High-throughput sequence analysis of variants of human cytomegalovirus strains Towne and AD169.

The genomes of commonly used variants of human cytomegalovirus (HCMV) strains Towne and AD169 each contain a substantial mutation in which a region (U(L)/b') at the right end of the long unique region has been replaced by an inverted duplication of a region from the left end of the genome. Using high-throughput technology, we have sequenced HCMV strain Towne (ATCC VR-977) and confirmed the presence of two variants, one exhibiting the replacement in U(L)/b' and the other intact in this region. Both variants are mutated in genes RL13, UL1, UL40, UL130, US1 and US9. We have also sequenced a novel AD169 variant (varUC) that is intact in U(L)/b' except for a small deletion that affects genes UL144, UL142, UL141 and UL140. Like other AD169 variants, varUC is mutated in genes RL5A, RL13, UL36 and UL131A. A subpopulation of varUC contains an additional deletion affecting genes IRS1, US1 and US2.

Genotypic analysis of two hypervariable human cytomegalovirus genes.

Most human cytomegalovirus (HCMV) genes are highly conserved in sequence among strains, but some exhibit a substantial degree of variation. Two of these genes are UL146, which encodes a CXC chemokine, and UL139, which is predicted to encode a membrane glycoprotein. The sequences of these genes were determined from a collection of 184 HCMV samples obtained from Africa, Australia, Asia, Europe, and North America. UL146 is hypervariable throughout, whereas variation in UL139 is concentrated in a sequence encoding a potentially highly glycosylated region. The UL146 sequences fell into 14 genotypes, as did all previously reported sequences. The UL139 sequences grouped into 8 genotypes, and all previously reported sequences fell into a subset of these. There were minor differences among continents in genotypic frequencies for UL146 and UL139, but no clear geographical separation, and identical nucleotide sequences were represented among communities distant from each other. The frequent detection of multiple genotypes indicated that mixed infections are common. For both genes, the degree of divergence was sufficient to preclude reliable sequence alignments between genotypes in the most variable regions, and the mode of evolution involved in generating the genotypes could not be discerned. Within genotypes, constraint appears to have been the predominant mode, and positive selection was detected marginally at best. No evidence was found for linkage disequilibrium. The emerging scenario is that the HCMV genotypes developed in early human populations (or even earlier), becoming established via founder or bottleneck effects, and have spread, recombined and mixed worldwide in more recent times.

Small-molecule complement inhibitors cannot prevent the development of the platelet storage lesion.

BACKGROUND: Suppression of the platelet (PLT) storage lesion would maintain PLT quality over longer storage times. An increased storage period would greatly improve the ability of blood agencies and hospitals to manage PLT inventories and minimize product wastage. Activation of the complement system has been proposed to play a role in initiating or potentiating the PLT storage lesion. This study examines the effect of complement inhibition on the development of the PLT storage lesion. STUDY DESIGN AND METHODS: Leukofiltered PLT concentrates (PCs) were split into miniunits containing the complement inhibitors N-acetylaspartylglutamic acid (NAAGA) or compstatin, a control peptide, or saline. Samples were collected up to Day 11 of storage. Complement activation was monitored as C3a generation. PLT quality was assessed by morphology, CD62 and CD63 expression, fibrinogen binding, pH, mean PLT volume, annexin V binding, and PLT viability. Caspase-3 activity served as a measure of PLT apoptosis. RESULTS: At concentrations of NAAGA required to achieve approximately 50 percent complement inhibition, PLT activation, and caspase-3 activity were increased. Complement inhibition by compstatin was highly variable. Compstatin addition consistently resulted in a 37 to 55 percent inhibition of PLT caspase-3 activity, but PLT quality and viability were no different between compstatin PCs and control PCs over the storage time. CONCLUSIONS: Neither NAAGA nor compstatin provided complete inhibition of complement over the storage period. Addition of these small-peptide inhibitors to PCs did not slow PLT storage lesion development, in spite of the partial inhibition of caspase-3 activity in the compstatin-treated PCs.

Immune complex binding by immunocamouflaged [poly(ethylene glycol)-grafted] erythrocytes.

Immune complexes (IC) are constantly formed at low levels in normal individuals. In humans, the red blood cell (RBC) complement receptor 1 (CR1) plays the dominant role in the IC binding and clearance. Over the last several years, we have investigated the potential utility of immunocamouflaged (methoxypoly(ethylene glycol) [mPEG] grafted) RBC to attenuate the risk of alloimmunization. Because the grafted polymer nonspecifically camouflages membrane proteins, its effects on CR1 detection and IC binding were assessed. The dose dependent (0-2.5 mM) effects of activated mPEG (CmPEG, 5 kDa; and BTCmPEG, 5 and 20 kDa) on CR1 detection and the binding of artificially generated IC [C3b coated alkaline phosphatase and antialkaline phosphatase complexes] to control and pegylated RBC was investigated by flow cytometry. In contrast to selected non-ABO blood group antigens, grafted mPEG did not effectively camouflage CR1. Surprisingly, however, even very low grafting concentrations of mPEG (>or=0.3 mM) resulted in a >or=95% loss in IC binding. Further reductions in grafting concentration (0.15 and 0.03 mM mPEG) still yielded decreased IC binding of approximately 60 and 40%, respectively. Importantly, unactivated mPEG had minimal effects on IC binding. These data demonstrate that even small amounts of grafted mPEG interfere with the multivalent CR1-IC interaction necessary for high affinity IC binding, hence large volume transfusions of mPEG-RBC may be contraindicated in patients with pre-existing IC disease. Whether this concern is of clinical significance in healthy humans is less clear due to dilutional effects and the presence of secondary clearance pathways.

Separation and purification of methoxypoly(ethylene glycol) grafted red blood cells via two-phase partitioning.

Alloimmunization to donor blood group antigens remains a significant problem in transfusion medicine. To attenuate the risk of alloimmunization, we have pioneered the membrane grafting of methoxypoly(ethylene glycol) (mPEG) to produce immunocamouflaged red blood cells (RBC). Grafting of the mPEG was accomplished using cyanuric chloride activated mPEG (CmPEG; M(r) = 5000), benzotriazole carbonate methoxyPEG (BTCmPEG; M(r) = 2000, 5000 or 20000); or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPAmPEG; M(r) = 5000, or 20000). Because of the heterogeneity of grafting, a crucial tool in developing the stealth RBC is an ability to purify the modified RBC from unmodified (immunogenic) donor cells. As demonstrated, a (5, 4) dextran:PEG aqueous two-phase polymer partitioning system cleanly separated the immunologically silent mPEG-grafted human RBC from control or lightly modified cells. Cell mixing experiments employing varying ratios of mPEG-modified and control RBC confirmed the purification efficacy of the phase partitioning system. Proportional changes in PEG-rich phase partitioning were achieved by increasing either the quantity of surface mPEG or the mPEG molecular weight. The biological viability of purified mPEG-RBC (BTCmPEG; [M(r) = 20000) was demonstrated by their normal in vivo survival at immunoprotective grafting concentrations (

Biophysical consequences of linker chemistry and polymer size on stealth erythrocytes: size does matter.

Immunocamouflaged red blood cells (RBC) are produced by cell surface derivatization with methoxypolyethylene glycol (mPEG). These immunologically attenuated cells may reduce the risk of allosensitization in chronically transfused patients. To characterize the effects of differing linker chemistries and polymer lengths, RBC were modified with cyanuric chloride activated mPEG (C-mPEG 5 kDa), benzotriazole carbonate methoxyPEG (BTC-mPEG; 5 or 20 kDa) or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPA-mPEG; 2, 5 or 20 kDa). Biophysical methods including particle electrophoresis and aqueous two-phase polymer partitioning were employed to compare the PEG derivatives. While C-mPEG was faster reacting, both BTC-mPEG and SPA-mPEG gave comparable findings after 1 h. Both PEG surface density and molecular mass had a large effect on RBC surface properties. Proportional changes in electrophoretic mobility and preferential phase partitioning were achieved by increasing either the quantity of surface PEG or the PEG molecular mass. In addition, two-phase partitioning may provide a means for efficiently removing unmodified or lightly modified (hence potentially immunogenic) RBC in the clinical setting. Furthermore, mPEG modification significantly inhibits cell-cell interaction as evidenced by loss of Rouleaux formation and, consequently, sedimentation rate. Importantly, BTC-mPEG 20 kDa RBC showed normal in vivo survival in mice at immunoprotective concentrations (up to 2 mM).