Douglas M. Lublin

Transfusion medicine service policies for recombinant factor VIIa administration

Recombinant FVIIa (rFVIIa) has been approved for treatment of bleeding in hemophilia patients with inhibitors. It has also been successfully used in nonhemophilia patients with acquired antibodies against FVIII (acquired hemophilia). Pharmacological doses of rFVIIa have been found to enhance the thrombin generation on already activated platelets and, therefore, may also likely be of benefit in providing hemostasis in other situations characterized by profuse bleeding and impaired thrombin generation, 1 such as patients with thrombocytopenia and in those with functional platelet defects. 2,3 Additionally, it has been used successfully in a variety of less well‐characterized bleeding situations, 4–7 as well as in patients with impaired liver function. 8,9

The efficacy of photopheresis for bronchiolitis obliterans syndrome after lung transplantation

BACKGROUND Extracorporeal photopheresis (ECP) has been used to treat acute and chronic rejection after solid organ transplantation. However, data supporting the use of ECP for bronchiolitis obliterans syndrome (BOS) after lung transplantation are limited. METHODS We retrospectively analyzed the efficacy and safety of ECP for progressive BOS at our institution. Between January 1, 2000, and December 31, 2007, 60 lung allograft recipients were treated with ECP for progressive BOS. RESULTS During the 6-month period before the initiation of ECP, the average rate of decline in forced expiratory volume in 1 second (FEV(1)) was -116.0 ml/month, but the slope decreased to -28.9 ml/month during the 6-month period after the initiation of ECP, and the mean difference in the rate of decline was 87.1 ml/month (95% confidence interval, 57.3-116.9; p < 0.0001). The FEV(1) improved in 25.0% of patients after the initiation of ECP, with a mean increase of 20.1 ml/month. CONCLUSIONS ECP is associated with a reduction in the rate of decline in lung function associated with progressive BOS.

A review of transfusion risks and optimal management of perioperative bleeding with cardiac surgery

A pproximately 10 to 14 million red blood cell (RBC) units and 1.5 million platelet (PLT) transfusions (approx. 85%, single-donor plateletpheresis products; the remainder, pools of six whole-blood-donor PLT concentrates) are administered in the United States each year. Transfusion-related adverse (20% of transfusions) and serious adverse (0.5%) events were estimated by Walker in the 1980s. More recently, serious adverse events have been estimated to occur in 0.1 percent of RBCs and 0.04 percent of PLT transfusions. With early estimates, transfusion-associated adverse events were thought to lead to a short-term (i.e., not including disease transmission–related deaths) mortality of 1 to 1.2 per 100,000 patients, or approximately 35 transfusion-related deaths per year in the United States. With more recent estimates, long-term or total mortality (i.e., including disease transmission–related deaths) is probably higher due to unrecognized or unreported transfusion-related deaths. Perioperative signs and symptoms (e.g., fever, hypotension, tachycardia, hypoxemia, microvascular bleeding, hemoglobinuria, low urine output) can accompany any of several serious transfusion-related complications such as acute hemolytic transfusion reactions (HTRs), bacterial contamination, anaphylaxis, transfusion-related acute lung injury (TRALI), or other events (e.g. hyperkalemia, fluid overload, air embolus). Current risks associated with blood and blood component transfusion can be characterized as immune-mediated versus non–immunemediated, hemolytic versus nonhemolytic, or acute versus delayed transfusion reactions, as well as bloodborne disease transmission. Another approach would involve subdividing transfusion risks into those that are the leading causes of mortality versus those that are uncommon causes of transfusion-related mortality.

Distinct Tyrosine Kinase Activation and Triton X-100 Insolubility upon FcγRII or FcγRIIIB Ligation in Human Polymorphonuclear Leukocytes. IMPLICATIONS FOR IMMUNE COMPLEX ACTIVATION OF THE RESPIRATORY BURST

Two tyrosine kinase-dependent pathways exist for activation of the respiratory burst by polymorphonuclear leukocyte (PMN) immunoglobulin G Fc receptors. Direct ligation of FcγRII activates the respiratory burst, but ligation of the glycan phosphoinositol-linked FcγRIIIB does not. Instead, this receptor and the integrin complement receptor CR3 synergize in activation of the respiratory burst (Zhou, M.-J., and Brown, E. J. (1994) J. Cell Biol. 125, 1407-1416). Here we show that direct ligation of FcγRII leads to activation and Triton X-100 insolubility of the Src family kinase Fgr, without effect on the related myeloid Src family member Hck. In contrast, adhesion of PMN via FcγRIIIB leads to activation and Triton X-100 insolubility of Hck but not Fgr. The exclusive association of FcγRIIIB with Hck activation and Triton insolubility is not solely a result of its glycan phosphoinositol anchor, since decay accelerating factor (CD55), another prominent glycan phosphoinositol-anchored PMN protein, is associated with Fgr insolubility to a greater extent than Hck. Ligation of decay accelerating factor, with or without coligation of CR3, does not activate the PMN respiratory burst. Coligation of FcγRIIIB with FcγRII overcomes the pertussis toxin inhibition of H2O2 production in response to direct ligation of FcγRII. These data support the hypothesis that activation of Hck upon FcγRIIIB ligation has a role in generation of the synergistic respiratory burst.

The Dually Acylated NH2-terminal Domain of Gi1α Is Sufficient to Target a Green Fluorescent Protein Reporter to Caveolin-enriched Plasma Membrane Domains PALMITOYLATION OF CAVEOLIN-1 IS REQUIRED FOR THE RECOGNITION OF DUALLY ACYLATED G-PROTEIN α SUBUNITS IN VIVO

Here we investigate the molecular mechanisms that govern the targeting of G-protein α subunits to the plasma membrane. For this purpose, we used Gi1α as a model dually acylated G-protein. We fused full-length Gi1α or its extreme NH2-terminal domain (residues 1–32 or 1–122) to green fluorescent protein (GFP) and analyzed the subcellular localization of these fusion proteins. We show that the first 32 amino acids of Gi1α are sufficient to target GFP to caveolin-enriched domains of the plasma membrane in vivo, as demonstrated by co-fractionation and co-immunoprecipitation with caveolin-1. Interestingly, when dual acylation of this 32-amino acid domain was blocked by specific point mutations (G2A or C3S), the resulting GFP fusion proteins were localized to the cytoplasm and excluded from caveolin-rich regions. The myristoylated but nonpalmitoylated (C3S) chimera only partially partitioned into caveolin-containing fractions. However, both nonacylated GFP fusions (G2A and C3S) no longer co-immunoprecipitated with caveolin-1. Taken together, these results indicate that lipid modification of the NH2-terminal of Gi1α is essential for targeting to its correct destination and interaction with caveolin-1. Also, a caveolin-1 mutant lacking all three palmitoylation sites (C133S, C143S, and C156S) was unable to co-immunoprecipitate these dually acylated GFP-G-protein fusions. Thus, dual acylation of the NH2-terminal domain of Gi1α and palmitoylation of caveolin-1 are both required to stabilize and perhaps regulate this reciprocal interaction at the plasma membrane in vivo. Our results provide the first demonstration of a functional role for caveolin-1 palmitoylation in its interaction with signaling molecules.

Recruitment of CD55 and CD66e Brush Border-Associated Glycosylphosphatidylinositol-Anchored Proteins by Members of the Afa/Dr Diffusely Adhering Family of Escherichia coli That Infect the Human Polarized Intestinal Caco-2/TC7 Cells

ABSTRACT The Afa/Dr family of diffusely adhering Escherichia coli (Afa/Dr DAEC) includes bacteria expressing afimbrial adhesins (AFA), Dr hemagglutinin, and fimbrial F1845 adhesin. We show that infection of human intestinal Caco-2/TC7 cells by the Afa/Dr DAEC strains C1845 and IH11128 is followed by clustering of CD55 around adhering bacteria. Mapping of CD55 epitopes involved in CD55 clustering by Afa/Dr DAEC was conducted using CD55 deletion mutants expressed by stable transfection in CHO cells. Deletion in the short consensus repeat 1 (SCR1) domain abolished Afa/Dr DAEC-induced CD55 clustering. In contrast, deletion in the SCR4 domain does not modify Afa/Dr DAEC-induced CD55 clustering. We show that the brush border-associated glycosylphosphatidylinositol (GPI)-anchored protein CD66e (carcinoembryonic antigen) is recruited by the Afa/Dr DAEC strains C1845 and IH11128. This conclusion is based on the observations that (i) infection of Caco-2/TC7 cells by Afa/Dr DAEC strains is followed by clustering of CD66e around adhering bacteria and (ii) Afa/Dr DAEC strains bound efficiently to stably transfected HeLa cells expressing CD66e, accompanied by CD66e clustering around adhering bacteria. Inhibition assay using monoclonal antibodies directed against CD55 SCR domains, and polyclonal anti-CD55 and anti-CD66e antibodies demonstrate that CD55 and CD66e function as a receptors for the C1845 and IH11128 bacteria. Moreover, using structural draE gene mutants, we found that a mutant in which cysteine replaced aspartic acid at position 54 displayed conserved binding capacity but failed to induce CD55 and CD66e clustering. Taken together, these data give new insights into the mechanisms by which Afa/Dr DAEC induces adhesin-dependent cross talk in the human polarized intestinal epithelial cells by mobilizing brush border-associated GPI-anchored proteins known to function as transducing molecules.

Role of Decay-Accelerating Factor Domains and Anchorage in Internalization of Dr-Fimbriated Escherichia coli

ABSTRACT Dr-fimbriated Escherichia coli capable of invading epithelial cells recognizes human decay-accelerating factor (DAF) as its cellular receptor. The role of extracellular domains and the glycosylphosphatidylinositol anchor of DAF in the process of internalization of Dr+E. coli was characterized in a cell-cell interaction model. Binding of Dr+E. coli to the short consensus repeat 3 domain of DAF expressed by Chinese hamster ovary cells was critical for internalization to occur. Deletion of short consensus repeat 3 domain or replacement of Ser165 by Leu in this domain, or the use of a monoclonal antibody to this region abolished internalization. Replacing the glycosylphosphatidylinositol anchor of DAF with the transmembrane anchor of membrane cofactor protein or HLA-B44 resulted in abolition or reduction of internalization respectively. Cells expressing glycosylphosphatidylinositol-anchored DAF but not the transmembrane-anchored DAF internalized Dr+E. coli through a glycolipid pathway, since the former cells were more sensitive to inhibition by methyl-β-cyclodextrin, a sterol-chelating agent. Electron microscopic studies revealed that the intracellular vacuoles containing the internalized Dr+E. coli were morphologically distinct between the anchor variants of DAF. The cells expressing glycosylphosphatidylinositol-anchored DAF contained a single bacterium in tight-fitting vacuoles, while the cells expressing transmembrane-anchored DAF contained multiple (two or three) bacteria in spacious phagosomes. This finding suggests that distinct postendocytic events operate in the cells expressing anchor variants of DAF. We provide direct evidence for the DAF-mediated internalization of Dr+E. coli and demonstrate the significance of the glycosylphosphatidylinositol anchor, which determines the ability and efficiency of the internalization event.

Human C3b- and C4b-regulatory proteins: a new multi-gene family

The complement cascade is regulated to prevent inappropriate activation. This regulation is targeted not only at the initiation of the cascade but also at the amplification andjunctional (C3) steps. Five glycoproteins with both complement regulatory activity and binding affinity for C3b/C4b have been characterized. In plasma these molecules are factor H(H) and C4-binding protein (C4-bp) and, on cells, they are the C3b/C4b receptor (CR1), decay-accelerating factor (DAF), and gp45-70. Here Michael Holers and his colleagues review structural, functional and genetic studies of these proteins and discuss the evidence for a new multi-gene family with a common ancestral protein.

Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets

Although caveolins normally reside in caveolae, they can accumulate on the surface of cytoplasmic lipid droplets (LDs). Here, we first provided support for our model that overaccumulation of caveolins in the endoplasmic reticulum (ER) diverts the proteins to nascent LDs budding from the ER. Next, we found that a mutant H-Ras, present on the cytoplasmic surface of the ER but lacking a hydrophobic peptide domain, did not accumulate on LDs. We used the fact that wild-type caveolin-1 accumulates in LDs after brefeldin A treatment or when linked to an ER retrieval motif to search for mutants defective in LD targeting. The hydrophobic domain, but no specific sequence therein, was required for LD targeting of caveolin-1. Certain Leu insertions blocked LD targeting, independently of hydrophobic domain length, but dependent on their position in the domain. We propose that proper packing of putative hydrophobic helices may be required for LD targeting of caveolin-1.

Induction of endothelial cell decay-accelerating factor by vascular endothelial growth factor: a mechanism for cytoprotection against complement-mediated injury during inflammatory angiogenesis.

OBJECTIVE Decay-accelerating factor (DAF) is a widely expressed, multifunctional cell surface protein involved in complement regulation and cell signaling. Previous studies have demonstrated that endothelial cell (EC) DAF is up-regulated by tumor necrosis factor alpha and inhibits complement binding. Because vascular endothelial growth factor (VEGF) is cytoprotective to endothelium and is expressed at sites of chronic inflammation, we hypothesized that VEGF may induce DAF expression during inflammatory angiogenesis. METHODS Human umbilical vein and dermal microvascular EC were isolated using routine procedures, and the regulation and function of DAF, as well as other complement-regulatory proteins (membrane cofactor protein and CD59), were analyzed following stimulation with VEGF. RESULTS Incubation of large- or small-vessel EC with VEGF led to increased expression of DAF, with maximal expression after 48-72 hours of stimulation. This effect depended on the activation of protein kinase C (PKC) and required increased steady-state messenger RNA levels and de novo protein synthesis. Although VEGF-induced EC proliferation was inhibited by both p38 and p42/44 mitogen-activated protein kinase (MAPK) antagonists, DAF up-regulation in response to VEGF was only sensitive to inhibition of p38 MAPK. VEGF-stimulated EC showed a 60% reduction in C3 deposition following complement activation, and this resulted in a marked reduction in complement-mediated EC lysis. These protective effects were abolished by anti-DAF monoclonal antibody 1H4. CONCLUSION This study confirms the importance of PKC for the regulation of DAF expression by EC and reveals VEGF to be a physiologic agonist for this pathway. The up-regulation of DAF expression by VEGF may represent an important mechanism for the protection of EC from complement-mediated injury during angiogenesis in inflammatory rheumatic diseases.