Long noncoding RNA FAM157C contributes to clonal proliferation in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal disease of hematopoietic stem cells (HSCs). Long noncoding RNAs (lncRNAs) perform a wide range of biological functions, including the regulation of gene expression, cell differentiation, and proliferation, but their role in PNH remains unclear.CD59- and CD59+ granulocytes and monocytes from 35 PNH patients were sorted. High-throughput sequencing was analyzed in 5 PNH patients, and differentially expressed lncRNAs and mRNAs were identified. The mRNAs with fragments per kilobase of exon model per million mapped fragments (FPKM) > 10 in at least 3 patients were selected, and experiments were performed to identify their upstream regulatory lncRNAs. The expression of selected mRNAs and lncRNAs was verified by qRT‒PCR, and the correlation of these expression patterns with clinical data from other 30 PNH patients was analyzed. Then, the functions of the lncRNAs were studied in the PIGA-KO-THP-1 cell line.Transcription analysis revealed 742 upregulated and 1376 downregulated lncRNAs and 3276 upregulated and 213 downregulated mRNAs. After deep screening, 8 highly expressed mRNAs that were related to the NF-κB pathway were analyzed to determine coexpression patterns. LINC01002, FAM157C, CTD-2530H12.2, XLOC-064331 and XLOC-106677 were correlated with the 8 mRNAs. After measuring the expression of these molecules in 30 PNH patients by qRT‒PCR, lncRNA FAM157C was verified to be upregulated in the PNH clone, and its expression levels were positively correlated with the LDH levels and CD59- granulated and monocyte cell ratios. After knockdown of the FAM157C gene in the PIGA-KO-THP-1 cell line, we found that the cells were arrested in the G0/G1 phase and S phase, the apoptosis rate increased, and the cell proliferation decreased.LncRNA FAM157C was proven to promote PNH clone proliferation, and this is the first study to explore the role of lncRNAs in PNH.

Plasma glycated CD59 predicts postpartum glucose intolerance after gestational diabetes.

AIM: To assess whether in women with gestational diabetes mellitus (GDM), postpartum plasma glycated CD59 (pGCD59) levels predict conversion to glucose intolerance diagnosed with an oral glucose tolerance test (OGTT). METHODS: Blood levels of pGCD59 were measured in a case-control study of 105 women with GDM who underwent a 75 g OGTT 3 months postpartum. The 35 postpartum glucose intolerant cases were individually matched for age, BMI, ethnic origin, and parity with 70 women with GDM but normal postpartum OGTT (controls). The GDM cohort (105) was also matched with 105 normal glucose tolerant women during pregnancy. pGCD59 was measured by ELISA in standard peptide units (SPU). RESULTS: Mean pGCD59 postpartum was significantly higher in cases than in controls (1.5 ± 0.6 SPU vs 1.0 ± 0.6 SPU, P < 0.001). The area under the receiving operating characteristic curve (AUC) in cases vs controls was 0.72 (95% CI: 0.62-0.83) for postpartum pGCD59 and 0.50 (95% CI: 0.36-0.61) for postpartum HbA1c. A 0.5-unit increase in postpartum pGCD59 was associated with an odds ratio (OR) of 3.3 (95% CI: 1.82-6.16, P < 0.001) for glucose intolerance postpartum. A pGCD59 cut-off postpartum of 0.9 SPU had a sensitivity of 85.7% (95% CI: 69.7-95.2%), specificity of 47.8% (95% CI: 35.6-60.2%), positive predictive value of 45.4% (95% CI: 33.1-58.2%), and negative predictive value of 86.8% (95% CI: 71.9-95.6%). pGCD59 in pregnancy was a poor predictor for glucose intolerance postpartum (AUC of 0.61 (95% CI: 0.50-0.72)). CONCLUSION: pGCD59 might identify women at low risk for glucose intolerance postpartum and could help to avoid an OGTT.

Selected CD molecules and the phagocytosis of microvesicles released from erythrocytes ex vivo.

BACKGROUND AND OBJECTIVES: The accumulation of microvesicles in erythrocyte concentrates during storage or irradiation may be responsible for clinical symptoms such as inflammation, coagulation and immunization. Our aim was to determine whether any of the cluster of differentiation (CD) molecules responsible for important functions are present on microvesicles, and if their expression level is dependent on the storage period of erythrocyte concentrates. MATERIAL AND METHODS: Erythrocyte microvesicles were isolated from 'fresh' (2nd day) and 'old' (42nd day) stored erythrocyte concentrates. Qualitative cytometric analysis of 0·5 µm, erythrocyte-derived, PS-exposing vesicles was performed using the annexin V-FITC, anti-CD235a-PE antibody and calibrated beads. The microvesicles were also visualized under a confocal microscope. The expression of the molecules CD235a, CD44, CD47, CD55, CD59 and of phosphatidylserine (PS) was compared using flow cytometry. Measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. RESULTS: The analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about 0·5 µm in the 'fresh' and 'old' samples. At day 2, the microvesicles had elevated expression levels of CD47, reduced expression levels of PS, CD55 and CD59. The phagocytosis index was higher for the microvesicles isolated from the 42-day-old erythrocyte concentrates. CONCLUSION: This research may bring us closer to understanding the factors responsible for erythrocyte ageing and to evaluate the quality of stored red blood concentrates intended for transfusion.

Acetylcholinesterase provides new insights into red blood cell ageing in vivo and in vitro.

BACKGROUND: During its 120 days sojourn in the circulation, the red blood cell (RBC) remodels its membrane. Acetylcholinesterase (AChE) is a glycosylphosphatidylinositol (GPI)-linked enzyme that may serve as a marker for membrane processes occurring this ageing-associated remodelling process. MATERIALS AND METHODS: Expression and enzymatic activity of AChE were determined on RBCs of various ages, as obtained by separation based on volume and density (ageing in vivo), and on RBCs of various times of storage in blood bank conditions (ageing in vitro), as well as on RBC-derived vesicles. RESULTS: During ageing in vivo, the enzymatic activity of AChE decreases, but not the AChE protein concentration. In contrast, neither AChE activity nor concentration show a consistent, significant decrease during ageing in vitro. CD59, another GPI-linked protein that protects against complement-induced removal, also remains constant during storage. The cellular content of the integral membrane protein glycophorin A, however, decreases with storage time in the more dense RBC fractions. The latter are enriched in echinocytes and other misshapen cells during storage. DISCUSSION: Our findings suggest that, during RBC ageing, GPI-linked proteins and integral membrane proteins are differentially sorted. Also, the vesicles that are generated in vitro show a fast and extensive loss of AChE activity, but not of AChE expression. Thus, AChE characteristics may constitute sensitive biomarkers of RBC ageing in vivo, and a source of information on the structural and functional changes that GPI-linked proteins undergo during ageing in vivo and in vitro. This information may help to understand RBC homeostasis and the effects of transfusion, especially in immunologically compromised patients.

In Vivo Pig-a gene mutation assay: Guidance for 3Rs-friendly implementation.

The rodent Pig-a assay is an in vivo method for the detection of gene mutation, where lack of glycosylphosphatidylinositol-anchored proteins on the surface of circulating red blood cells (RBCs) serves as a reporter for Pig-a gene mutation. In the case of rats, the frequency of mutant phenotype RBCs is measured via fluorescent anti-CD59 antibodies and flow cytometry. The Pig-a assay meets the growing expectations for novel approaches in animal experimentation not only focusing on the scientific value of the assay but also on animal welfare aspects (3Rs principles), for example, amenable to integration into pivotal rodent 28-day general toxicology studies. However, as recommended in the Organisation for Economic Co-operation and Development Test Guidelines for genotoxicity testing, laboratories are expected to demonstrate their proficiency. While this has historically involved the extensive use of animals, here we describe an alternative approach based on a series of blood dilutions covering a range of mutant frequencies. The experiments described herein utilized either non-fluorescent anti-CD59 antibodies to provide elevated numbers of mutant-like cells, or a low volume blood sample from a single N-ethyl-N-nitrosourea treated animal. Results from these so-called reconstruction experiments from four independent laboratories showed good overall precision (correlation coefficients: 0.9979-0.9999) and accuracy (estimated slope: 0.71-1.09) of mutant cell scoring, which was further confirmed by Bland-Altman analysis. These data strongly support the use of reconstruction experiments for training purposes and demonstrating laboratory proficiency with very few animals, an ideal situation given the typically conflicting goals of demonstrating laboratory proficiency and reducing the use of animals. Environ. Mol. Mutagen. 57:678-686, 2016. © 2016 Wiley Periodicals, Inc.

Raft-based interactions of gangliosides with a GPI-anchored receptor.

Gangliosides, glycosphingolipids containing one or more sialic acid(s) in the glyco-chain, are involved in various important physiological and pathological processes in the plasma membrane. However, their exact functions are poorly understood, primarily because of the scarcity of suitable fluorescent ganglioside analogs. Here, we developed methods for systematically synthesizing analogs that behave like their native counterparts in regard to partitioning into raft-related membrane domains or preparations. Single-fluorescent-molecule imaging in the live-cell plasma membrane revealed the clear but transient colocalization and codiffusion of fluorescent ganglioside analogs with a fluorescently labeled glycosylphosphatidylinisotol (GPI)-anchored protein, human CD59, with lifetimes of 12 ms for CD59 monomers, 40 ms for CD59's transient homodimer rafts in quiescent cells, and 48 ms for engaged-CD59-cluster rafts, in cholesterol- and GPI-anchoring-dependent manners. The ganglioside molecules were always mobile in quiescent cells. These results show that gangliosides continually and dynamically exchange between raft domains and the bulk domain, indicating that raft domains are dynamic entities.

Drug-induced exposure of Schistosoma mansoni antigens SmCD59a and SmKK7.

BACKGROUND: Schistosomiasis is a serious health problem especially in developing countries and affects more than 243 million people. Only few anthelmintic drugs are available up to now. A major obstacle for drug treatment is the different developmental stages and the varying host compartments during worm development. Anthelmintic drugs have been tested mainly on adult schistosomes or freshly transformed cercariae. Knowledge concerning the larval stages is lacking. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we used in vitro-grown schistosomula (aged between 2 to 14 days) to investigate drug effects of the three anthelmintics praziquantel, artemether, and oxamniquine. Further, we analyzed the antibody accessibility of two exemplary schistosome antigens SmCD59a and SmKK7, before and after drug treatment. Our results demonstrated that praziquantel applied at a concentration of 1 µM inhibited development of all life stages. Application of 10 µM praziquantel led to dramatic morphological changes of all schistosomula. Artemether at 1 and 10 µM had differential effects depending on whether it was applied to 2-day as compared to 7- and 14-day schistosomula. While 2-day schistosomula were not killed but inhibited from further development, severe morphological damage was seen in 7- and 14-day schistosomula. Oxamniquine (1 and 10 µM) led to severe morphological impairment in all life stages. Analyzing the accessibility of the antigens SmCD59a and SmKK7 before drug treatment showed no antibody binding on living intact schistosomula. However, when schistosomula were treated with anthelmintics, both antigens became exposed on the larvae. Oxamniquine turned out to be most effective in promoting antibody binding to all schistosomula stages. CONCLUSION: This study has revealed marked differences in anthelmintic drug effects against larvae. Drug treatment increases surface antigen presentation and renders larvae accessible to antibody attack.

Evaluation of the in vivo mutagenicity of isopropyl methanesulfonate in acute and 28-day studies.

Understanding the mutagenic dose response could prove beneficial in the management of pharmaceutically relevant impurities. For most alkyl ester impurities, such as isopropyl methanesulfonate (IPMS), little in vivo mutagenicity data exist for dose analysis. The likelihood of a sublinear dose response for IPMS was assessed by comparing the Swain Scott constant, the SN 1/SN 2 reaction mechanism and the O(6) :N(7) guanine adduct ratio to that of more well-known alkyl esters. Based on available information, IPMS was predicted to have a mutagenic profile most like ethyl nitrosourea. To test this hypothesis, mature male Wistar Han rats were administered IPMS using acute (single administration at 3.5 to 56 mg/kg) or subchronic (28 days at 0.125 to 2 mg/kg/day) exposures. The in vivo Pig-a mutation assay was used to identify mutant phenotype reticulocyte (Ret) and red blood cell (RBC) populations. The maximum mutant response occurred approximately 15 and 28 days after the last dose administration in the mutant Ret and RBC populations respectively in the acute study and on Day 29 and 56 in the mutant Ret and RBC populations, respectively, in the subchronic study. A comparison of RBC mutant frequencies from acute and subchronic protocols suggests a sublinear response; however, this was not substantiated by statistical analysis. A No Observed Effect Level (NOEL) of 0.25 mg/kg/day resulted in a Permitted Daily Exposure equivalent to the Threshold of Toxicological Concern. An estimate of the NOEL based on the previously mentioned factors, in practice, would have pre-empted further investigation of the potent mutagen IPMS.

Site-specific chemoenzymatic labeling of aerolysin enables the identification of new aerolysin receptors.

Aerolysin is a secreted bacterial toxin that perforates the plasma membrane of a target cell with lethal consequences. Previously explored native and epitope-tagged forms of the toxin do not allow site-specific modification of the mature toxin with a probe of choice. We explore sortase-mediated transpeptidation reactions (sortagging) to install fluorophores and biotin at three distinct sites in aerolysin, without impairing binding of the toxin to the cell membrane and with minimal impact on toxicity. Using a version of aerolysin labeled with different fluorophores at two distinct sites we followed the fate of the C-terminal peptide independently from the N-terminal part of the toxin, and show its loss in the course of intoxication. Making use of the biotinylated version of aerolysin, we identify mesothelin, urokinase plasminogen activator surface receptor (uPAR, CD87), glypican-1, and CD59 glycoprotein as aerolysin receptors, all predicted or known to be modified with a glycosylphosphatidylinositol anchor. The sortase-mediated reactions reported here can be readily extended to other pore forming proteins.

Systemic changes in neovascular age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of visual impairment in the aged population worldwide. The mechanisms underlying this multifactorial and heterogenic disease are complex and incompletely understood. There is increasing evidence to suggest that regulatory differences in the immune system are involved in the development of the various subtypes of AMD. The purpose of this thesis was to identify some of these potential differences in patients with early or late (wet, dry, or fibrotic) AMD. Specifically, we sought to determine differences in 1) expression of regulators of the complement pathway (CD46, CD55, and CD59) on circulating leukocytes; 2) expression of microglia-inhibitory proteins (CD200 and CD200R) on circulating leukocytes; and 3) plasma concentrations of 25-hydroxyvitamin D, a factor known to inhibit angiogenesis, fibrosis, inflammation, and oxidation. All participants underwent a semi-structured interview and detailed retinal imaging. Fresh venous blood was obtained and the frequency of cells expressing the proteins in question was determined using flow cytometry. Plasma 25-hydroxyvitamin D was measured using liquid chromatography-tandem mass spectrometry. Also, genotyping as performed in order to determine the frequency of certain single-nucleotide polymorphisms in the vitamin D metabolism. Patients with wet AMD were found to have statistically significant lower frequencies of CD46 and CD59 on CD14+monocytes and higher frequencies of CD200 on CD11b+ monocytes compared to control individuals without AMD (p = 0.0070 and p = 0.047, respectively). Moreover, we found a lower frequency of CD46 on CD45+lymphocytes in patients with wet AMD and subretinal fibrosis compared to patients with wet AMD without fibrosis (p = 0.010). Vitamin D status was not different across AMD subgroups; however, the presence of subretinal fibrosis in patients with wet AMD was associated with a statistically significant lower concentration of 25-hydroxyvitamin D (p < 0.001). Our results suggest that inadequate systemic immune modulation is an important pathogenic mechanism in the aetiology of AMD. Moreover, some differences in protein expression and vitamin D status appear to be related to the phenotypical diversity of AMD, proposing that different mechanisms may underlie the different subtypes of AMD.

Simultaneous multi-species tracking in live cells with quantum dot conjugates.

Quantum dots are available in a range of spectrally separated emission colors and with a range of water-stabilizing surface coatings that offers great flexibility for enabling bio-specificity. In this study, we have taken advantage of this flexibility to demonstrate that it is possible to perform a simultaneous investigation of the lateral dynamics in the plasma membrane of i) the transmembrane epidermal growth factor receptor, ii) the glucosylphospatidylinositol-anchored protein CD59, and iii) ganglioside GM1-cholera toxin subunit B clusters in a single cell. We show that a large number of the trajectories are longer than 50 steps, which we by simulations show to be sufficient for robust single trajectory analysis. This analysis shows that the populations of the diffusion coefficients are heterogeneously distributed for all three species, but differ between the different species. We further show that the heterogeneity is decreased upon treating the cells with methyl-ß-cyclodextrin.

Molecular mechanism of inhibitory effects of C-phycocyanin combined with all-trans-retinoic acid on the growth of HeLa cells in vitro.

We studied the effects of all-trans-retinoic acid (ATRA), C-phycocyanin (C-PC), or ATRA+C-PC on the growth of cervical cells (HeLa cells), cell cycle distribution, and apoptosis. The anticancer mechanism of the drug combination was revealed. MTT assay was adopted to determine the effects of C-PC and ATRA on the growth of HeLa cells. The expression quantities of cyclin-dependent kinase (CDK) 4, cyclin D1, Bcl-2, caspase-3, and CD59 were determined by in situ hybridization, immunofluorescence, immunohistochemistry staining, Western blot, and RT-PCR. TUNEL assay was adopted to determine the cellular apoptosis levels. Both C-PC and ATRA could inhibit the growth of HeLa cells, and the combination of ATRA+C-PC functioned cooperatively to induce apoptosis in HeLa cells. The dosage of ATRA was reduced when it cooperated with C-PC to reduce the toxicity. ATRA treated with C-PC could induce more cell cycle arrests than the single drug used by decrease in cyclin D1 and CDK4 expression. The combination of the two drugs could upregulate caspase-3 and downregulate the Bcl-2 gene and induce cell apoptosis. Moreover, the combination therapy has an important immunological significance in decreased expression of the CD59 protein. Singly, C-PC or ATRA could inhibit the growth of HeLa cells, and the effects of treatment were further enhanced in the combination group. In combination with C-PC, the dosage of ATRA was effectively reduced. The C-PC + ATRA combination might take effect by inhibiting the progress of the cell cycle, inducing cell apoptosis and promoting complement-mediated cytolysis.

[Ex vivo expansion and clonal variation of CD34(+)CD59(+) cells from bone marrow in children with paroxysmal nocturnal hemoglobinuria].

OBJECTIVE: To investigate the isolation, purification and ex vivo expansion of CD34(+)CD59(+) cells from the bone marrow of children with paroxysmal nocturnal hemoglobinuria (PNH), to evaluate the capability of long-term hematopoietic reconstruction of the expanded CD34(+)CD59(+) cells, and to provide a laboratory basis for novel treatment of PNH. METHODS: CD34(+)CD59(+) cells were isolated from the bone marrow mononuclear cells of children with PNH using immunomagnetic beads and flow cytometer in sequence. The isolated cells were subjected to ex vivo expansion in the presence of different combinations of hematopoietic growth factors for two weeks. The colony-forming cells and long-term culture-initiating cells (LTC-ICs) were cultured and counted. RESULTS: The optimal combination of hematopoietic growth factors for ex vivo expansion was stem cell factor+interleukin (IL)-3+IL-6+FLT3 ligand+thrombopoietin+ery-thropoietin, and maximum expansion (30.4 ± 6.7 folds) was seen on day 7 of days 4 to 14 of ex vivo expansion. After ex vivo expansion, CD34(+)CD59(+) cells remained CD59-positive, retained strong capability of forming colony-forming units, and could still form LTC-ICs. There was no significant difference in capability of forming LTC-ICs between CD34(+)CD59(+) cells before and after expansion. The expansion capability of CD34(+)CD59(+) cells from children with PNH was significantly lower than that of CD34(+) cells from normal controls (P<0.01). CONCLUSIONS: The CD34(+)CD59(+) cells from children with PNH can be expanded in vitro. Post-expansion CD34(+)CD59(+) cells retain capability of long-term hematopoietic reconstruction. CD34(+)CD59(+) cells showed no trend towards PNH clone during culture. Ex vivo expansion of CD34(+)CD59(+) cells from children with PNH might be practical in performing autologous transplantation clinically for these children.

Interlaboratory trial of the rat Pig-a mutation assay using an erythroid marker HIS49 antibody.

The peripheral blood Pig-a assay has shown promise as a tool for evaluating in vivo mutagenicity. In this study five laboratories participated in a collaborative trial that evaluated the transferability and reproducibility of a rat Pig-a assay that uses a HIS49 antibody reacts with an antigen found on erythrocytes and erythroid progenitors. In preliminary work, flow cytometry methods were established that enabled all laboratories to detect CD59-negative erythrocyte frequencies (Pig-a mutant frequencies) of <10×10(-6) in control rats. Four of the laboratories (the in-life labs) then treated male rats with a single oral dose of N-nitroso-N-ethylurea, 7,12-dimethylbenz[a]anthracene (DMBA), or 4-nitroquinoline-1-oxide (4NQO). Blood samples were collected up to 4 weeks after the treatments and analyzed by flow cytometry for the frequency of CD59-negative cells among total red blood cells (RBCs; RBC Pig-a assay). RBC Pig-a assays were conducted in the four in-life laboratories, plus a fifth laboratory that received blood samples from the other laboratories. In addition, three of the five laboratories performed a Pig-a assay on reticulocytes (RETs; PIGRET assay), using blood from the rats treated with DMBA and 4NQO. The four in-life laboratories detected consistent, time- and dose-related increases in RBC Pig-a mutant frequency (MF) for all three test articles. Furthermore, comparable results were obtained in the fifth laboratory that received blood samples from other laboratories. The three laboratories conducting the PIGRET assay also detected consistent, time- and dose-related increases in Pig-a MF, with the RET MFs increasing more rapidly with time than RBC MFs. These results indicate that rat Pig-a assays using a HIS49 antibody were transferable between laboratories and that data generated by the assays were reproducible. The findings also suggest that the PIGRET assay may detect the in vivo mutagenicity of test compounds earlier than the RBC Pig-a assay.

Maternal dietary omega-3 fatty acid intake increases resolvin and protectin levels in the rat placenta.

Placental inflammation is associated with several pregnancy disorders. Inflammation is limited by anti-inflammatory and proresolving mechanisms, the latter partly mediated by resolvins and protectins derived from omega-3 polyunsaturated fatty acids (n-3PUFA). We examined effects of dietary n-3PUFAs on levels of resolvins, protectins, and lipoxygenase (ALOX) enzymes in the rat placenta. Rats consumed standard (Std) or high n-3PUFA (Hn3) diets from day 1 of pregnancy; tissues were collected on day 17 or 22 (term = day 23). Maternal Hn3 diet increased resolvin and protectin precursors, 18R/S-HEPE (P < 0.001), and 17R/S-HDHA (P < 0.01) at both days. Resolvins (17R-RvD1 and RvD1) increased at day 22 (P < 0.001) after Hn3 consumption, coincident with higher Alox15b and Alox5 mRNA expression, while RvD2 increased at both days (P < 0.05). Protectins, PD1, and 10S,17S-DiHDHA increased over late gestation (P < 0.001), coincident with higher Alox15 mRNA expression (P < 0.001) and further increased with Hn3 diet (P < 0.05). Maternal systemic and placental proinflammatory mediators were not suppressed by Hn3 diet; systemic IL1ß, placental Il1ß, and Il6 mRNA expression increased marginally with Hn3 at day 22 (P < 0.001), while Ptgs1 (Cox1) expression increased both days (P < 0.05). Our data indicate that maternal n-3PUFA supplementation enhances expression of enzymes in the n-3PUFA metabolic pathway and increases placental levels of resolvins and protectins.

Mesenchymal stem cells engineered to inhibit complement-mediated damage.

Mesenchymal stem cells (MSC) preferentially migrate to damaged tissues and, due to their immunomodulatory and trophic properties, contribute to tissue repair. Although MSC express molecules, such as membrane cofactor protein (CD46), complement decay-accelerating factor (CD55), and protectin (CD59), which confer protection from complement-mediated lysis, MSC are recruited and activated by anaphylatoxins after transplantation, potentially causing MSC death and limiting therapeutic benefit. We have previously demonstrated that transduction of MSC with a retrovirus encoding HCMV-US proteins resulted in higher levels of MSC engraftment due to decreased HLA-I expression. Here, we investigate whether engineering MSC to express US2 (MSC-US2), US3 (MSC-US3), US6 (MSC-US6), or US11 (MSC-US11) HCMV proteins can alter complement recognition, thereby better protecting MSC from complement attack and lysis. HCMV-US proteins increased MSC CD59 expression at different levels as determined by flow cytometric evaluation of the median fluorescence intensity ratio (MFI). A significant increase in CD59 expression was seen in MSC-US2, MSC-US3, and MSC-US6, but not in MSC-US11. Only MSC-US2 displayed increased expression of CD46, while US2 and US3 proteins were both able to augment the percentage of MSC expressing this molecule. Regardless of the HCMV protein expressed, none changed CD55 MFI; however, expression of US6, US11, and US2 each increased the percentage of MSC that were positive for this molecule. Because US2 protein was the most efficient in up-regulating all three complement regulatory proteins, we used a functional complement-mediated cytotoxicity assay to investigate whether MSC-US2 were protected from complement-mediated lysis. We demonstrated that over-expression of the US2 protein reduced complement lysis by 59.10±12.89% when compared to untransduced MSC. This is the first report, to our knowledge, describing a role of HCMV-US proteins in complement evasion, and our data shows that over-expression of US2 protein on MSC could serve as a strategy to protect these cells from complement lysis.

Flow cytometric analysis of erythrocytes in paroxysmal nocturnal hemoglobinuria reveals superiority of CD59 as a diagnostic marker compared to CD55.

CONTEXT: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by complement-mediated hemolysis due to reduced expression of glycosyl phosphatidylinositol-anchored complement deactivating proteins such as CD55 and CD59 on RBC. Flow cytometric analysis of CD55 and CD59 expression by RBC is a reliable tool for the diagnosis of PNH. AIMS: Detection and quantification of PNH clone and comparison of the relative role of CD55 and CD59 expression by RBC in the diagnosis of PNH. MATERIALS AND METHODS: Flow cytometric analysis of RBC was performed in blood samples of 239 patients by direct immunofluorescence using monoclonal anti-CD55 and anti-CD59 antibodies. CD55 and CD59 expressions by RBC were compared in 54 cases in which PNH clones were detected. RESULTS: Out of 54 cases, 85% and 72% revealed CD59 and CD55 negative populations, respectively. Various combinations of type II and III erythrocytes could be identified in all cases having CD59 deficient RBC. In contrast, distinct populations of CD55-deficient RBC were seen in only 33% cases. In the remaining (67%) cases, CD55 negative RBC caused sloping of the ascending limb of the histogram resulting in difficulties in interpretation. Fifteen percent cases had false CD55-deficient RBC and in 23% cases anti-CD55 antibody failed to identify PNH clones which were detected by CD59. CONCLUSION: CD59 is a better marker for the diagnosis of PNH. Although CD55 negativity supported the diagnosis of PNH in cases with CD59-deficient RBC, its role as an independent diagnostic marker for PNH is questionable due to its lower sensitivity and specificity.

Multiparameter flow cytometry for the diagnosis and monitoring of small GPI-deficient cellular populations.

BACKGROUND: Glycosyl-phosphatidylinositol (GPI)-negative blood cells are diagnostic for Paroxysmal Nocturnal Hemoglobinuria (PNH). Marrow failure states are often associated with GPI-negative cell populations. Quantification of small clonal populations of GPI-negative cells influences clinical decisions to administer immunosuppressive therapy in marrow failure states (aplastic anemia or myelodysplastic syndrome) and to monitor minimal residual disease after allogeneic blood or marrow transplantation (BMT). We studied the reliability of high-resolution flow cytometry markers operating at the limits of detection. METHODS: We performed serial quantification of the PNH clone size in 38 samples using multiparameter flow cytometry. Granulocytes, monocytes, and RBCs were gated using forward and side scatter as well as lineage-specific markers. The GPI-linked markers fluorescent aerolysin (FLAER), CD55, and CD59 were comparatively evaluated. We also evaluated CD16 on granulocytes and CD14 on monocytes. The sensitivity of detection by each marker was further defined by serial dilution experiments on a flow-sorted sample. Two patients had quantification of their GPI-negative clones before and after allogeneic BMT. RESULTS: FLAER was the most discriminant marker and allowed identification of 0.1% of GPI-negative cells despite other markers having superior signal-to-noise characteristics. CD14 and CD16 were inferior to CD55 at lower concentrations and in clinical application. CONCLUSIONS: Multiparameter flow cytometry permits quantification of small GPI-negative clones with a sensitivity limit of about 0.1%. The single most reliable marker to monitor small granulocyte or monocyte PNH clones is FLAER, especially in conditions such as myelodysplastic syndromes or BMT, when traditional GPI-linked surface marker expression can be significantly altered. © 2010 International Clinical Cytometry Society.

Evidence that Gag facilitates HIV-1 envelope association both in GPI-enriched plasma membrane and detergent resistant membranes and facilitates envelope incorporation onto virions in primary CD4+ T cells.

HIV-1 particle assembly mediated by viral Gag protein occurs predominantly at plasma membrane. While colocalization of HIV-1 envelope with lipid rich microenvironment have been shown in T cells, the significance of viral proteins modulating envelope association in such microdomains in plasma membrane enriched in glycosylphosphatidylinositol-anchored proteins in primary CD4+ T cells that are natural targets of HIV-1 is poorly understood. Here we show that in primary CD4+ T cells that are natural targets of HIV-1 in vivo, Gag modulates HIV-1 envelope association with GM1 ganglioside and CD59 rich cellular compartments as well as with detergent resistant membranes. Our data strengthen evidence that Gag-Env interaction is important in envelope association with lipid rafts containing GPI-anchored proteins for efficient assembly onto mature virions resulting in productive infection of primary CD4+ T cells.

Use of a FLAER-based WBC assay in the primary screening of PNH clones.

Diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) with flow cytometry traditionally involves the analysis of CD55 and CD59 on RBCs and neutrophils. However, the ability to accurately detect PNH RBCs is compromised by prior hemolysis and/or transfused RBCs. Patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS) can also produce PNH clones. We recently described a multiparameter fluorescent aerolysin (FLAER)-based flow assay using CD45, CD33, and CD14 that accurately identified PNH monocyte and neutrophil clones in PNH, AA, and MDS. Here, we compared the efficiency of this WBC assay with a CD59-based assay on RBCs during a 3-year period. PNH clones were detected with the FLAER assay in 63 (11.8%) of 536 samples tested, whereas PNH RBCs were detected in only 33 (6.2%), and always with a smaller clone size. The FLAER assay on WBCs is a more sensitive and robust primary screening assay for detecting PNH clones in clinical samples.