwanted catch in the form of discards. Declines in global catches and legislation to ban discarding will significantly reduce discards, but this subsidy supports a large scavenger community. Understanding the potential impact of declining discards for scavengers should feature in an eco-system based approach to fisheries management, but requires greater knowledge of scavenger/fishery interactions. Here we use bird-borne cameras, in tandem with GPS loggers, to provide a unique view of seabird/fishery interactions. 20,643 digital images (one min(-1)) from ten bird-borne cameras deployed on central place northern gannets Morus bassanus revealed that all birds photographed fishing vessels. These were large (>15 m) boats, with no small-scale vessels. Virtually all vessels were trawlers, and gannets were almost always accompanied by other scavenging birds. All individuals exhibited an Area-Restricted Search (ARS) during foraging, but only 42% of ARS were associated with fishing vessels, indicating much 'natural' foraging. The proportion of ARS behaviours associated with fishing boats were higher for males (81%) than females (30%), although the reasons for this are currently unclear. Our study illustrates that fisheries form a very important component of the prey-landscape for foraging gannets and that a discard ban, such as that proposed under reforms of the EU Common Fisheries Policy, may have a significant impact on gannet behaviour, particularly males. However, a continued reliance on 'natural' foraging suggests the ability to switch away from scavenging, but only if there is sufficient food to meet their needs in the absence of a discard subsidy.EU INTERREG project CHARM-IIINatural Environment Research CouncilAssociation for the Study of Animal Behaviour research gran

Analogue peptides for the immunotherapy of human acute myeloid leukemia

Accepted manuscript. The final publication is available at: http://link.springer.com/article/10.1007%2Fs00262-015-1762-9The use of peptide vaccines, enhanced by adjuvants, has shown some efficacy in clinical trials. However, responses are often short-lived and rarely induce notable memory responses. The reason is that self-antigens have already been presented to the immune system as the tumor develops, leading to tolerance or some degree of host tumor cell destruction. To try to break tolerance against self-antigens, one of the methods employed has been to modify peptides at the anchor residues to enhance their ability to bind major histocompatibility complex molecules, extending their exposure to the T-cell receptor. These modified or analogue peptides have been investigated as stimulators of the immune system in patients with different cancers with variable but sometimes notable success. In this review we describe the background and recent developments in the use of analogue peptides for the immunotherapy of acute myeloid leukemia describing knowledge useful for the application of analogue peptide treatments for other malignancies