Controlling intrathoracic hemorrhage on ECMO: help from Factor VIIa and Virchow.

OBJECTIVE: Thoracentesis with chest tube placement is often needed to decompress a clinically significant pneumothorax or pleural effusion. The risks of such a procedure may be considered too great to perform on a systemically anticoagulated patient supported by extracorporeal membrane oxygenation (ECMO). RESULTS: An 8-year-old child with respiratory failure due to necrotizing pneumonia and autoimmune vasculitis, on veno-venous ECMO, developed a severe tension pneumothorax that required emergent decompression with a chest tube. Post-procedure, the patient developed a hemothorax that was approaching non-sustainability. We developed a strategy based on Virchow's triad to favor homeostasis in the patient while avoiding thrombosis in the ECMO circuit. We employed selective lung ventilation, passive pleural drainage, high flow ECMO, and aggressive coagulation cascade control, including the use of aminocaproic acid and activated factor VIIa. Following this strategy, the hemorrhage was controlled and, later, the patient was able to successfully come off ECMO. CONCLUSIONS: With careful coagulation cascade manipulation, complete lung rest for the affected lung, control of ECMO blood flow, and prudent hemothorax drainage, we were able to facilitate hemostasis that was required for the successful recovery of our patient while avoiding critical ECMO circuit thrombosis. Even with today's highly advanced medical technologies, centuries-old basic medical principles can still assist in the care of our sickest and most complex patients. Chest tube placement while on ECMO is rare and, although necessary, may be a risky procedure. With precise coagulation control, it can be a successful procedure on ECMO.

Targeting regulatory T cells in the treatment of type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is a T cell-mediated autoimmune disease resulting in islet ß cell destruction, hypoinsulinemia, and severely altered glucose homeostasis. T1DM has classically been attributed to the pathogenic actions of auto-reactive effector T cells(Teffs) on the ß cell. Recent literature now suggests that a failure of a second T cell subtype, known as regulatory T cells (Tregs), plays a critical role in the development of T1DM. During immune homeostasis, Tregs counterbalance the actions of autoreactive Teff cells, thereby participating in peripheral tolerance. An imbalance in the activity between Teff and Tregs may be crucial in the breakdown of peripheral tolerance, leading to the development of T1DM. In this review, we summarize our current understanding of Treg function in health and in T1DM, and examine the effect of experimental therapies for T1DM on Treg cell number and function in both mice and humans.

NK cells rapidly reject allogeneic bone marrow in the spleen through a perforin- and Ly49D-dependent, but NKG2D-independent mechanism.

We have used a sensitive and specific in vivo killing assay to monitor the kinetics, anatomic location and mechanisms controlling NK-mediated rejection of Balb/c bone marrow by C57BL/6 natural killer (NK) cells. We find that NK killing of fully allogeneic bone marrow is a rapid, highly efficient process, leading to substantial rejection of transplanted marrow within 6 h of transplant and elimination of 85% of the transplanted cells within 2 days. NK-mediated rejection occurred predominantly in the spleen, with sparing of rejection in the bone marrow and lymph nodes. Rejection was dependent on Perforin gene function, but was independent of interferon-gamma. Finally, rejection of Balb/c bone marrow by B6 NK cells required signaling through the Ly49D receptor, but occurred despite blockade of NKG2D, which distinguishes these results from previous studies using semiallogeneic transplant pairs. These results identify NK cells as highly active mediators of bone marrow rejection, and suggest that inhibiting NK function early during transplantation may increase the efficiency of engraftment and allow successful engraftment of limiting doses of donor bone marrow.

Induction of chimerism in rhesus macaques through stem cell transplant and costimulation blockade-based immunosuppression.

A strategy for producing high-level hematopoietic chimerism after non-myeloablative conditioning has been established in the rhesus macaque. This strategy relies on hematopoietic stem cell transplantation after induction with a non-myeloablative dose of busulfan and blockade of the IL2-receptor in the setting of mTOR inhibition with sirolimus and combined CD28/CD154 costimulation blockade. Hematopoietic stem cells derived from bone marrow and leukopheresis products both were found to be successful in inducing high-level chimerism. Mean peripheral blood peak donor chimerism was 81% with a median chimerism duration of 145 days. Additional immune modulation strategies, such as pre-transplant CD8 depletion, donor-specific transfusion, recipient thymectomy or peritransplant deoxyspergualin treatment did not improve the level or durability of chimerism. Recipient immunologic assessment suggested that chimerism occurred amidst donor-specific down-regulation of alloreactive T cells, and the reappearance of vigorous T-mediated alloreactivity accompanied rejection of the transplants. Furthermore, viral reactivation constituted a significant transplant-related toxicity and may have negatively impacted the ability to achieve indefinite survival of transplanted stem cells. Nevertheless, this chimerism-induction regimen induced amongst the longest-lived stem cell chimerism reported to date for non-human primates and thus represents a platform upon which to evaluate emerging tolerance-induction strategies.

Nicotinamide adenine dinucleotide (NAD) and its metabolites inhibit T lymphocyte proliferation: role of cell surface NAD glycohydrolase and pyrophosphatase activities.

The presence of NAD-metabolizing enzymes (e.g., ADP-ribosyltransferase (ART)2) on the surface of immune cells suggests a potential immunomodulatory activity for ecto-NAD or its metabolites at sites of inflammation and cell lysis where extracellular levels of NAD may be high. In vitro, NAD inhibits mitogen-stimulated rat T cell proliferation. To investigate the mechanism of inhibition, the effects of NAD and its metabolites on T cell proliferation were studied using ART2a+ and ART2b+ rat T cells. NAD and ADP-ribose, but not nicotinamide, inhibited proliferation of mitogen-activated T cells independent of ART2 allele-specific expression. Inhibition by P2 purinergic receptor agonists was comparable to that induced by NAD and ADP-ribose; these compounds were more potent than P1 agonists. Analysis of the NAD-metabolizing activity of intact rat T cells demonstrated that ADP-ribose was the predominant metabolite, consistent with the presence of cell surface NAD glycohydrolase (NADase) activities. Treatment of T cells with phosphatidylinositol-specific phospholipase C removed much of the NADase activity, consistent with at least one NADase having a GPI anchor; ART2- T cell subsets contained NADase activity that was not releasable by phosphatidylinositol-specific phospholipase C treatment. Formation of AMP from NAD and ADP-ribose also occurred, a result of cell surface pyrophosphatase activity. Because AMP and its metabolite, adenosine, were less inhibitory to rat T cell proliferation than was NAD or ADP-ribose, pyrophosphatases may serve a regulatory role in modifying the inhibitory effect of ecto-NAD on T cell activation. These data suggest that T cells express multiple NAD and adenine nucleotide-metabolizing activities that together modulate immune function.

Characterization of a novel sphingosine 1-phosphate receptor, Edg-8.

Three G protein-coupled receptors (Edg-1, Edg-3, and Edg-5) for the lysolipid phosphoric acid mediator sphingosine 1-phosphate have been described by molecular cloning. Using a similar sequence that we found in the expressed sequence tag data base, we cloned and characterized of a fourth, high affinity, rat brain sphingosine 1-phosphate receptor, Edg-8. When HEK293T cells were co-transfected with Edg-8 and G protein DNAs, prepared membranes showed sphingosine 1- phosphate-dependent increases in [(35)S]guanosine 5'-(3-O-thio)triphosphate binding with an EC(50) of 90 nm. In a rat hepatoma Rh7777 cell line that exhibits modest endogenous responses to sphingosine 1-phosphate, this lipid mediator inhibited forskolin-driven rises in cAMP by greater than 90% when the cells were transfected with Edg-8 DNA (IC(50) 0.7 nm). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through G(i/o)alpha proteins. Furthermore, Xenopus oocytes exhibit a calcium response to sphingosine 1-phosphate after injection of Edg-8 mRNA, but only when oocytes are co-injected with chimeric G(q/i)alpha protein mRNA. Membranes from HEK293T and Rh7777 cell cultures expressing Edg-8 exhibited high affinity (K(D) = 2 nm) binding for radiolabeled sphingosine 1-phosphate. Rat Edg-8 RNA is expressed in spleen and throughout adult rat brain where in situ hybridization revealed it to be associated with white matter. Together our data demonstrate that Edg-8 is a high affinity sphingosine 1-phosphate receptor that couples to G(i/o)alpha proteins and is expressed predominantly by oligodendrocytes and/or fibrous astrocytes in the rat brain.

theta, a novel gamma-aminobutyric acid type A receptor subunit.

gamma-Aminobutyric acid type A (GABA-A) receptors are a major mediator of inhibitory neurotransmission in the mammalian central nervous system, and the site of action of a number of clinically important drugs. These receptors exist as a family of subtypes with distinct temporal and spatial patterns of expression and distinct properties that presumably underlie a precise role for each subtype. The newest member of this gene family is the theta subunit. The deduced polypeptide sequence is 627 amino acids long and has highest sequence identity (50.5%) with the beta1 subunit. Within the rat striatum, this subunit coassembles with alpha2, beta1, and gamma1, suggesting that gamma-aminobutyric acid type A receptors consisting of arrangements other than alpha beta + gamma, delta, or epsilon do exist. Expression of alpha2beta1gamma1theta in transfected mammalian cells leads to the formation of receptors with a 4-fold decrease in the affinity for gamma-aminobutyric acid compared with alpha2beta1gamma1. This subunit has a unique distribution, with studies so far suggesting significant expression within monoaminergic neurons of both human and monkey brain.

Molecular and functional diversity of the expanding GABA-A receptor gene family.

Fast inhibitory neurotransmission in the mammalian CNS is mediated primarily by the neurotransmitter gamma-aminobutyric acid (GABA), which, upon binding to its receptor, leads to opening of the intrinsic ion channel, allowing chloride to enter the cell. Over the past 10 years it has become clear that a family of GABA-A receptor subtypes exists, generated through the coassembly of polypeptides selected from alpha 1-alpha 6, beta 1-beta 3, gamma 1-gamma 3, delta, epsilon, and pie to form what is most likely a pentomeric macromolecule. The gene transcripts, and indeed the polypeptides, show distinct patterns of temporal and spatial expression, such that the GABA-A receptor subtypes have a defined localization that presumably reflects their physiological role. A picture is beginning to emerge of the properties conferred to receptor subtypes by the different subunits; these include different functional properties, differential modulation by protein kinases, and the targeting to different membrane compartments. These properties presumably underlie the different physiological roles of the various receptor subtypes. Recently we have identified a further member of the GABA-A receptor gene family, which we have termed theta, which appears to be most closely related to the beta subunits. The structure, function, and distribution of theta-containing receptors, and receptors containing the recently reported epsilon subunit, are described.

Neuronally restricted RNA splicing regulates the expression of a novel GABAA receptor subunit conferring atypical functional properties [corrected; erratum to be published].

We report the isolation and characterization of a cDNA encoding a novel member of the GABA receptor gene family, epsilon. This polypeptide is 506 amino acids in length and exhibits its greatest amino acid sequence identity with the GABAA receptor gamma3 subunit (47%), although this degree of homology is not sufficient for it to be classified as a fourth gamma subunit. The epsilon subunit coassembles with GABAA receptor alpha and beta subunits in Xenopus laevis oocytes and transfected mammalian cells to form functional GABA-gated channels. alpha1beta1epsilon GABAA receptors, like alpha1beta1gamma2s receptors, are modulated by pentobarbital and the steroid 5alpha-pregnan-3alpha-ol-20-one but, unlike alpha1beta1gamma2s receptors, are insensitive to flunitrazepam. Additionally, alpha1beta1epsilon receptors exhibit rapid desensitization kinetics, as compared with alpha1beta1 or alpha1beta1gamma2s. Northern analysis demonstrates widespread expression of a large epsilon subunit transcript in a variety of non-neuronal tissues and expression of a smaller transcript in brain and spinal cord. Sequence analysis demonstrated that the large transcript contained an unspliced intron, whereas the small transcript represents the mature mRNA, suggesting regulation of expression of the epsilon subunit via neuronally restricted RNA splicing. In situ hybridization and immunocytochemistry reveal a pattern of expression in the brain restricted primarily to the hypothalamus, suggesting a role in neuroendocrine regulation, and also to subfields of the hippocampus, suggesting a role in the modulation of long term potentiation and memory.

The rat T-cell surface protein RT6 is associated with src family tyrosine kinases and generates an activation signal.

RT6 is a glycosyl-phosphatidylinositol-linked surface molecule present on most mature rat T-cells. RT6+ T-cells can prevent the expression of autoimmune diabetes in the BB rat, but the mechanism is unknown. Because cross-linking of other glycosyl-phosphatidylinositol-linked T-cell proteins is known to activate T-cells, we investigated the signaling properties of RT6. Antibody cross-linking of RT6 enhanced expression of the alpha subunit of the interleukin-2 (IL-2) receptor and potentiated the proliferation of rat T-cells cultured in the presence of phorbol ester plus recombinant IL-2 (rIL-2) and/or rIL-4. RT6 was found to coimmunoprecipitate with five tyrosine phosphorylated proteins including p60fyn and p56lck, members of the src tyrosine kinase family. Pretreatment of T-cells with phorbol ester increased the phosphorylation of proteins that coimmunoprecipitated with RT6, altered the electrophoretic mobility of several of these coimmunoprecipitated phosphoproteins, and increased the amount of p60fyn and p56lck coimmunoprecipitated with RT6. These data indicate that RT6-mediated signaling events may prime T-cells to respond to exogenous cytokines, suggesting a possible mechanism by which surface RT6 may influence T-cell function.

Cloning and expression of a novel neuropeptide Y receptor.

The neuropeptide Y family of peptides, which includes neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP), are found in the central and peripheral nervous system and display a wide array of biological activities. These actions are believed to be mediated through pharmacologically distinct G protein-coupled receptors, and, to date, three members of the NPY receptor family have been cloned. In this study we describe the cloning and expression of a novel NPY receptor from mouse genomic DNA. This receptor, designated NPY Y5, shares 60% amino acid identity to the murine NPY Y1 receptor. The pharmacology of this novel receptor resembles that of the NPY Y1 receptor and is distinct from that described for the NPY Y2, Y3, and Y4 receptors. In situ hybridization of mouse brain sections reveals expression of this receptor within discrete regions of the hypothalamus including the suprachiasmatic nucleus, anterior hypothalamus, bed nucleus stria terminalis, and the ventromedial nucleus with no localization apparent elsewhere in the brain.

Rat RT6.2 and mouse Rt6 locus 1 are NAD+: arginine ADP ribosyltransferases with auto-ADP ribosylation activity.

RT6 is a glycosylphosphatidylinositol-linked protein found on the surface of mature rat T lymphocytes. Cells that express RT6 have an immunoregulatory function and modulate the expression of autoimmune diabetes mellitus in the BioBreeding rat. A homologue of the rat RT6 gene, designated Rt6, has been identified in the mouse, but expression of mouse Rt6 protein has not been documented. Rat RT6 is known to be a nicotinamide adenine dinucleotide (NAD+) glycohydrolase. We now report that rat RT6.2 and recombinant mouse Rt6 locus 1 proteins possess auto-ADP ribosylation activity. In addition, mouse Rt6 but not rat RT6, catalyzes the ADP ribosylation of exogenous acceptors such as histones. The ADP-ribosyl-protein bonds in auto-ADP-ribosylated rat RT6.2, auto-ADP-ribosylated mouse Rt6, and ADP-ribosylhistone synthesized by Rt6 were stable to HgCl2 and HCl, but labile to NH2OH, consistent with ADP ribosylarginine linkages. To determine if these enzymatic activities could affect the function of rat T cells, the effect of substrate availability on lymphocyte proliferation was examined. An inverse correlation was observed between NAD+ concentration in the medium and the ability of rat T cells to respond to anti-CD3, ConA, and PMA plus ionomycin. The data suggest that lymphocyte surface ADP ribosyltransferases could be involved in signaling and immunoregulatory processes.

Heterogeneous expression of four MAP kinase isoforms in human tissues.

Mitogen-activated protein kinases (MAP kinases) are a group of closely related enzymes implicated in signal transduction pathways. We report the molecular cloning of four human proteins (p40mapk, p41mapk, p44mapk and p63mapk) with high homology to members of the MAP kinase family. Sequence analysis demonstrated that p44mapk and p63mapk were the products of distinct genes. However, the p40mapk and p41mapk were found to be related, and are likely to result from alternative processing of transcripts from a single gene. The heterogeneous expression of these human MAP kinase isoforms in different tissues may reflect the diversity of signal transduction pathways in differentiated cells.

Urography during pregnancy.

A review of 22,971 pregnancies from 1969-74 reveals that the reasons for requesting excretory urography were renal colic of pregnancy 11, pyelonephritis 8, renal contusion 2, threatened abortion 1, and suspected degeneration of fibroid 1. The final diagnoses were similar except that two patients with torsion of ovarian cysts and a renal calculus were discovered. The only intravenous pyelographic examination that was decisive for diagnosis was in a patient with a ureteric calculus. Careful clinical correlation should reduce excretory urography during pregnancy.