Red cell alloimmunisation in oncology patients: A study from eastern India.

PubMed

Dhar, Supriya; Basu, Sabita

2015-06-01

Red cell alloimmunisation is an important complication in multi-transfused patients with haematologic and surgical malignancies. Antibody screening with identification is necessary to ensure transfusion safety. Data on the prevalence of alloimmunisation in oncology patients is limited. In this study we assessed multitransfused haematology-oncology patients for red cell alloimmunisation. This was a retrospective analysis undertaken to assess the alloantibody prevalence and determine the antibody specificity. Retrospective analysis of antibody screening data was done for haematopoietic stem cell transplant (HSCT) patients as well as surgical oncology patients, from April 2013 to May 2014. This included the antibody screening done prior to surgery, antibody screening prior to HSCT and any antibody screening performed for these patients at cross match. Antibody screening was done using the three cell panel (surgiscreen) and if positive, further identification performed using the 11 cell panel (Resolve Panel A). If the antibody screen (three cell panel) was positive, an autocontrol was performed using reverse diluent (Ortho Biovue System) card. Patients with autoantibodies were excluded from this study. Our overall red cell alloimmunisation rate was 2.5%. Alloimmunisation rate among HSCT transplant patients was 1.6% as compared to the 2.4% in patients with solid organ malignancies. Keeping in view the low alloimmunisation rate, the justification of repeating antibody screening 72 hours post transfusion in this category of patients needs to be re-assessed. Copyright © 2015. Published by Elsevier Ltd.

Red blood cell sedimentation of Apheresis Granulocytes.

PubMed

Lodermeier, Michelle A; Byrne, Karen M; Flegel, Willy A

2017-10-01

Sedimentation of Apheresis Granulocyte components removes red blood cells. It is used to increase the blood donor pool when blood group-compatible donors cannot be recruited for a patient because of a major ABO incompatibility or incompatible red blood cell antibodies in the recipient. Because granulocytes have little ABO and few other red blood cell antigens on their membrane, such incompatibility lies mostly with the contaminating red blood cells. Video Clip S1 shows the process of red blood cell sedimentation of an Apheresis Granulocyte component. This video was filmed with a single smart phone attached to a commercial tripod and was edited on a tablet computer with free software by an amateur videographer without prior video experience. © 2017 AABB.

Red cell exchange to mitigate a delayed hemolytic transfusion reaction in a patient transfused with incompatible red blood cells.

PubMed

Irani, Mehraboon S; Karafin, Matthew S; Ernster, Luke

2017-02-01

A red cell exchange was performed to prevent a potentially fatal hemolytic transfusion reaction in a patient with anti-e who was transfused with e-antigen unscreened red blood cells during liver transplant surgery. A 64-year-old woman with cirrhosis due to hepatitis C was scheduled to receive a liver transplant. She had a previously documented anti-e, an antibody to the Rh(e)-antigen that is known to cause delayed hemolytic transfusion reactions. Pre-operatively and intra-operatively, she had massive hemorrhage which required transfusion of 34 e-antigen unscreened red blood cells (RBCs) most of which were incompatible. The hemoglobin dropped from 9.1 g/dL on post-operative day (POD)1 to 6.6 g/dL on POD6, with no evidence of blood loss. The bilirubin also increased from 5.0 mg/dL on POD 1 to 11.0 mg/dL on POD 6. As she was also becoming more hemodynamically unstable, a red cell exchange with 10 units of e-negative RBCs was performed on POD 6. She improved clinically and was extubated the following day. A few residual transfused e-positive red cells were detected after the red cell exchange until POD 13. This case illustrates how a red cell exchange can mitigate the potentially harmful effects of a delayed hemolytic transfusion reaction caused by red cell antibodies. With massive intraoperative blood loss it may not be possible to have antigen-negative RBCs immediately available, particularly for the e-antigen, which is present in 98% of the donor population. The ability to perform such a procedure may be life-saving in such patients. J. Clin. Apheresis 32:59-61, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Red Cell Alloimmunization In Multitransfused Thalassaemia Major Patients.

PubMed

Moeen, Shazia; Farooq, Nazish; Irshad, Romana; Ashfaq, Muhammad; Farooq, Umer; Idris, Muhammad

2018-01-01

Lifelong transfusions are life savers for thalassaemia patients but are associated with many complications. Alloimmunization is a major problem for blood banks. Antigens of foreign red blood cells induce the formation of antibodies in patients suffering from thalassaemia. The purpose of this study was to examine the frequency of red cell alloantibodies and to express the type of these antibodies in thalassaemia patients. Patients that have received multiple transfusions were included in this study. Those with the positive Coombs test (DAT) results were excluded from the study and remaining patients were screened for antibodies. A panel of known blood group antigens was used for the patients who had a positive antibody screening test because they had alloantibodies in their serum. First, three cell panel was applied. If the screen was positive then eleven cell panels was used to identify the specific antibody. Both the cell panels were applied at room-temperature, liss (low ionic strength saline) and coombs phase. Three hundred & two patients were selected out of which 65.6% (n=198) were males and 34.4% (n=104) females. Patient's age ranged from 1.5 years to 26 years ±5.40 years. All of the patients were given regular red cell transfusion at 2-4 weeks interval. They were given non leukodepleted transfusions. It is not the practice in any thalassaemia Centre in Pakistan to give phenotypically matched blood for Kell, Kidd, Duffy or any other minor group antigens to patients on regular blood transfusion. Alloimmunization was positive in 12 (4.0%) of the 302 patients studied. Male were 66.67% (n=8) and female were 33.33% (n=4). Samples of these positive patients were further tested to determine specificity of alloantibodies. Anti Cw was most common, detected in 4 out of 12 (1.3%) patients. Anti K, k, S and Lua were detected in 2 out of 12 (0.7%) each. Thalassemia major patients on regular blood transfusions can develop red cell alloantibodies. Detailed pretransfusion

Enhanced Neutralization Potency of Botulinum Neurotoxin Antibodies Using a Red Blood Cell-Targeting Fusion Protein

PubMed Central

Adekar, Sharad P.; Segan, Andrew T.; Chen, Cindy; Bermudez, Rodney; Elias, M. D.; Selling, Bernard H.; Kapadnis, B. P.; Simpson, Lance L.; Simon, Paul M.; Dessain, Scott K.

2011-01-01

Botulinum neurotoxin (BoNT) potently inhibits cholinergic signaling at the neuromuscular junction. The ideal countermeasures for BoNT exposure are monoclonal antibodies or BoNT antisera, which form BoNT-containing immune complexes that are rapidly cleared from the general circulation. Clearance of opsonized toxins may involve complement receptor-mediated immunoadherence to red blood cells (RBC) in primates or to platelets in rodents. Methods of enhancing immunoadherence of BoNT-specific antibodies may increase their potency in vivo. We designed a novel fusion protein (FP) to link biotinylated molecules to glycophorin A (GPA) on the RBC surface. The FP consists of an scFv specific for murine GPA fused to streptavidin. FP:mAb:BoNT complexes bound specifically to the RBC surface in vitro. In a mouse model of BoNT neutralization, the FP increased the potency of single and double antibody combinations in BoNT neutralization. A combination of two antibodies with the FP gave complete neutralization of 5,000 LD50 BoNT in mice. Neutralization in vivo was dependent on biotinylation of both antibodies and correlated with a reduction of plasma BoNT levels. In a post-exposure model of intoxication, FP:mAb complexes gave complete protection from a lethal BoNT/A1 dose when administered within 2 hours of toxin exposure. In a pre-exposure prophylaxis model, mice were fully protected for 72 hours following administration of the FP:mAb complex. These results demonstrate that RBC-targeted immunoadherence through the FP is a potent enhancer of BoNT neutralization by antibodies in vivo. PMID:21399689

Red Cell Alloantibodies in Multiple Transfused Thalassaemia Patients.

PubMed

Chaudhari, C N

2011-01-01

Thalassaemia major patients require lifelong transfusion support due to which they are prone for alloimmunization to foreign RBCs. Alloimmunization can be prevented by extended phenotype match blood transfusion. The study was conducted to know the extent of problem of alloimmunization and to find important red cell antibodies in thalassaemia patients. A cross-sectional study was conducted. A total of 32 thalassaemia patients were enrolled. The specimen was subjected to red cell alloantibody and autoantibody by column gel agglutination technique. R 1 (w) R 1 , R 2 R 2 , rr (papaine and non papain) and 11 cell panel reagent cells were used in screening and identification of alloantibodies respectively. Six (18.8 %) subjects were alloimmunized. All alloimmunized subjects were recipient of more than 20 units of transfusion. Total seven clinically significant alloantibodies were identified. Anti E and anti c were commonest antibodies in four (12.5%) patients. Red cell alloimmunization is an important risk in thalassaemia patient. 71.4% of alloantibodies were anti E and anti c type. Extended phenotype match blood transfusion for Rh-c and Rh-E antigens or level 2 antigen matching stringency needs to be explored in preventing alloimmunization in thalassaemia patients.

Influence of antigenic competition on the development of antibody-forming cell clones.

PubMed Central

Pritchard-Briscoe, H; McDougall, C; Inchley, C J

1977-01-01

Isoelectric focusing in polyacrylamide gels was used to investigate the anti-sheep red blood cell antibody responses of mice subjected to antigenic competition. A reduction in the number and intensity of antibody bands was found, even in situations where the suppression of IgG antibody titres was minimal, while with large reductions in titre, antibody bands were rarely seen. It thus appeared that the output of individual B-cell clones was severely depressed during competition. It was concluded that inhibition of clonal expansion is an important feature of competition, and that this may reflect a normal regulatory activity which acts to limit cellular proliferation during immune responses. This conclusion was supported by observations on the level of DNA synthesis, following immunization with sheep red cells, in the spleens of normal and suppressed mice. Images Fig. 2 Fig. 3 Fig. 4 PMID:300313

Red cell surface changes in cold agglutination

PubMed Central

Salsbury, A. J.; Clarke, J. A.; Shand, W. S.

1968-01-01

Surface changes in red blood cells undergoing cold agglutination have been investigated using the Cambridge Stereoscan electron microscope. On incubation of red cells with a cold agglutinin of anti-I specificity at 4°C, circular shadows on the red cell membrane developed within 2 min. At the same time the membrane showed a granularity and processes began to develop on the surface. These processes increased in length, the processes of contiguous cells became interlinked and agglutination was complete after incubation of 1 hr. On warming an agglutinated specimen, the process was reversed with separation of red cells and retraction of the finger-like processes to yield discrete red cells of normal appearance. The addition of heparin in vivo prevented agglutination but did not inhibit surface changes completely. Complement appeared to play no part in the production of cold agglutination due to these antibodies or in the reversal of agglutination by warming. The significance of the surface changes described in relation to previous information on the mechanism of agglutination, has been discussed. ImagesFig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8Fig. 9Fig. 10Fig. 11 PMID:5655472

Characterisation of a monoclonal antibody detecting Atlantic salmon endothelial and red blood cells, and its association with the infectious salmon anaemia virus cell receptor.

PubMed

Aamelfot, Maria; Weli, Simon C; Dale, Ole B; Koppang, Erling O; Falk, Knut

2013-05-01

Endothelial cells (ECs) line the luminal surfaces of the cardiovascular system and play an important role in cardiovascular functions such as regulation of haemostasis and vasomotor tone. A number of fish and mammalian viruses target these cells in the course of their infection. Infectious salmon anaemia virus (ISAV) attacks ECs and red blood cells (RBCs) of farmed Atlantic salmon (Salmo salar L.), producing the severe disease of infectious salmon anaemia (ISA). The investigation of ISA has up to now been hampered by the lack of a functional marker for ECs in Atlantic salmon in situ. In this study, we report the characterisation and use of a novel monoclonal antibody (MAb) detecting Atlantic salmon ECs (e.g. vessel endothelium, endocardial cells and scavenger ECs) and RBCs. The antibody can be used with immunohistochemistry, IFAT and on Western blots. It appears that the epitope recognised by the antibody is associated with the ISAV cellular receptor. Besides being a tool to identify ECs in situ, it could be useful in further studies of the pathogenicity of ISA. Finally, the detection of an epitope shared by ECs and RBCs agrees with recent findings that these cells share a common origin, thus the MAb can potentially be used to study the ontogeny of these cells in Atlantic salmon. © 2013 Anatomical Society.

Transfusion of prion-filtered red cells does not increase the rate of alloimmunization or transfusion reactions in patients: results of the UK trial of prion-filtered versus standard red cells in surgical patients (PRISM A).

PubMed

Elebute, Modupe O; Choo, Louise; Mora, Ana; MacRury, Coral; Llewelyn, Charlotte; Purohit, Shilpi; Hicks, Vicky; Casey, Caroline; Malfroy, Moira; Deary, Alison; Reed, Tania; Meredith, Sarah; Manson, Lynn; Williamson, Lorna M

2013-03-01

This study, conducted for the UK Blood Transfusion Services (UKBTS), evaluated the clinical safety of red cells filtered through a CE-marked prion removal filter (P-Capt™). Patients requiring blood transfusion for elective procedures in nine UK hospitals were entered into a non-randomized open trial to assess development of red cell antibodies to standard red cell (RCC) or prion-filtered red cell concentrates (PF-RCC) at eight weeks and six months post-transfusion. Patients who received at least 1 unit of PF-RCC were compared with a control cohort given RCC only. About 917 PF-RCC and 1336 RCC units were transfused into 299 and 291 patients respectively. Twenty-six new red cell antibodies were detected post-transfusion in 10 patients in each arm, an overall alloimmunization rate of 4.4%. Neither the treatment arm [odds ratio (OR) 0.93, 95% confidence interval (CI) 0.3, 2.5] nor number of units transfused (OR 0.95, 95% CI 0.8, 1.1) had a significant effect on the proportion of patients who developed new alloantibodies. No pan-reactive antibodies or antibodies specifically against PF-RCC were detected. There was no difference in transfusion reactions between arms, and no novel transfusion-related adverse events clearly attributable to PF-RCC were seen. These data suggest that prion filtration of red cells does not reduce overall transfusion safety. This finding requires confirmation in large populations of transfused patients. © 2013 Blackwell Publishing Ltd.

Paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

PubMed

Yeow, Natasha; McLiesh, Heather; Guan, Liyun; Shen, Wei; Garnier, Gil

2016-07-01

A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. Graphical abstract A rapid and simple paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

DOEpatents

Jensen, R.H.; Vanderlaan, M.; Bigbee, W.L.; Stanker, L.H.; Branscomb, E.W.; Grabske, R.J.

1984-11-29

The present invention provides monoclonal antibodies specific to and distinguishing between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype. 4 figs.

Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

DOEpatents

Jensen, Ronald H.; Vanderlaan, Martin; Bigbee, William L.; Stanker, Larry H.; Branscomb, Elbert W.; Grabske, Robert J.

1988-01-01

The present invention provides monoclonal antibodies specific to and distinguish between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype.

Sperm, nuclear, phospholipid, and red blood cell antibodies and isotype RF in infertile couples and patients with autoimmune rheumatic diseases.

PubMed

Fichorova, R; Nakov, L; Baleva, M; Nikolov, K; Gegova, I

1996-12-01

To determine if measuring of nonorgan-specific autoantibodies is useful for better understanding and management of unexplained infertility. Sera were obtained from 70 infertile couples, 57 rheumatic patients, and 76 fertile donors. Sperm antibodies (SA) were detected by the tests of Kibrick and Friberg, anti-histones, anti-cardiolipin antibodies, and RF isotypes by ELISA, antinuclear antibodies by indirect immunofluorescence, and anti-red blood cell antibodies by Capture-R. Multiple autoimmune reactivity (both partners positive and/or more than one type of autoantibody involved), higher than naturally occurring in fertile individuals, was found in 55% of the idiopathically infertile couples. IgA-RF was the dominant autoimmune marker. SA revealed similar rates in patients with rheumatic diseases and in infertiles with or without other autoantibodies. Although no single autoimmunity marker could predict occurrence of SA, the coincidence of enhanced polyclonal autoimmunity in both partners of infertile couples might potentiate their negative effect on reproduction.

Clearance of Human IgG1-Sensitised Red Blood Cells In Vivo in Humans Relates to the In Vitro Properties of Antibodies from Alternative Cell Lines

PubMed Central

Armour, Kathryn L.; Smith, Cheryl S.; Ip, Natasha C. Y.; Ellison, Cara J.; Kirton, Christopher M.; Wilkes, Anthony M.; Williamson, Lorna M.; Clark, Michael R.

2014-01-01

We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance. PMID:25302805

Clearance of human IgG1-sensitised red blood cells in vivo in humans relates to the in vitro properties of antibodies from alternative cell lines.

PubMed

Armour, Kathryn L; Smith, Cheryl S; Ip, Natasha C Y; Ellison, Cara J; Kirton, Christopher M; Wilkes, Anthony M; Williamson, Lorna M; Clark, Michael R

2014-01-01

We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance.

A single-chain fragment variable recombinant antibody against F5 fimbria of enterotoxigenic Escherichia coli inhibits agglutination of horse red blood cells induced by F5 protein.

PubMed

Bhaskaran, S; Jay, C M; Berghman, L R; Wagner, G G; Waghela, S D

2005-08-01

Bovine colibacillosis caused by enterotoxigenic Escherichia coli (ETEC) is a worldwide problem. Adhesion of ETEC to intestinal cell receptors mediated by the surface protein F5 fimbriae is the initial step in the establishment of colibacillosis. Prevention of ETEC F5(+) adhesion to enterocytes protects newborn calves against collibacillosis. On the enterocytes, the F5 fimbriae bind to a ganglioside that is also found on horse red blood cells. Thus, the presence of F5 fimbriae induces haemagglutination, which is useful as an indicator in a functional assay system. In this study, recombinant anti-F5 scFv antibody fragment produced in E. coli HB2151 reacted with F5 fimbriae in ELISA and Western immunoblot, and prevented haemagglutination induced by the binding of the F5 fimbriae to its natural host receptors on horse red blood cells. Given the ease with which recombinant antibodies can be mass-produced, the presently described scFv may hold promise as a prophylactic agent for colibacillosis.

Avian influenza virus antibodies in Pacific Coast Red Knots (Calidris canutus rufa)

USGS Publications Warehouse

Johnson, James A.; DeCicco, Lucas H.; Ruthrauff, Daniel R.; Krauss, Scott; Hall, Jeffrey S.

2014-01-01

Prevalence of avian influenza virus (AIV) antibodies in the western Atlantic subspecies of Red Knot (Calidris canutus rufa) is among the highest for any shorebird. To assess whether the frequency of detection of AIV antibodies is high for the species in general or restricted only to C. c. rufa, we sampled the northeastern Pacific Coast subspecies of Red Knot (Calidris canutus roselaari) breeding in northwestern Alaska. Antibodies were detected in 90% of adults and none of the chicks sampled. Viral shedding was not detected in adults or chicks. These results suggest a predisposition of Red Knots to AIV infection. High antibody titers to subtypes H3 and H4 were detected, whereas low to intermediate antibody levels were found for subtypes H10 and H11. These four subtypes have previously been detected in shorebirds at Delaware Bay (at the border of New Jersey and Delaware) and in waterfowl along the Pacific Coast of North America.

Antibodies to Rickettsia spp. and Borrelia burgdorferi in Spanish Wild Red Foxes (Vulpes vulpes).

PubMed

Lledó, Lourdes; Serrano, José Luis; Isabel Gegúndez, María; Giménez-Pardo, Consuelo; Saz, José Vicente

2016-01-01

We examined 314 red foxes (Vulpes vulpes) from the province of Soria, Spain, for Rickettsia typhi, Rickettsia slovaca, and Borrelia burgdorferi infection. Immunofluorescence assays showed 1.9% had antibodies to R. typhi, 6.7% had antibodies to R. slovaca, and 8.3% had antibodies to B. burgdorferi. Serostatus was not correlated with sex or age. Because red foxes can be infected by Rickettsiae and B. burgdorferi, presence of red foxes may be and indicator for the presence of these pathogens.

Red Blood Cell Antibody Identification

MedlinePlus

... cause a transfusion reaction (is not clinically significant). Examples of RBC antibodies and their clinical significance are ... online at http://chapters.redcross.org/ca/socal/research/circular.html. Accessed June 2009. Oren, E. and ...

Method using CO for extending the useful shelf-life of refrigerated red blood cells

DOEpatents

Bitensky, Mark W.

1995-01-01

Method using CO for extending the useful shelf-life of refrigerated red blood cells. Carbon monoxide is utilized for stabilizing hemoglobin in red blood cells to be stored at low temperature. Changes observed in the stored cells are similar to those found in normal red cell aging in the body, the extent thereof being directly related to the duration of refrigerated storage. Changes in cell buoyant density, vesiculation, and the tendency of stored cells to bind autologous IgG antibody directed against polymerized band 3 IgG, all of which are related to red blood cell senescence and increase with refrigerated storage time, have been substantially slowed when red blood cells are treated with CO. Removal of the carbon monoxide from the red blood cells is readily and efficiently accomplished by photolysis in the presence of oxygen so that the stored red blood cells may be safely transfused into a recipient.

Red Blood Cell Antibody Screen

MedlinePlus

... Internet]. Ann Arbor (MI): The Regents of the University of Michigan; c1995-2017. Coombs Antibody Test (Indirect ... gov/health/health-topics/topics/bdt/with NorthShore University Health System [Internet]. NorthShore University Health System; c2017. ...

Red cell alloantibodies in thalassemia major. Results of an Italian cooperative study.

PubMed

Sirchia, G; Zanella, A; Parravicini, A; Morelati, F; Rebulla, P; Masera, G

1985-01-01

Clinical and serological data on 1435 Italian thalassemia major patients were collected during a cooperative study involving 19 centers in 10 regions. The main findings were as follows: 18 percent of the patients were under 6 years of age, 63 percent between 6 and 15, and 19 percent over 15. Forty-one percent had undergone splenectomy. Sixty-two percent of the patients were maintained at pretransfusion hemoglobin levels higher than 10 g per dl, 36 percent between 8 and 10 g per dl, and 2 percent below 8 g per dl. Overall, 5.2 percent of the patients had clinically significant red cell alloantibodies (136 alloantibodies in 74 patients). One-half of the immunized patients had more than one and one-fourth had more than two alloantibodies. The specificities of the 136 alloantibodies were almost exclusively confined to the common antigens of the Rh, Kell, Kidd, and Duffy systems, in that decreasing order of frequency. The antibody screening procedure, using a low-ionic-strength solution antiglobulin test against a three-red-cell panel and the patient's own red cells (autocontrol) with a serum to cell ratio of 100 to 1 was shown to be an adequate technique for red cell antibody detection.

Method using CO for extending the useful shelf-life of refrigerated red blood cells

DOEpatents

Bitensky, M.W.

1995-12-19

A method is disclosed using CO for extending the useful shelf-life of refrigerated red blood cells. Carbon monoxide is utilized for stabilizing hemoglobin in red blood cells to be stored at low temperature. Changes observed in the stored cells are similar to those found in normal red cell aging in the body, the extent thereof being directly related to the duration of refrigerated storage. Changes in cell buoyant density, vesiculation, and the tendency of stored cells to bind autologous IgG antibody directed against polymerized band 3 IgG, all of which are related to red blood cell senescence and increase with refrigerated storage time, have been substantially slowed when red blood cells are treated with CO. Removal of the carbon monoxide from the red blood cells is readily and efficiently accomplished by photolysis in the presence of oxygen so that the stored red blood cells may be safely transfused into a recipient. 5 figs.

Decreased complement mediated binding of antibody//sup 3/-dsDNA immune complexes to the red blood cells of patients with systemic lupus erythematosus, rheumatoid arthritis, and hematologic malignancies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taylor, R.P.; Horgan, C.; Buschbacher, R.

1983-06-01

The complement mediated binding of prepared antibody//sup 3/H-dsDNA immune complexes to the red blood cells obtained from a number of patient populations has been investigated. Patients with solid tumors have binding activity similar to that seen in a normal group of individuals. However, a significant fraction of patients with systemic lupus erythematosus, rheumatoid arthritis, and hematologic malignancies have lowered binding activity compared with normal subjects. Quantitative studies indicate the lowered activity probably arises due to a decrease in complement receptors on the respective red blood cells. The potential importance and implications of these findings are briefly discussed.

Nature of a Red Cell Sensitizing Substance from Streptococci1

PubMed Central

Jackson, Robert W.; Moskowitz, Merwin

1966-01-01

Jackson, Robert W. (Purdue University, Lafayette, Ind.), and Merwin Moskowitz. Nature of a red cell sensitizing substance from streptococci. J. Bacteriol. 91:2205–2209. 1966.—A method for purifying a streptococcal antigen which sensitizes red cells to agglutination by antiserum is described. The antigen, when purified by this method, is almost exclusively composed of glycerophosphate and d-alanine. The ratio of alanine to glycerophosphate varies from 1:5 to 1:3. The glycerophosphate is polymerized and is thus a teichoic acid. The polyglycerophosphate appears to be the antigenic determinant for agglutination. d-Alanine is readily removed by mild base and appears to be necessary for the attachment of the teichoic acid to red cells. Quantitative removal of alanine does not affect the ability of the polymer to absorb antibody from serum. PMID:5329284

Hyperreactive malarial splenomegaly is associated with low levels of antibodies against red blood cell and Plasmodium falciparum derived glycolipids in Yanomami Amerindians from Venezuela.

PubMed

Vivas, Livia; O'Dea, Kieran P; Noya, Oscar; Pabon, Rosalba; Magris, Magda; Botto, Carlos; Holder, Anthony A; Brown, K Neil

2008-03-01

The immunological basis of the aberrant immune response in hyperreactive malarial splenomegaly (HMS) is poorly understood, but believed to be associated with polyclonal B cell activation by an unidentified malaria mitogen, leading to unregulated immunoglobulin and autoantibody production. HMS has been previously reported in Yanomami communities in the Upper Orinoco region of the Venezuelan Amazon. To investigate a possible association between antibody responses against Plasmodium falciparum and uninfected red blood cell (URBC) glycolipids and splenomegaly, a direct comparison of the parasite versus host anti-glycolipid antibody responses was made in an isolated community of this area. The anti-P. falciparum glycolipid (Pfglp) response was IgG3 dominated, whereas the uninfected red blood cell glycolipid (URBCglp) response showed a predominance of IgG1. The levels of IgG1 against Pfglp, and of IgG4 and IgM against URBCglp were significantly higher in women, while the anti-Pfglp or URBCglp IgM levels were inversely correlated with the degree of splenomegaly. Overall, these results suggest differential regulation of anti-parasite and autoreactive responses and that these responses may be linked to the development and evolution of HMS in this population exposed to endemic malaria. The high mortality rates associated with HMS point out that its early diagnosis together with the implementation of malaria control measures in these isolated Amerindian communities are a priority.

Neospora caninum and Toxoplasma gondii antibodies in red foxes (Vulpes vulpes) in the Czech Republic.

PubMed

Bártová, Eva; Slezáková, Radka; Nágl, Ivan; Sedlák, Kamil

2016-01-01

Neospora caninum and Toxoplasma gondii are worldwide spread parasites, causing serious illnesses in sensitive animals; toxoplasmosis is also important zoonosis. Although neosporosis is not considered as a zoonosis, it leads to aborted births in cattle, as well as paresis and paralysis in dogs. The aim of this study was to discover the prevalence of N. caninum and T. gondii antibodies in red foxes (Vulpes vulpes) in the Czech Republic. Sera of 80 foxes from 8 regions of the Czech Republic were tested for antibodies to N. caninum and T. gondii by competitive enzyme linked immunosorbent assay (cELISA) and indirect ELISA. All samples were simultaneously tested by indirect fluorescent antibody test (IFAT) to detect both N. caninum and T. gondii antibodies. Antibodies to N. caninum were found by IFAT in 3 (3.8%) red foxes with titre 50 and in 2 (2.5%) red foxes with inhibition 42.7% and 30.2 %. Antibodies to T. gondii were found in all tested animals in both IFAT (titres 50-6400) and in ELISA (S/P ranging from 34%-133%). This is the first prevalence study of N. caninum and T. gondii antibodies in red foxes in the Czech Republic. The results obtained show that red foxes are exposed at different levels to both protozoan infections, and thus could play an important role in the transmission cycle of N. caninum and T. gondii in sylvatic cycle.

Passive potassium transport in low potassium sheep red cells: dependence upon cell volume and chloride.

PubMed Central

Dunham, P B; Ellory, J C

1981-01-01

The major pathway of passive K influx (ouabain-insensitive) was characterized in low-K type (LK) red cells of sheep. 1. Passive K transport in these cells was highly sensitive to variations in cell volume; it increased threefold or more in cells swollen osmotically by 10%, and decreased up to twofold in cells shrunken 5-10%. Active K influx was insensitive to changes in cell volume. Three different methods for varying cell volume osmotically all gave similar results. 2. The volume-sensitive pathway was specific for K in that Na influx did not vary with changes in cell volume. 3. The volume-sensitive K influx was a saturable function of external K concentration. It was slightly inhibited by Na, whereas K influx in shrunken cells was unaffected by Na. 4. Passive K influx was dependent on the major anion in the medium in that replacement of Cl with any of six other anions resulted in a reduction of K influx by 50-80% (replacement of Cl by Br caused an increase in K influx). The activation of K influx by Cl followed sigmoid kinetics. 5. Passive K influx is inhibited by anti-L antibody. The antibody affected only that portion of influx which was Cl-dependent and volume-sensitve. Of the subfractions of the antibody, it is anti-L1 which inhibits passive K transport. 6. Pretreatment of cells with iodoacetamide reduced the sensitivity of K influx to cell volume in that the influx was reduced in swollen IAA-treated cells and increased in shrunken IAA-cells. 7. Intracellular Ca has no role in altering passive K transport in LK sheep cells. Therefore, the major pathway of passive K transport in LK sheep red cells is sensitive to changes in cell volume, specific for K, dependent on Cl, and inhibited by anti-L1 antibody, The minor pathway, observed in shrunken cells, has none of these properties. PMID:6798197

Detection of adult T-cell leukemia virus (ATLV) bearing lymphocytes in concentrated red blood cells derived from ATL associated antibody (ATLA-Ab) positive donors.

PubMed

Morishima, Y; Ohya, K; Ueda, R; Fukuda, T

1986-01-01

Adult T cell leukemia associated antibody (ATLA-Ab) positive persons were screened by indirect immunofluorescence (IF) testing. Their lymphocytes were collected from concentrated red blood cells (CRC), and cultured in vitro with and without phytohemagglutinin (PHA) for 10 days. The expression of ATL virus (ATLV) positive lymphocytes during the in vitro culture was then analyzed by IF assay using mouse monoclonal antibody ATL-19 reactive to p19 core protein of ATLV. 97% of ATLA-Ab positive CRC (36 cases) demonstrated ATLV positive lymphocytes after being cultured for more than 10 days with PHA, whereas, none of ATLA-Ab negative CRC (22 cases) demonstrated ATLV positive lymphocytes. All of the 10 ATLA-Ab positive CRC that were stored for 2, 4, and 7 days contained lymphocytes which expressed ATLV after in vitro culture, while 7 of 10 CRC stored for 14 days and only 1 of 10 CRCs stored for 20 days, expressed ATLV positive lymphocytes. This data indicates that almost all of the ATLA-Ab positive blood contained ATLV positive lymphocytes, and that the in vitro appearance of these ATLV positive lymphocytes was reduced by storing the CRC for more than 14 days.

Human monoclonal antibodies reactive with human myelomonocytic leukemia cells.

PubMed

Posner, M R; Santos, D J; Elboim, H S; Tumber, M B; Frackelton, A R

1989-04-01

Peripheral blood mononuclear cells from a patient with chronic myelogenous leukemia (CML), in remission, were depleted of CD8-positive T-cells and cultured with Epstein-Barr virus. Four of 20 cultures (20%) secreted human IgG antibodies selectively reactive with the cell surfaces of certain human leukemia cell lines. Three polyclonal, Epstein-Barr virus-transformed, B-cell lines were expanded and fused with the human-mouse myeloma analogue HMMA2.11TG/O. Antibody from secreting clones HL 1.2 (IgG1), HL 2.1 (IgG3), and HL 3.1 (IgG1) have been characterized. All three react with HL-60 (promyelocytic), RWLeu4 (CML promyelocytic), and U937 (monocytic), but not with KG-1 (myeloblastic) or K562 (CML erythroid). There is no reactivity with T-cell lines, Burkitt's cell lines, pre-B-leukemia cell lines, or an undifferentiated CML cell line, BV173. Leukemic cells from two of seven patients with acute myelogenous leukemia and one of five with acute lymphocytic leukemia react with all three antibodies. Normal lymphocytes, monocytes, polymorphonuclear cells, red blood cells, bone marrow cells, and platelets do not react. Samples from patients with other diverse hematopoietic malignancies showed no reactivity. Immunoprecipitations suggest that the reactive antigen(s) is a lactoperoxidase iodinatable series of cell surface proteins with molecular weights of 42,000-54,000 and a noniodinatable protein with a molecular weight of 82,000. Based on these data these human monoclonal antibodies appear to react with myelomonocytic leukemic cells and may detect a leukemia-specific antigen or a highly restricted differentiation antigen.

Anti-erythrocyte antibodies may contribute to anaemia in Plasmodium vivax malaria by decreasing red blood cell deformability and increasing erythrophagocytosis.

PubMed

Mourão, Luiza Carvalho; Roma, Paula Magda da Silva; Sultane Aboobacar, Jamila da Silva; Medeiros, Camila Maia Pantuzzo; de Almeida, Zélia Barbosa; Fontes, Cor Jesus Fernandes; Agero, Ubirajara; de Mesquita, Oscar Nassif; Bemquerer, Marcelo Porto; Braga, Érika Martins

2016-08-04

Plasmodium vivax accounts for the majority of human malaria infections outside Africa and is being increasingly associated in fatal outcomes with anaemia as one of the major complications. One of the causes of malarial anaemia is the augmented removal of circulating non-infected red blood cells (nRBCs), an issue not yet fully understood. High levels of auto-antibodies against RBCs have been associated with severe anaemia and reduced survival of nRBCs in patients with falciparum malaria. Since there are no substantial data about the role of those antibodies in vivax malaria, this study was designed to determine whether or not auto-antibodies against erythrocytes are involved in nRBC clearance. Moreover, the possible immune mechanisms elicited by them that may be associated to induce anaemia in P. vivax infection was investigated. Concentrations of total IgG were determined by sandwich ELISA in sera from clinically well-defined groups of P. vivax-infected patients with or without anaemia and in healthy controls never exposed to malaria, whereas the levels of specific IgG to nRBCs were determined by cell-ELISA. Erythrophagocytosis assay was used to investigate the ability of IgGs purified from each studied pooled sera in enhancing nRBC in vitro clearance by THP-1 macrophages. Defocusing microscopy was employed to measure the biomechanical modifications of individual nRBCs opsonized by IgGs purified from each group. Anaemic patients had higher levels of total and specific anti-RBC antibodies in comparison to the non-anaemic ones. Opsonization with purified IgG from anaemic patients significantly enhanced RBCs in vitro phagocytosis by THP-1 macrophages. Auto-antibodies purified from anaemic patients decreased the nRBC dynamic membrane fluctuations suggesting a possible participation of such antibodies in the perturbation of erythrocyte flexibility and morphology integrity maintenance. These findings revealed that vivax-infected patients with anaemia have increased

Direct measurement of IgM-Antigen interaction energy on individual red blood cells.

PubMed

Yeow, Natasha; Tabor, Rico F; Garnier, Gil

2017-07-01

Most blood grouping tests rely on the principle of red blood cells (RBCs) agglutination. Agglutination is triggered by the binding of specific blood grouping antibodies to the corresponding RBC surface antigen on multiple cells. The interaction energies between blood grouping antibodies and antigens have been poorly defined in immunohaematology. Here for the first time, we functionalized atomic force microscope (AFM) cantilevers with the IgM form of blood grouping antibodies to probe populations of individual RBCs of different groups under physiological conditions. The force-mapping mode of AFM allowed us to measure specific antibody - antigen interactions, and simultaneously localize and quantify antigen sites on the scanned cell surface. This study provides a new insight of the interactions between IgM antibodies and its corresponding antigen. The technique and information can be translated to develop better blood typing diagnostics and optimize target-specific drug delivery for medical applications. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Comparison of non-invasive approaches to red marrow dosimetry for radiolabelled monoclonal antibodies.

PubMed

Plaizier, M A; Roos, J C; Teule, G J; van Dieren, E B; den Hollander, W; Haisma, H J; DeJager, R L; van Lingen, A

1994-03-01

Red marrow is usually the dose-limiting organ during radioimmunotherapy. Several non-invasive approaches to calculate the red marrow dose have been proposed. We compared four approaches to analyse the differences in calculated red marrow doses. The data were obtained from immunoscintigraphy of two antibodies with different red marrow kinetics [iodine-131-16.88 IgM and indium-111-OV-TL-3 F(ab')2]. The approaches are based on, respectively, homogeneously distributed activity in the body, a red marrow-blood activity concentration ratio of 0.3, scintigraphic quantification, and a combination of the second and third approaches. This fourth approach may be more adequate because of its independence from the chosen antibody. In addition, the influence of activity accumulation in liver, kidneys or cancellous bone on red marrow dose was studied. The calculated red marrow dose varied between 0.14 and 0.42 mGy/MBq for 111In-OV-TL-3 and between 0.13 and 0.68 mGy/MBq for 131I-16.88. If the radiopharmaceutical shows high affinity for cancellous bone or another organ situated near the red marrow, the activity in these organs must be included in dose calculations. This study shows a large variation in calculated red marrow dose and selection of the definitive non-invasive approach awaits validation.

Interaction forces between red cells agglutinated by antibody. II. Measurement of hydrodynamic force of breakup.

PubMed Central

Tha, S P; Shuster, J; Goldsmith, H L

1986-01-01

The expressions derived in the previous paper for the respective normal, F3, and shear forces, Fshear, acting along and perpendicular to the axis of a doublet of rigid spheres, were used to determine the hydrodynamic forces required to separate two red cell spheres of antigenic type B crosslinked by the corresponding antibody. Cells were sphered and swollen in isotonic buffered glycerol containing 8 X 10(-5) M sodium dodecyl sulfate, fixed in 0.085% glutaraldehyde, and suspended in aqueous glycerol (viscosity: 15-34 mPa s), containing 0.15 M NaCl and anti-B antibody from human hyperimmune antiserum at concentrations from 0.73 to 3.56 vol%. After incubating and mixing for 12 h, doublets were observed through a microscope flowing in a 178-micron tube by gravity feed between two reservoirs. Using a traveling microtube apparatus, the doublets were tracked in a constantly accelerating flow and the translational and rotational motions were recorded on videotape until breakup occurred. From a frame by frame replay of the tape, the radial position, velocity and orientation of the doublet were obtained and the normal and shear forces of separation at breakup computed. Both forces increased significantly with increasing antiserum concentration, the mean values of F3 increasing from 0.060 to 0.197 nN, and Fshear from 0.023 to 0.072 nN. There was no significant effect of glycerol viscosity on the forces of separation. It was not possible to determine whether the shear or normal force was responsible for doublet separation. Measurements of the mean dimensionless period of rotation, TG, of doublets in suspensions containing 0.73 and 2.40% antiserum undergoing steady flow were also made to test whether the spheres were rigidly linked or capable of some independent rotation. A fairly narrow distribution in TG about the value 15.64, predicted for rigidly-linked doublets, was obtained at both antiserum concentrations. Images FIGURE 1 PMID:3801572

Phosphatidylserine exposure and red cell viability in red cell aging and in hemolytic anemia.

PubMed

Boas, F E; Forman, L; Beutler, E

1998-03-17

Phosphatidylserine (PS) normally localizes to the inner leaflet of cell membranes but becomes exposed in abnormal or apoptotic cells, signaling macrophages to ingest them. Along similar lines, it seemed possible that the removal of red cells from circulation because of normal aging or in hemolytic anemias might be triggered by PS exposure. To investigate the role of PS exposure in normal red cell aging, we used N-hydroxysuccinimide-biotin to tag rabbit red cells in vivo, then used phycoerythrin-streptavidin to label the biotinylated cells, and annexin V-fluorescein isothiocyanate (FITC) to detect the exposed PS. Flow cytometric analysis of these cells drawn at 10-day intervals up to 70 days after biotinylation indicated that older, biotinylated cells expose more PS. Furthermore, our data match a simple model of red cell senescence that assumes both an age-dependent destruction of senescent red cells preceded by several hours of PS exposure and a random destruction of red cells without PS exposure. By using this model, we demonstrated that the exposure of PS parallels the rate at which biotinylated red cells are removed from circulation. On the other hand, using an annexin V-FITC label and flow cytometry demonstrates that exposed PS does not cause the reduced red cell life span of patients with hemolytic anemia, with the possible exception of those with unstable hemoglobins or sickle cell anemia. Thus, in some cases PS exposure on the cell surface may signal the removal of red cells from circulation, but in other cases some other signal must trigger the sequestration of cells.

Long Term Maintenance of Polysaccharide-specific Antibodies by IgM Secreting Cells1

PubMed Central

Foote, Jeremy B.; Mahmoud, Tamer I.; Vale, Andre M.; Kearney, John F.

2011-01-01

Many bacteria-associated polysaccharides induce long-lived antibody responses that protect against pathogenic microorganisms. The maintenance of polysaccharide-specific antibody titers may be due to long-lived plasma cells or ongoing antigen-driven B cell activation due to polysaccharide persistence. BALB/c and VHJ558.3 transgenic (TG) mice respond to α 1→3-dextran (DEX) by generating a peak anti-DEX response at 7 days, followed by maintenance of serum antibody levels for up to 150 days. Analysis of the cellular response to DEX identified a population of short-lived, cyclophosphamide sensitive DEX-specific plasmablasts in the spleen, and a quiescent, cyclophosphamide resistant DEX-specific antibody-secreting population in the bone marrow. BrdU pulse-chase experiments demonstrated the longevity of the DEX-specific antibody-secreting population in the bone marrow. Splenic DEX-specific plasmablasts were located in the red pulp with persisting DEX-associated CD11c+ dendritic cells 90 days after immunization, whereas DEX was not detected in the bone marrow after 28 days. Selective depletion of short-lived DEX-specific plasmablasts and memory B1b B cells using cyclophosphamide and anti-CD20 treatment had a minimal impact on the maintenance of serum anti-DEX antibodies. Collectively, these findings demonstrate that the maintenance of serum polysaccharide-specific antibodies is the result of continuous antigen-driven formation of short-lived plasmablasts in the spleen and a quiescent population of antibody-secreting cells maintained in the bone marrow for a long duration. PMID:22116821

Similar Idiotypes in Antibody-Forming Cells and in Cells Synthesizing Immunoglobulins Without Detectable Antibody Function

PubMed Central

Cazenave, P. -A.; Ternynck, T.; Avrameas, S.

1974-01-01

The occurrence of immunoglobulins with and without antibody specificity and with and without idiotypic specificity was studied, by use of enzyme-labeled antigen and antibodies, in lymph node cells of rabbits immunized with horse-radish peroxidase and hen ovalbumin. Some cells, containing immunoglobulins without detectable antibody function, were shown to contain idiotypes similar to those found in antibody-producing cells. PMID:4140504

Observations on the antibody-dependent cytotoxic cell by scanning electron microscopy.

PubMed Central

Inglis, J R; Penhale, W J; Farmer, A; Irvine, W J; Williams, A E

1975-01-01

The cytotoxic effect of human peripheral blood leucocytes on antibody-coated sheep erythrocyte monolayers has been investigated using scanning electron microscopy. Only a small proportion of leucocytes were found to adhere to the monolayers. A progressive destruction was observed beginning as small plaque-like areas of erythrocyte clearing which later became confluent. Three distinct cell types were found to be associated with the areas of lysis. No destruction was observed in control monolayers incubated for a similar period in the absence of either antibody of leucocytes. Surface changes in the erthrocytes adjacent to the leucocytes suggest that mechanical factors may be involved in erythrocyte lysis in this system. It is concluded that more than one leucocyte type may damage antibody-coated erythrocytes, possibly by a mechanism involving attachment to and mechanical disruption of the red cell membrane. Images FIG. 5 FIG. 2 FIG. 3 FIG. 1 FIG. 2 FIG. 4 PMID:1191386

Serologic survey of antibodies to Trypanosoma cruzi in coyotes and red foxes from Pennsylvania and Tennessee.

PubMed

Rosypal, Alexa C; Smith, Trynecia; Alexander, Andrew; Weaver, Melanie; Stewart, Richard; Houston, Allan; Gerhold, Richard; Van Why, Kyle; Dubey, Jitender P

2014-12-01

Trypanosoma cruzi is a zoonotic parasite of humans and other mammalian hosts with distribution throughout the Americas. Domestic and wild canine species are reservoirs for human T. cruzi infections. The present study examined the prevalence of antibodies to T. cruzi in wild canids from the United States. Sera from 13 red foxes (Vulpes vulpes) and 263 coyotes (Canis latrans), originating in Pennsylvania and Tennessee, were assayed for antibodies to T. cruzi with immunochromatographic tests. Antibodies to T. cruzi were found in 2 of 276 (0.72%) of all wild canids tested. Both T. cruzi-positive wild canids were coyotes and represented 2 of 21 (9.52%) wild canids assayed from Tennessee. Antibodies to T. cruzi were not detected in red fox. Anti-T. cruzi antibodies were not found in any wild canids from Pennsylvania. These results suggest that coyotes are exposed to T. cruzi in Tennessee but not in Pennsylvania.

Red blood cell production

MedlinePlus Videos and Cool Tools

... body's tissues in exchange for carbon dioxide, which is carried to and eliminated by the lungs. Red blood cells are formed in the red bone marrow ... 2 days. The body makes about two million red blood cells every second. Blood is made up of both cellular and liquid components. ...

Sickle red cell-endothelium interactions.

PubMed

Kaul, Dhananjay K; Finnegan, Eileen; Barabino, Gilda A

2009-01-01

Periodic recurrence of painful vaso-occlusive crisis is the defining feature of sickle cell disease. Among multiple pathologies associated with this disease, sickle red cell-endothelium interaction has been implicated as a potential initiating mechanism in vaso-occlusive events. This review focuses on various interrelated mechanisms involved in human sickle red cell adhesion. We discuss in vitro and microcirculatory findings on sickle red cell adhesion, its potential role in vaso-occlusion, and the current understanding of receptor-ligand interactions involved in this pathological phenomenon. In addition, we discuss the contribution of other cellular interactions (leukocytes recruitment and leukocyte-red cell interaction) to vaso-occlusion, as observed in transgenic sickle mouse models. Emphasis is given to recently discovered adhesion molecules that play a predominant role in mediating human sickle red cell adhesion. Finally, we analyze various therapeutic approaches for inhibiting sickle red cell adhesion by targeting adhesion molecules and also consider therapeutic strategies that target stimuli involved in endothelial activation and initiation of adhesion.

Frequency and specificity of red blood cell alloimmunization in chilean transfused patients.

PubMed

Caamaño, José; Musante, Evangelina; Contreras, Margarita; Ulloa, Hernán; Reyes, Carolina; Inaipil, Verónica; Saavedra, Nicolás; Guzmán, Neftalí

2015-01-01

Alloimmunization is an adverse effect of blood transfusions. In Chile, alloimmunization frequency is not established, and for this reason the aim of this study was to investigate the prevalence and specificity of red blood cell (RBC) alloantibodies in Chilean transfused subjects. Records from 4,716 multi-transfused patients were analyzed. In these patients, antibody screening was carried out prior to cross-matching with a commercially available two-cell panel by the microcolum gel test, and samples with a positive screen were analyzed for the specificity of the alloantibody with a 16-cell identification panel. The incidence of RBC alloimmunization in transfused patients was 1.02% (48/4,716) with a higher prevalence in women (40/48). We detected 52 antibodies, the most frequent specificities identified were anti-E (30.8%), anti-K (26.9%), anti-D (7.7%), and anti-Fy(a) (5.8%). The highest incidence of alloantibodies was observed in cancer and gastroenterology patients. The data demonstrated a low alloimmunization frequency in Chilean transfused patients, principally associated with antibodies anti-E, anti-K, anti-D, and anti-Fy(a).

Role of enzyme-treated cells in RBC antibody screening using the gel test: a study of anti-RH1, -RH2, and -RH3 antibodies.

PubMed

Conne, Jocelyne; Schneider, Philippe; Tissot, Jean-Daniel

2007-01-01

The role of enzyme-treated cells (ETCs) in red blood cell (RBC) antibody screening has been the subject of controversy, and its place in the clinical routine remains to be determined. In this work, plasma samples containing anti-RH1 (anti-D; N = 10), anti-RH2 (anti-C; N = 10), or anti-RH3 (anti-E; N = 10) antibodies were studied. The samples were diluted in nonbuffered or buffered normal saline, as well as in a pool of AB plasma samples. Titers and scores were determined by means of the gel test, using the indirect antiglobulin test (IAT) as well as ETCs, with R(0)r, r'r, or r''r test cells. Our results showed that compared to the IAT, ETCs allowed a clearer detection of anti-RH2 and anti-RH3, but not of anti-RH1 antibodies. Based on our study, it is not clear whether the ETC phase of the gel test should be maintained for RBC antibody screening. 2007 Wiley-Liss, Inc.

Red cell metabolism studies on Skylab

NASA Technical Reports Server (NTRS)

Mengel, C. E.

1977-01-01

Blood samples from Spacelab crewmembers were studied for possible environment effects on red cell components. Analysis involved peroxidation of red cell lipids, enzymes of red cell metabolism, and levels of 2,3-diphosphoglyceric acid and adenosine triphosphate. Results show that there is no evidence of lipid peroxidation, that biochemical effect known to be associated with irreversible red cell damage. Changes observed in glycolytic intermediates and enzymes cannot be directly implicated as indicating evidence of red cell damage.

Red blood cells in sports: effects of exercise and training on oxygen supply by red blood cells

PubMed Central

Mairbäurl, Heimo

2013-01-01

During exercise the cardiovascular system has to warrant substrate supply to working muscle. The main function of red blood cells in exercise is the transport of O2 from the lungs to the tissues and the delivery of metabolically produced CO2 to the lungs for expiration. Hemoglobin also contributes to the blood's buffering capacity, and ATP and NO release from red blood cells contributes to vasodilation and improved blood flow to working muscle. These functions require adequate amounts of red blood cells in circulation. Trained athletes, particularly in endurance sports, have a decreased hematocrit, which is sometimes called “sports anemia.” This is not anemia in a clinical sense, because athletes have in fact an increased total mass of red blood cells and hemoglobin in circulation relative to sedentary individuals. The slight decrease in hematocrit by training is brought about by an increased plasma volume (PV). The mechanisms that increase total red blood cell mass by training are not understood fully. Despite stimulated erythropoiesis, exercise can decrease the red blood cell mass by intravascular hemolysis mainly of senescent red blood cells, which is caused by mechanical rupture when red blood cells pass through capillaries in contracting muscles, and by compression of red cells e.g., in foot soles during running or in hand palms in weightlifters. Together, these adjustments cause a decrease in the average age of the population of circulating red blood cells in trained athletes. These younger red cells are characterized by improved oxygen release and deformability, both of which also improve tissue oxygen supply during exercise. PMID:24273518

Monoclonal antibodies targeting non-small cell lung cancer stem-like cells by multipotent cancer stem cell monoclonal antibody library.

PubMed

Cao, Kaiyue; Pan, Yunzhi; Yu, Long; Shu, Xiong; Yang, Jing; Sun, Linxin; Sun, Lichao; Yang, Zhihua; Ran, Yuliang

2017-02-01

Cancer stem cells (CSCs) are a rare subset of cancer cells that play a significant role in cancer initiation, spreading, and recurrence. In this study, a subpopulation of lung cancer stem-like cells (LCSLCs) was identified from non-small cell lung carcinoma cell lines, SPCA-1 and A549, using serum-free suspension sphere-forming culture method. A monoclonal antibody library was constructed using immunized BLAB/c mice with the multipotent CSC cell line T3A-A3. Flow cytometry analysis showed that 33 mAbs targeted antigens can be enriched in sphere cells compared with the parental cells of SPCA-1 and A549 cell lines. Then, we performed functional antibody screening including sphere-forming inhibiting and invasion inhibiting assay. The results showed that two antibodies, 12C7 and 9B8, notably suppressed the self-renewal and invasion of LCSLCs. Fluorescence-activated cell sorting (FACs) found that the positive cells recognized by mAbs, 12C7 or 9B8, displayed features of LCSLCs. Interestingly, we found that these two antibodies recognized different subsets of cells and their combination effect was superior to the individual effect both in vitro and in vivo. Tissue microarrays were applied to detect the expression of the antigens targeted by these two antibodies. The positive expression of 12C7 and 9B8 targeted antigen was 84.4 and 82.5%, respectively, which was significantly higher than that in the non-tumor lung tissues. In conclusion, we screened two potential therapeutic antibodies that target different subsets of LCSLCs.

21 CFR 640.10 - Red Blood Cells.

Code of Federal Regulations, 2014 CFR

2014-04-01

... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.10 Red Blood Cells. The proper name of this product shall be Red Blood Cells. The product is defined as red blood cells remaining... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Red Blood Cells. 640.10 Section 640.10 Food and...

21 CFR 640.10 - Red Blood Cells.

Code of Federal Regulations, 2013 CFR

2013-04-01

... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.10 Red Blood Cells. The proper name of this product shall be Red Blood Cells. The product is defined as red blood cells remaining... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Red Blood Cells. 640.10 Section 640.10 Food and...

21 CFR 640.10 - Red Blood Cells.

Code of Federal Regulations, 2011 CFR

2011-04-01

... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.10 Red Blood Cells. The proper name of this product shall be Red Blood Cells. The product is defined as red blood cells remaining... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Red Blood Cells. 640.10 Section 640.10 Food and...

21 CFR 640.10 - Red Blood Cells.

Code of Federal Regulations, 2012 CFR

2012-04-01

... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.10 Red Blood Cells. The proper name of this product shall be Red Blood Cells. The product is defined as red blood cells remaining... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Red Blood Cells. 640.10 Section 640.10 Food and...

21 CFR 640.10 - Red Blood Cells.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Red Blood Cells. 640.10 Section 640.10 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.10 Red Blood Cells. The proper name of this product shall be Red Blood Cells. The product is defined as red blood cells remaining...

Red Blood Cell Susceptibility to Pneumolysin

PubMed Central

Bokori-Brown, Monika; Petrov, Peter G.; Khafaji, Mawya A.; Mughal, Muhammad K.; Naylor, Claire E.; Shore, Angela C.; Gooding, Kim M.; Casanova, Francesco; Mitchell, Tim J.; Titball, Richard W.; Winlove, C. Peter

2016-01-01

This study investigated the effect of the biochemical and biophysical properties of the plasma membrane as well as membrane morphology on the susceptibility of human red blood cells to the cholesterol-dependent cytolysin pneumolysin, a key virulence factor of Streptococcus pneumoniae, using single cell studies. We show a correlation between the physical properties of the membrane (bending rigidity and surface and dipole electrostatic potentials) and the susceptibility of red blood cells to pneumolysin-induced hemolysis. We demonstrate that biochemical modifications of the membrane induced by oxidative stress, lipid scrambling, and artificial cell aging modulate the cell response to the toxin. We provide evidence that the diversity of response to pneumolysin in diabetic red blood cells correlates with levels of glycated hemoglobin and that the mechanical properties of the red blood cell plasma membrane are altered in diabetes. Finally, we show that diabetic red blood cells are more resistant to pneumolysin and the related toxin perfringolysin O relative to healthy red blood cells. Taken together, these studies indicate that the diversity of cell response to pneumolysin within a population of human red blood cells is influenced by the biophysical and biochemical status of the plasma membrane and the chemical and/or oxidative stress pre-history of the cell. PMID:26984406

Suppression of Red Blood Cell Autofluorescence for Immunocytochemistry on Fixed Embryonic Mouse Tissue.

PubMed

Whittington, Niteace C; Wray, Susan

2017-10-23

Autofluorescence is a problem that interferes with immunofluorescent staining and complicates data analysis. Throughout the mouse embryo, red blood cells naturally fluoresce across multiple wavelengths, spanning the emission and excitation spectra of many commonly used fluorescent reporters, including antibodies, dyes, stains, probes, and transgenic proteins, making it difficult to distinguish assay fluorescence from endogenous fluorescence. Several tissue treatment methods have been developed to bypass this issue with varying degrees of success. Sudan Black B dye has been commonly used to quench autofluorescence, but can also introduce background fluorescence. Here we present a protocol for an alternative called TrueBlack Lipofuscin Autofluorescence Quencher. The protocol described in this unit demonstrates how TrueBlack efficiently quenches red blood cell autofluorescence across red and green wavelengths in fixed embryonic tissue without interfering with immunofluorescent signal intensity or introducing background staining. We also identify optimal incubation, concentration, and multiple usage conditions for routine immunofluorescence microscopy. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

The effects of antibodies on cells

PubMed Central

Dumonde, D. C.; Bitensky, Lucille; Cunningham, G. J.; Chayen, J.

1965-01-01

Biochemical and histochemical methods were used to study the interaction of antibodies and complement with mouse Ehrlich ascites tumour cells. In the presence of complement, both iso- and hetero-antibodies caused cell lysis with penetration of antibodies into the damaged cells, as detected by immunofluorescence; the cells were then unable to support aerobic glycolysis though they retained their ability to consume oxygen in the presence of succinate. Under these conditions there was unmasking of phospholipid particularly at the cell surface, together with lysosomal changes resulting in diffuse staining for lysosomal acid—phosphatase. In the absence of complement, antibodies did not appear to penetrate the cells which respired normally and were not lysed. However, in these cells there was intense lysosomal activation accompanied by unmasking of cytoplasmic phospholipid; it appeared that an immune reaction confined to the cell surface was able to induce changes in the cytoplasm without acutely impairing the viability of the cell. ImagesFIGS. 1-8FIGS. 9-14 PMID:14245309

Red blood cell alloimmunization in transfused patients in sub-Saharan Africa: A systematic review and meta-analysis.

PubMed

Ngoma, Alain M; Mutombo, Paulin B; Ikeda, Kazuhiko; Nollet, Kenneth E; Natukunda, Bernard; Ohto, Hitoshi

2016-04-01

Previous studies of Sub-Saharan Africans show significant alloimmunization to red blood cell (RBC) antigens, but country-specific data are limited. Thus, the aim of this study was to estimate, by meta-analysis, the overall proportion of red blood cell alloantibodies among transfused patients. We systematically searched Medline, Embase, and the Africa-Wide Information database to identify relevant studies in any language. Case reports, comments, letters, conference abstracts, editorials, and review articles were excluded. Of the 269 potentially relevant articles, 11 studies fulfilled our selection criteria. Overall proportions of alloimmunization were 6.7 (95% CI: 5.7, 7.8) per 100 transfused patients. With regard to antibody specificity, among clinically significant antibodies, anti-E ranked as the most common, followed by anti-K, anti-C and anti-D. Meta-analysis of available literature quantifies and qualifies the clinical challenge of RBC alloimmunization among transfused patients in Sub-Saharan Africa. These results should drive policy decisions in favour of routine testing of RBC antigens and irregular antibodies for transfused patients as a standard of care throughout Sub-Saharan Africa. Copyright © 2015 Elsevier Ltd. All rights reserved.

Red blood cells inhibit activation-induced cell death and oxidative stress in human peripheral blood T lymphocytes.

PubMed

Fonseca, A M; Porto, G; Uchida, K; Arosa, F A

2001-05-15

Red blood cells (RBCs) are known to perform one prominent function: to carry and deliver oxygen to the tissues. Earlier studies, however, suggested a role for RBCs in potentiating T-cell proliferation in vitro. Here it is shown that the presence of RBCs in cultures of stimulated human peripheral blood lymphocytes strengthens T-cell proliferation and survival. Analysis of phosphatidylserine externalization and DNA fragmentation showed that RBCs inhibit T-cell apoptosis. This inhibition correlated with a reduction in CD71 but not CD95 expression. RBCs enhanced T-cell proliferation and survival upon activation with phytohemagglutinin and with OKT3 antibodies. Studies aimed at characterizing the cellular and molecular basis of the protection afforded to T cells by RBCs showed that (1) optimal protection required intact RBCs and red cell/T-cell contact but not monocytes; (2) RBCs markedly reduced the level of intracellular reactive oxygen species; and (3) RBCs inhibited the formation of protein-bound acrolein, a peroxidation adduct in biologic systems. Overall, these data indicate that human RBCs protect T cells from activation-induced cell death, at least in part by reducing the pro-oxidant state, and suggest a role for RBCs as conceivable modulators of T-cell homeostasis.

Rejuvenation of allogenic red cells: benefits and risks.

PubMed

Aujla, H; Woźniak, M; Kumar, T; Murphy, G J

2018-06-04

To review preclinical and clinical studies that have evaluated the effects of red cell rejuvenation in vivo and in vitro and to assess the potential risks and benefits from their clinical use. A systematic review and narrative synthesis of the intervention of red cell rejuvenation using a red cell processing solution containing inosine, pyruvate, phosphate and adenine. Outcomes of interest in vitro were changes in red cell characteristics including adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG), deformability and the accumulation of oxidized lipids and other reactive species in the red cell supernatant. Outcomes in vivo were 24-h post-transfusion survival and the effects on oxygen delivery, organ function and inflammation in transfused recipients. The literature search identified 49 studies evaluating rejuvenated red cells. In vitro rejuvenation restored cellular properties including 2,3-DPG and ATP to levels similar to freshly donated red cells. In experimental models, in vivo transfusion of rejuvenated red cells improved oxygen delivery and myocardial, renal and pulmonary function when compared to stored red cells. In humans, in vivo 24-h survival of rejuvenated red cells exceeded 75%. In clinical studies, rejuvenated red cells were found to be safe, with no reported adverse effects. In one adult cardiac surgery trial, transfusion of rejuvenated red cells resulted in improved myocardial performance. Transfusion of rejuvenated red cells reduces organ injury attributable to the red cell storage lesion without adverse effects in experimental studies in vivo. The clinical benefits of this intervention remain uncertain. © 2018 International Society of Blood Transfusion.

[Screening serum response special antibodies of U251 cell line from surface display phage antibody library].

PubMed

Yu, Min; Tan, De-Yong; Qian, Wei; Lai, Jian-Hua; Sun, Gui-Lin

2004-05-01

U251 cell is a sensitive cell line to serum, which stops at G0 phase of cell cycle in no-serum medium, and recovers growth when the serum is added into no-serum medium. The cell can express corresponding proteins in different phase of cell cycle. Therefore it is very signification for the study of cell cycle regulation mechanism that explores these proteins. In this paper, the mouse antibody phage display library was added into the bottle in which the serum starvation U251 cells had been cultured, and the special antibody phages were absorbed. Then the absorbed antibody phages were amplified by adding E. coli TG1 and helper phage M13K07. Amplified antibody phages were added into bottle in which the serum cultured cell after serum starvation (follow named as serum recovered cells) were incubated, so that the cell absorbed the no-special antibody phages for the serum starvation cell and the special antibody phages were in supernatant. The remaining no-special antibody phages in the supernatant were discarded by repeating above program 3-4 times. The pure special antibody phages were gotten, and amplified by adding the host cell E. coli TG1 and helper phage M13K07. Then the host bacterium infected special antibody phage was spread on the plate medium with ampicillin, and the monoclonal antibody phages were gotten. Using same as above program, the monoclonal antibody phages absorbed specially for serum recovered U251 cells were obtained when the serum recovered cells instead of serum starvation cells and serum starvation cells instead of serum recovered cells. In this study, ninety-six positive monoclonal antibody phages that absorbed specially the serum starvation cells and eighty-two positive monoclonal antibody phages that absorbed specially the serum recovered cells were obtained. By using cell immunochemistry assay, two special signification antibodies were obtained. one (No.11) was the strong response in serum starvation cells, the other (No.2) was the strong

Simplified spectraphotometric method for the detection of red blood cell agglutination.

PubMed

Ramasubramanian, Melur; Anthony, Steven; Lambert, Jeremy

2008-08-01

Human error is the most significant factor attributed to incompatible blood transfusions. A spectrophotometric approach to blood typing has been developed by examining the spectral slopes of dilute red blood cell (RBC) suspensions in saline, in the presence and absence of various antibodies, offering a technique for the quantitative determination of agglutination intensity [Transfusion39, 1051, 1999TRANAT0041-113210.1046/j.1537-2995.1999.39101051.x]. We offer direct theoretical prediction of the observed change in slope in the 660-1000 nm range through the use of the T-matrix approach and Lorenz-Mie theory for light scattering by dilute RBC suspensions. Following a numerical simulation using the T-matrix code, we present a simplified sensing method for detecting agglutination. The sensor design has been prototyped, fully characterized, and evaluated through a complete set of tests with over 60 RBC samples and compared with the full spectrophotometric method. The LED and photodiode pairs are found to successfully reproduce the spectroscopic determination of red blood cell agglutination.

Red blood cell antibody-induced anemia causes differential degrees of tissue hypoxia in kidney and brain.

PubMed

Mistry, Nikhil; Mazer, C David; Sled, John G; Lazarus, Alan H; Cahill, Lindsay S; Solish, Max; Zhou, Yu-Qing; Romanova, Nadya; Hare, Alexander G M; Doctor, Allan; Fisher, Joseph A; Brunt, Keith R; Simpson, Jeremy A; Hare, Gregory M T

2018-04-01

Moderate anemia is associated with increased mortality and morbidity, including acute kidney injury (AKI), in surgical patients. A red blood cell (RBC)-specific antibody model was utilized to determine whether moderate subacute anemia could result in tissue hypoxia as a potential mechanism of injury. Cardiovascular and hypoxic cellular responses were measured in transgenic mice capable of expressing hypoxia-inducible factor-1α (HIF-1α)/luciferase activity in vivo. Antibody-mediated anemia was associated with mild intravascular hemolysis (6 h) and splenic RBC sequestration ( day 4), resulting in a nadir hemoglobin concentration of 89 ± 13 g/l on day 4. At this time point, renal tissue oxygen tension (P t O 2 ) was decreased in anemic mice relative to controls (13.1 ± 4.3 vs. 20.8 ± 3.7 mmHg, P < 0.001). Renal tissue hypoxia was associated with an increase in HIF/luciferase expression in vivo ( P = 0.04) and a 20-fold relative increase in renal erythropoietin mRNA transcription ( P < 0.001) but no increase in renal blood flow ( P = 0.67). By contrast, brain P t O 2 was maintained in anemic mice relative to controls (22.7 ± 5.2 vs. 23.4 ± 9.8 mmHg, P = 0.59) in part because of an increase in internal carotid artery blood flow (80%, P < 0.001) and preserved cerebrovascular reactivity. Despite these adaptive changes, an increase in brain HIF-dependent mRNA levels was observed (erythropoietin: P < 0.001; heme oxygenase-1: P = 0.01), providing evidence for subtle cerebral tissue hypoxia in anemic mice. These data demonstrate that moderate subacute anemia causes significant renal tissue hypoxia, whereas adaptive cerebrovascular responses limit the degree of cerebral tissue hypoxia. Further studies are required to assess whether hypoxia is a mechanism for acute kidney injury associated with anemia.

Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells.

PubMed

Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

2015-01-01

Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.

In vivo studies of sickle red blood cells.

PubMed

Kaul, Dhananjay K; Fabry, Mary E

2004-03-01

The defining clinical feature of sickle cell anemia is periodic occurrence of painful vasoocclusive crisis. Factors that promote trapping and sickling of red cells in the microcirculation are likely to trigger vasoocclusion. The marked red cell heterogeneity in sickle blood and abnormal adhesion of sickle red cells to vascular endothelium would be major disruptive influences. Using ex vivo and in vivo models, the authors show how to dissect the relative contribution of heterogeneous sickle red cell classes to adhesive and obstructive events. These studies revealed that (1) both rheological abnormalities and adhesion of sickle red cells contribute to their abnormal hemodynamic behavior, (2) venules are the sites of sickle cell adhesion, and (3) sickle red cell deformability plays an important role in adhesive and obstructive events. Preferential adhesion of deformable sickle red cells in postcapillary venules followed by selective trapping of dense sickle red cells could result in vasoocclusion. An updated version of this 2-step model is presented. The multifactorial nature of sickle red cell adhesion needs to be considered in designing antiadhesive therapy in vivo.

Red blood cell decreases of microgravity

NASA Technical Reports Server (NTRS)

Johnson, P. C.

1985-01-01

Postflight decreases in red blood cell mass (RBCM) have regularly been recorded after exposure to microgravity. These 5-25 percent decreases do not relate to the mission duration, workload, caloric intake or to the type of spacecraft used. The decrease is accompanied by normal red cell survivals, increased ferritin levels, normal radioactive iron studies, and increases in mean red blood cell volume. Comparable decreases in red blood cell mass are not found after bed rest, a commonly used simulation of the microgravity state. Inhibited bone marrow erythropoiesis has not been proven to date, although reticulocyte numbers in the peripheral circulation are decreased about 50 percent. To date, the cause of the microgravity induced decreases in RBCM is unknown. Increased splenic trapping of circulating red blood cells seem the most logical way to explain the results obtained.

A NEW SENSITIVE ASSAY FOR ANTIBODY AGAINST CELL SURFACE ANTIGENS BASED ON INHIBITION OF CELL-DEPENDENT ANTIBODY-MEDIATED CYTOTOXICITY

PubMed Central

Halloran, Phil; Schirrmacher, Volker; Festenstein, Hilliard

1974-01-01

Inhibition of cell-dependent antibody-mediated cytotoxicity has been investigated as a new assay for antibody against cell surface antigens. The cytotoxicity system consisted of effector cells (normal mouse spleen cells), target cells (61Cr-labeled chicken erythrocytes), and antitarget cell antibody. Addition of antibody against cell surface antigens in the effector cell population regularly inhibited the cytotoxicity measured in this system. This cytotoxicity inhibition assay (CIA) detected antibody with a variety of specificities: anti-H-2, anti-Thy 1.2, anti-immunoglobulin, and antimouse bone marrow-derived lymphocyte antigen. When the inhibition by anti-H-2 sera was analyzed using effector cells from congenic mice, the activity was found to be directed against specificities mapping in the H-2K, H-2D, and I regions of the H-2 complex, correlating well with the specificities characterized by complement-dependent assays. A comparison between the sensitivity of the CIA and complement-dependent lysis revealed that the CIA was 2–11 times more sensitive for anti-H-2 antisera and 20–780 times more sensitive for certain antisera against subpopulations of the spleen cells (i.e., T cells or B cells). The CIA proved to be precise, sensitive, and reliable. It may become a very useful antibody assay in various species including man. PMID:4547657

Shape-Shifted Red Blood Cells: A Novel Red Blood Cell Stage?

PubMed Central

Chico, Verónica; Puente-Marin, Sara; Ciordia, Sergio; Mena, María Carmen; Carracedo, Begoña; Mercado, Luis; Coll, Julio

2018-01-01

Primitive nucleated erythroid cells in the bloodstream have long been suggested to be more similar to nucleated red cells of fish, amphibians, and birds than the red cells of fetal and adult mammals. Rainbow trout Ficoll-purified red blood cells (RBCs) cultured in vitro undergo morphological changes, especially when exposed to stress, and enter a new cell stage that we have coined shape-shifted RBCs (shRBCs). We have characterized these shRBCs using transmission electron microscopy (TEM) micrographs, Wright–Giemsa staining, cell marker immunostaining, and transcriptomic and proteomic evaluation. shRBCs showed reduced density of the cytoplasm, hemoglobin loss, decondensed chromatin in the nucleus, and striking expression of the B lymphocyte molecular marker IgM. In addition, shRBCs shared some features of mammalian primitive pyrenocytes (extruded nucleus surrounded by a thin rim of cytoplasm and phosphatidylserine (PS) exposure on cell surface). These shRBCs were transiently observed in heat-stressed rainbow trout bloodstream for three days. Functional network analysis of combined transcriptomic and proteomic studies resulted in the identification of proteins involved in pathways related to the regulation of cell morphogenesis involved in differentiation, cellular response to stress, and immune system process. In addition, shRBCs increased interleukin 8 (IL8), interleukin 1 β (IL1β), interferon ɣ (IFNɣ), and natural killer enhancing factor (NKEF) protein production in response to viral hemorrhagic septicemia virus (VHSV). In conclusion, shRBCs may represent a novel cell stage that participates in roles related to immune response mediation, homeostasis, and the differentiation and development of blood cells. PMID:29671811

Shape-Shifted Red Blood Cells: A Novel Red Blood Cell Stage?

PubMed

Chico, Verónica; Puente-Marin, Sara; Nombela, Iván; Ciordia, Sergio; Mena, María Carmen; Carracedo, Begoña; Villena, Alberto; Mercado, Luis; Coll, Julio; Ortega-Villaizan, María Del Mar

2018-04-19

Primitive nucleated erythroid cells in the bloodstream have long been suggested to be more similar to nucleated red cells of fish, amphibians, and birds than the red cells of fetal and adult mammals. Rainbow trout Ficoll-purified red blood cells (RBCs) cultured in vitro undergo morphological changes, especially when exposed to stress, and enter a new cell stage that we have coined shape-shifted RBCs (shRBCs). We have characterized these shRBCs using transmission electron microscopy (TEM) micrographs, Wright⁻Giemsa staining, cell marker immunostaining, and transcriptomic and proteomic evaluation. shRBCs showed reduced density of the cytoplasm, hemoglobin loss, decondensed chromatin in the nucleus, and striking expression of the B lymphocyte molecular marker IgM. In addition, shRBCs shared some features of mammalian primitive pyrenocytes (extruded nucleus surrounded by a thin rim of cytoplasm and phosphatidylserine (PS) exposure on cell surface). These shRBCs were transiently observed in heat-stressed rainbow trout bloodstream for three days. Functional network analysis of combined transcriptomic and proteomic studies resulted in the identification of proteins involved in pathways related to the regulation of cell morphogenesis involved in differentiation, cellular response to stress, and immune system process. In addition, shRBCs increased interleukin 8 (IL8), interleukin 1 β (IL1β), interferon ɣ (IFNɣ), and natural killer enhancing factor (NKEF) protein production in response to viral hemorrhagic septicemia virus (VHSV). In conclusion, shRBCs may represent a novel cell stage that participates in roles related to immune response mediation, homeostasis, and the differentiation and development of blood cells.

Red blood cell microparticles and blood group antigens: an analysis by flow cytometry

PubMed Central

Canellini, Giorgia; Rubin, Olivier; Delobel, Julien; Crettaz, David; Lion, Niels; Tissot, Jean-Daniel

2012-01-01

Background The storage of blood induces the formation of erythrocytes-derived microparticles. Their pathogenic role in blood transfusion is not known so far, especially the risk to trigger alloantibody production in the recipient. This work aims to study the expression of clinically significant blood group antigens on the surface of red blood cells microparticles. Material and methods Red blood cells contained in erythrocyte concentrates were stained with specific antibodies directed against blood group antigens and routinely used in immunohematology practice. After inducing erythrocytes vesiculation with calcium ionophore, the presence of blood group antigens was analysed by flow cytometry. Results The expression of several blood group antigens from the RH, KEL, JK, FY, MNS, LE and LU systems was detected on erythrocyte microparticles. The presence of M (MNS1), N (MNS2) and s (MNS4) antigens could not be demonstrated by flow cytometry, despite that glycophorin A and B were identified on microparticles using anti-CD235a and anti-MNS3. Discussion We conclude that blood group antigens are localized on erythrocytes-derived microparticles and probably keep their immunogenicity because of their capacity to bind specific antibody. Selective segregation process during vesiculation or their ability to elicit an immune response in vivo has to be tested by further studies. PMID:22890266

Inflight Assay of Red Blood Cell Deformability

NASA Technical Reports Server (NTRS)

Ingram, M.; Paglia, D. E.; Eckstein, E. C.; Frazer, R. E.

1985-01-01

Studies on Soviet and American astronauts have demonstrated that red blood cell production is altered in response to low gravity (g) environment. This is associated with changes in individual red cells including increased mean cell volume and altered membrane deformability. During long orbital missions, there is a tendency for the red cell mass deficit to be at least partly corrected although the cell shape anomalies are not. Data currently available suggest that the observed decrease in red cell mass is the result of sudden suppression of erythropoieses and that the recovery trend observed during long missions reflects re-establishment of erythropoietic homeostasis at a "set point" for the red cell mass that is slightly below the normal level at 1 g.

Virus-specific antibody secreting cell, memory B-cell, and sero-antibody responses in the human influenza challenge model.

PubMed

Huang, Kuan-Ying Arthur; Li, Chris Ka-Fai; Clutterbuck, Elizabeth; Chui, Cecilia; Wilkinson, Tom; Gilbert, Anthony; Oxford, John; Lambkin-Williams, Rob; Lin, Tzou-Yien; McMichael, Andrew J; Xu, Xiao-Ning

2014-05-01

 Antibodies play a major role in the protection against influenza virus in human. However, the antibody level is usually short-lived and the cellular mechanisms underlying influenza virus-specific antibody response to acute infection remain unclear.  We studied the kinetics and magnitude of influenza virus-specific B-cell and serum antibody responses in relation to virus replication during the course of influenza infection in healthy adult volunteers who were previously seronegative and experimentally infected with seasonal influenza H1N1 A/Brisbane/59/07 virus.  Our data demonstrated a robust expansion of the virus-specific antibody-secreting cells (ASCs) and memory B cells in the peripheral blood, which correlated with both the throat viral load and the duration of viral shedding. The ASC response was obviously detected on day 7 post-infection when the virus was completely cleared from nasal samples, and serum hemagglutination-inhibition antibodies were still undetectable. On day 28 postinfection, influenza virus-specific B cells were further identified from the circulating compartment of isotype-switched B cells. Virus-specific ASCs could be the earliest marker of B-cell response to a new flu virus infection, such as H7N9 in humans.

Neocytolysis: physiological down-regulator of red-cell mass

NASA Technical Reports Server (NTRS)

Alfrey, C. P.; Rice, L.; Udden, M. M.; Driscoll, T. B.

1997-01-01

It is usually considered that red-cell mass is controlled by erythropoietin-driven bone marrow red-cell production, and no physiological mechanisms can shorten survival of circulating red cells. In adapting to acute plethora in microgravity, astronauts' red-cell mass falls too rapidly to be explained by diminished red-cell production. Ferrokinetics show no early decline in erythropolesis, but red cells radiolabelled 12 days before launch survive normally. Selective destruction of the youngest circulating red cells-a process we call neocytolysis-is the only plausible explanation. A fall in erythropoietin below a threshold is likely to initiate neocytolysis, probably by influencing surface-adhesion molecules. Recognition of neocytolysis will require re-examination of the pathophysiology and treatment of several blood disorders, including the anaemia of renal disease.

21 CFR 864.8185 - Calibrator for red cell and white cell counting.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Calibrator for red cell and white cell counting... Calibrator for red cell and white cell counting. (a) Identification. A calibrator for red cell and white cell counting is a device that resembles red or white blood cells and that is used to set instruments intended...

Human spleen and red blood cells

NASA Astrophysics Data System (ADS)

Pivkin, Igor; Peng, Zhangli; Karniadakis, George; Buffet, Pierre; Dao, Ming

2016-11-01

Spleen plays multiple roles in the human body. Among them is removal of old and altered red blood cells (RBCs), which is done by filtering cells through the endothelial slits, small micron-sized openings. There is currently no experimental technique available that allows us to observe RBC passage through the slits. It was previously noticed that people without a spleen have less deformable red blood cells, indicating that the spleen may play a role in defining the size and shape of red blood cells. We used detailed RBC model implemented within the Dissipative Particle Dynamics (DPD) simulation framework to study the filter function of the spleen. Our results demonstrate that spleen indeed plays major role in defining the size and shape of the healthy human red blood cells.

21 CFR 864.5300 - Red cell indices device.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Red cell indices device. 864.5300 Section 864.5300....5300 Red cell indices device. (a) Identification. A red cell indices device, usually part of a larger... corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC). The red cell indices...

21 CFR 864.5300 - Red cell indices device.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Red cell indices device. 864.5300 Section 864.5300....5300 Red cell indices device. (a) Identification. A red cell indices device, usually part of a larger... corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC). The red cell indices...

21 CFR 864.5300 - Red cell indices device.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Red cell indices device. 864.5300 Section 864.5300....5300 Red cell indices device. (a) Identification. A red cell indices device, usually part of a larger... corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC). The red cell indices...

21 CFR 864.5300 - Red cell indices device.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Red cell indices device. 864.5300 Section 864.5300....5300 Red cell indices device. (a) Identification. A red cell indices device, usually part of a larger... corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC). The red cell indices...

21 CFR 864.5300 - Red cell indices device.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red cell indices device. 864.5300 Section 864.5300....5300 Red cell indices device. (a) Identification. A red cell indices device, usually part of a larger... corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC). The red cell indices...

Single-Cell Droplet Microfluidic Screening for Antibodies Specifically Binding to Target Cells.

PubMed

Shembekar, Nachiket; Hu, Hongxing; Eustace, David; Merten, Christoph A

2018-02-20

Monoclonal antibodies are a main player in modern drug discovery. Many antibody screening formats exist, each with specific advantages and limitations. Nonetheless, it remains challenging to screen antibodies for the binding of cell-surface receptors (the most important class of all drug targets) or for the binding to target cells rather than purified proteins. Here, we present a high-throughput droplet microfluidics approach employing dual-color normalized fluorescence readout to detect antibody binding. This enables us to obtain quantitative data on target cell recognition, using as little as 33 fg of IgG per assay. Starting with an excess of hybridoma cells releasing unspecific antibodies, individual clones secreting specific binders (of target cells co-encapsulated into droplets) could be enriched 220-fold after sorting 80,000 clones in a single experiment. This opens the way for therapeutic antibody discovery, especially since the single-cell approach is in principle also applicable to primary human plasma cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

21 CFR 864.8540 - Red cell lysing reagent.

Code of Federal Regulations, 2013 CFR

2013-04-01

...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells for... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...

21 CFR 864.8540 - Red cell lysing reagent.

Code of Federal Regulations, 2014 CFR

2014-04-01

...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells for... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...

21 CFR 864.8540 - Red cell lysing reagent.

Code of Federal Regulations, 2012 CFR

2012-04-01

...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells for... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...

21 CFR 864.8540 - Red cell lysing reagent.

Code of Federal Regulations, 2011 CFR

2011-04-01

...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells for... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...

21 CFR 864.8540 - Red cell lysing reagent.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells for...

Lymphocyte-dependent antibody-mediated cytotoxicity in Hashimoto thyroiditis

PubMed Central

Calder, Elizabeth A.; Penhale, W. J.; McLeman, Dena; Barnes, E. W.; Irvine, W. J.

1973-01-01

In the presence of normal human lymphocytes, decomplemented sera from twentynine out of thirty-nine patients with Hashimoto thyroiditis caused significant lysis of thyroglobulin-coated chicken red blood cells, as estimated by the release of 51Cr; the mean% specific 51Cr release being 14·1 ± 1·9 (SEM). Serum from twenty-one control subjects studied concurrently caused no significant lysis of thyroglobulin-coated chicken red blood cells; the mean% specific 51Cr release being −1·6±0·7 (SEM). The degree of cytotoxicity correlated with the titre of thyroglobulin antibodies in the serum, determined by tanned red cell haemagglutination. The active component in the Hashimoto serum was localized in the 19S fraction, was unaffected by pre-absorption with anti-human IgM serum, but was neutralized by pre-absorption with anti-human IgG serum. These findings suggest that the cytotoxic activity of serum from patients with Hashimoto thyroiditis is due to the presence of thyroglobulin antibody of the IgG class in the form of complexes, either alone or with antigen. It is postulated that non-specific lymphocytes may play an important role in the pathogenesis of Hashimoto thyroiditis, being activated by the presence in the gland of thyroglobulin antibody, either alone or in the form of complexes attached to thyroid cells. PMID:4740445

Red blood cell alloimmunization among sickle cell Kuwaiti Arab patients who received red blood cell transfusion.

PubMed

Ameen, Reem; Al Shemmari, Salem; Al-Bashir, Abdulaziz

2009-08-01

Sickle cell disease (SCD) is common in the Arabian Gulf region. Most cases require a red blood cell (RBC) transfusion, increasing the potential for RBC alloantibody development. The incidence of RBC alloimmunization among Kuwaiti Arab SCD patients is not yet known. This study retrospectively assessed the effect of using two different matching protocols on the incidence of alloimmunization among multiply transfused Kuwaiti Arab SCD patients. A total of 233 Kuwaiti Arab SCD patients were divided into two groups: Group 1 (n = 110) received RBC transfusion through standard ABO- and D-matched nonleukoreduced blood; Group 2 (n = 123) received RBCs matched for ABO, Rh, and K1 poststorage-leukoreduced blood. Multivariate analysis was performed on the factors associated with RBC alloimmunization and antibody specificity. Sixty-five percent of patients in Group 1 developed clinically significant RBC alloantibody with an increased prevalence in females; in patients in Group 2, 23.6% developed RBC alloantibodies (p = 0.01). In Group 1, 72 patients (65.5%) had alloantibodies directed against Rh and Kell systems (p = 0.01). Multivariate analysis further confirmed the results, showing that blood transfusion type and sex have significant effects on the rate of alloimmunizations. This study confirms the importance of selecting RBCs matched for Rh and Kell to reduce the risk of alloimmunizations among Kuwaiti Arab SCD patients.

Clinical survey of antibodies against red blood cells in horses after homologous blood transfusion.

PubMed

Wong, P L; Nickel, L S; Bowling, A T; Steffey, E P

1986-12-01

Serum samples of 20 horses were evaluated for antibodies against RBC after homologous blood transfusion. Transfusion-associated antibodies against RBC were detected in 10 horses. Antibodies recognizing horse blood group antigens Aa, Ae, Db, and Dc were identified. Antibodies against Aa were found in all samples from Aa-negative horses that were transfused with Aa-positive RBC. Antibodies against Aa persisted for at least 1 year after transfusion. Antibodies against Ae were detected in 7 of 8 horses transfused with Ae-positive RBC. Initial appearance and persistence of antibodies against Ae differed among the horses; antibodies were initially detected 1 week to 154 weeks after transfusion and disappeared as early as 4 weeks after transfusion. Antibodies against Db or Dc were detected in less than or equal to 33% of the horses that lacked Db or Dc antigens and were transfused with Db- or Dc-positive RBC. Antibodies against Db and Dc were initially detected in sera later than were the A-system antibodies. Three mares with transfusion-associated antibodies subsequently produced healthy offspring. Two foals had RBC antigens corresponding to their dam's alloantibodies; maternal colostrum with antibodies against Aa was withheld from the Aa-positive foal. The Db-positive foal remained healthy after nursing the mare with serum antibodies against Db.

Neutrophils Kill Antibody-Opsonized Cancer Cells by Trogoptosis.

PubMed

Matlung, Hanke L; Babes, Liane; Zhao, Xi Wen; van Houdt, Michel; Treffers, Louise W; van Rees, Dieke J; Franke, Katka; Schornagel, Karin; Verkuijlen, Paul; Janssen, Hans; Halonen, Pasi; Lieftink, Cor; Beijersbergen, Roderick L; Leusen, Jeanette H W; Boelens, Jaap J; Kuhnle, Ingrid; van der Werff Ten Bosch, Jutte; Seeger, Karl; Rutella, Sergio; Pagliara, Daria; Matozaki, Takashi; Suzuki, Eiji; Menke-van der Houven van Oordt, Catharina Willemien; van Bruggen, Robin; Roos, Dirk; van Lier, Rene A W; Kuijpers, Taco W; Kubes, Paul; van den Berg, Timo K

2018-06-26

Destruction of cancer cells by therapeutic antibodies occurs, at least in part, through antibody-dependent cellular cytotoxicity (ADCC), and this can be mediated by various Fc-receptor-expressing immune cells, including neutrophils. However, the mechanism(s) by which neutrophils kill antibody-opsonized cancer cells has not been established. Here, we demonstrate that neutrophils can exert a mode of destruction of cancer cells, which involves antibody-mediated trogocytosis by neutrophils. Intimately associated with this is an active mechanical disruption of the cancer cell plasma membrane, leading to a lytic (i.e., necrotic) type of cancer cell death. Furthermore, this mode of destruction of antibody-opsonized cancer cells by neutrophils is potentiated by CD47-SIRPα checkpoint blockade. Collectively, these findings show that neutrophil ADCC toward cancer cells occurs by a mechanism of cytotoxicity called trogoptosis, which can be further improved by targeting CD47-SIRPα interactions. Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.

Specific targeting and toxicity of sulphonated aluminium phthalocyanine photosensitised liposomes directed to cells by monoclonal antibody in vitro.

PubMed Central

Morgan, J.; Gray, A. G.; Huehns, E. R.

1989-01-01

A partially purified fraction of the water soluble photosensitive dye sulphonated aluminium phthalocyanine (AlSPc) was encapsulated in liposomes which were then linked to a targeting monoclonal antibody 791T/36 using a heterobifunctional linking agent. The photocytotoxic effects of the liposomes were determined on two cell lines bearing an antigen with which the targeting antibody binds: 791T, an osteosarcoma and C170, a colorectal carcinoma; and a control cell line not bearing the antigen; DW-BCL, an Epstein-Barr virus immortalised B-cell line. Antibody dependent cytotoxicity was observed in 791T and C170 cells and was proportional to the number of antigens on the cells, the AlSPc concentration and the time of exposure to activating red light. No significant toxicity was seen using untargeted liposomes, control cells or free AlSPc fraction under similar conditions. Targeted cells and controls kept in the dark also showed no significant toxicity. A possible mechanism of action is postulated and simple adaptations which demonstrate the versatility of the model are discussed. Some suggestions as to the clinical situations to which this system might be applied in the form of photodynamic therapy (PDT) are made. PMID:2930700

Red cell changes in hyperthyroidism.

PubMed

How, J; Davidson, R J; Bewsher, P D

1979-10-01

The Coulter 'S' red cell profile was studied prospectively in 100 untreated non-anaemic hyperthyroid patients and followed up in 52 of them until they had become euthyroid with radio-iodine or carbimazole treatment. Serial haematological data were also obtained in 23 hyperthyroid patients during treatment with beta-adrenoreceptor blocking drug alone. The most significant finding was a low mean corpuscular volume (MCV) which was invariably present throughout the hyperthyroid state. Treatment with beta-adrenoreceptor blocking drugs did not significantly alter any of the red cell parameters. On the other hand, the MCV increased and was restored to normal with radio-iodine or carbimazole treatment although there was a lag period of about 6--8 weeks between achieving the euthyroid state and the normalisation of this red cell index. While none of the patients were aneaemic, the haemoglobin level rose significantly following effective anti-thyroid treatment. It is suggested that measurement of the MCV may have a useful role in the diagnosis of hyperthyroidism. 2 possible mechanisms leading to the observed red cell changes in hyperthyroidism are postulated.

Antibody distance from the cell membrane regulates antibody effector mechanisms

PubMed Central

Cleary, Kirstie L.S.; Chan, H.T. Claude; James, Sonja; Glennie, Martin J.; Cragg, Mark S.

2017-01-01

Immunotherapy using monoclonal antibodies (mAb) such as rituximab is an established means of treating haematological malignancies. Antibodies can elicit a number of mechanisms to delete target cells, including complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP). The inherent properties of the target molecule help define which of these mechanisms are more important for efficacy. However, why mAb binding to different epitopes within the same target elicits different levels of therapeutic activity, is often unclear. To specifically address whether distance from the target cell membrane influences the aforementioned effector mechanisms, a panel of fusion proteins consisting of a CD20 or CD52 epitope attached to various CD137 scaffold molecules were generated. The CD137 scaffold was modified through the removal or addition of cysteine-rich extracellular domains, to produce a panel of chimeric molecules which held the target epitope at different distances along the protein. It was shown that CDC and ADCC favoured a membrane proximal epitope, whilst ADCP favoured an epitope positioned further away. These findings were then confirmed using reagents targeting the membrane proximal or distal domains of CD137 itself before investigating these properties in vivo where a clear difference in the splenic clearance of transfected tumour cells was observed. Together, this work demonstrates how altering the position of the antibody epitope is able to change the effector mechanisms engaged and facilitates the selection of mAbs designed to delete target cells through specific effector mechanisms and provide more effective therapeutic agents. PMID:28404636

Red blood cell vesiculation in hereditary hemolytic anemia

PubMed Central

Alaarg, Amr; Schiffelers, Raymond M.; van Solinge, Wouter W.; van Wijk, Richard

2013-01-01

Hereditary hemolytic anemia encompasses a heterogeneous group of anemias characterized by decreased red blood cell survival because of inherited membrane, enzyme, or hemoglobin disorders. Affected red blood cells are more fragile, less deformable, and more susceptible to shear stress and oxidative damage, and show increased vesiculation. Red blood cells, as essentially all cells, constitutively release phospholipid extracellular vesicles in vivo and in vitro in a process known as vesiculation. These extracellular vesicles comprise a heterogeneous group of vesicles of different sizes and intracellular origins. They are described in literature as exosomes if they originate from multi-vesicular bodies, or as microvesicles when formed by a one-step budding process directly from the plasma membrane. Extracellular vesicles contain a multitude of bioactive molecules that are implicated in intercellular communication and in different biological and pathophysiological processes. Mature red blood cells release in principle only microvesicles. In hereditary hemolytic anemias, the underlying molecular defect affects and determines red blood cell vesiculation, resulting in shedding microvesicles of different compositions and concentrations. Despite extensive research into red blood cell biochemistry and physiology, little is known about red cell deformability and vesiculation in hereditary hemolytic anemias, and the associated pathophysiological role is incompletely assessed. In this review, we discuss recent progress in understanding extracellular vesicles biology, with focus on red blood cell vesiculation. Also, we review recent scientific findings on the molecular defects of hereditary hemolytic anemias, and their correlation with red blood cell deformability and vesiculation. Integrating bio-analytical findings on abnormalities of red blood cells and their microvesicles will be critical for a better understanding of the pathophysiology of hereditary hemolytic anemias. PMID

Growth and replication of red rain cells at 121°C and their red fluorescence

NASA Astrophysics Data System (ADS)

Gangappa, Rajkumar; Wickramasinghe, Chandra; Wainwright, Milton; Kumar, A. Santhosh; Louis, Godfrey

2010-09-01

We have shown that the red cells found in the Red Rain (which fell on Kerala, India, in 2001) survive and grow after incubation for periods of up to two hours at 121°C . Under these conditions daughter cells appear within the original mother cells and the number of cells in the samples increases with length of exposure to 121°C. No such increase in cells occurs at room temperature, suggesting that the increase in daughter cells is brought about by exposure of the Red Rain cells to high temperatures. This is an independent confirmation of results reported earlier by two of the present authors, claiming that the cells can replicate under high pressure at temperatures upto 300°C. The flourescence behaviour of the red cells is shown to be in remarkable correspondence with the extended red emission observed in the Red Rectagle planetary nebula and other galactic and extragalactic dust clouds, suggesting, though not proving an extraterrestrial origin.

21 CFR 660.30 - Reagent Red Blood Cells.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Reagent Red Blood Cells. 660.30 Section 660.30 Food... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall be Reagent Red...

21 CFR 864.8185 - Calibrator for red cell and white cell counting.

Code of Federal Regulations, 2014 CFR

2014-04-01

... counting is a device that resembles red or white blood cells and that is used to set instruments intended to count red cells, white cells, or both. It is a suspension of particles or cells whose size, shape... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Calibrator for red cell and white cell counting...

21 CFR 864.8185 - Calibrator for red cell and white cell counting.

Code of Federal Regulations, 2012 CFR

2012-04-01

... counting is a device that resembles red or white blood cells and that is used to set instruments intended to count red cells, white cells, or both. It is a suspension of particles or cells whose size, shape... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Calibrator for red cell and white cell counting...

21 CFR 864.8185 - Calibrator for red cell and white cell counting.

Code of Federal Regulations, 2011 CFR

2011-04-01

... counting is a device that resembles red or white blood cells and that is used to set instruments intended to count red cells, white cells, or both. It is a suspension of particles or cells whose size, shape... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Calibrator for red cell and white cell counting...

21 CFR 864.8185 - Calibrator for red cell and white cell counting.

Code of Federal Regulations, 2013 CFR

2013-04-01

... counting is a device that resembles red or white blood cells and that is used to set instruments intended to count red cells, white cells, or both. It is a suspension of particles or cells whose size, shape... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Calibrator for red cell and white cell counting...

Production of a Recombinant Antibody Specific for i Blood Group Antigen, a Mesenchymal Stem Cell Marker

PubMed Central

Suila, Heli; Tiitinen, Sari; Natunen, Suvi; Laukkanen, Marja-Leena; Kotovuori, Annika; Reinman, Mirka; Satomaa, Tero; Alfthan, Kaija; Laitinen, Saara; Takkinen, Kristiina; Räbinä, Jarkko; Valmu, Leena

2013-01-01

Abstract Multipotent mesenchymal stem/stromal cells (MSCs) offer great promise for future regenerative and anti-inflammatory therapies. Panels of functional and phenotypical markers are currently used in characterization of different therapeutic stem cell populations from various sources. The i antigen (linear poly-N-acetyllactosamine) from the Ii blood group system has been suggested as a marker for MSCs derived from umbilical cord blood (UCB). However, there are currently no commercially available antibodies recognizing the i antigen. In the present study, we describe the use of antibody phage display technology to produce recombinant antibodies recognizing a structure from the surface of mesenchymal stem cells. We constructed IgM phage display libraries from the lymphocytes of a donor with an elevated serum anti-i titer. Antibody phage display technology is not dependent on immunization and thus allows the generation of antibodies against poorly immunogenic molecules, such as carbohydrates. Agglutination assays utilizing i antigen–positive red blood cells (RBCs) from UCB revealed six promising single-chain variable fragment (scFv) antibodies, three of which recognized epitopes from the surface of UCB-MSCs in flow cytometric assays. The amino acid sequence of the VH gene segment of B12.2 scFv was highly similar to the VH4.21 gene segment required to encode anti-i specificities. Further characterization of binding properties revealed that the binding of B12.2 hyperphage was inhibited by soluble linear lactosamine oligosaccharide. Based on these findings, we suggest that the B12.2 scFv we have generated is a prominent anti-i antibody that recognizes i antigen on the surface of both UCB-MSCs and RBCs. This binder can thus be utilized in UCB-MSC detection and isolation as well as in blood group serology. PMID:24083089

Monoclonal antibodies to alphaVbeta3 (7E3 and LM609) inhibit sickle red blood cell-endothelium interactions induced by platelet-activating factor.

PubMed

Kaul, D K; Tsai, H M; Liu, X D; Nakada, M T; Nagel, R L; Coller, B S

2000-01-15

Abnormal interaction of sickle red blood cells (SS RBC) with the vascular endothelium has been implicated as a factor in the initiation of vasoocclusion in sickle cell anemia. Both von Willebrand factor (vWf) and thrombospondin (TSP) play important roles in mediating SS RBC-endothelium interaction and can bind to the endothelium via alphaVbeta3 receptors. We have used monoclonal antibodies (MoAb) directed against alphaVbeta3 and alphaIIbbeta3 (GPIIb/IIIa) integrins to dissect the role of these integrins in SS RBC adhesion. The murine MoAb 7E3 inhibits both alphaVbeta3 and alphaIIbbeta3 (GPIIb/IIIa), whereas MoAb LM609 selectively inhibits alphaVbeta3, and MoAb 10E5 binds only to alphaIIbbeta3. In this study, we have tested the capacity of these MoAbs to block platelet-activating factor (PAF)-induced SS RBC adhesion in the ex vivo mesocecum vasculature of the rat. Infusion of washed SS RBC in preparations treated with PAF (200 pg/mL), with or without a control antibody, resulted in extensive adhesion of these cells in venules, accompanied by frequent postcapillary blockage and increased peripheral resistance units (PRU). PAF also caused increased endothelial surface and interendothelial expression of endothelial vWf. Importantly, pretreatment ofthe vasculature with either MoAb 7E3 F(ab')(2) or LM609, but not 10E5 F(ab')(2), after PAF almost completely inhibited SS RBC adhesion in postcapillary venules, the sites of maximal adhesion and frequent blockage. The inhibition of adhesion with 7E3 or LM609 was accompanied by smaller increases in PRU and shorter pressure-flow recovery times. Thus, blockade of alphaVbeta3 may constitute a potential therapeutic approach to prevent SS RBC-endothelium interactions under flow conditions. (Blood. 2000;95:368-374)

Antibody-mediated immune suppression is improved when blends of anti-RBC monoclonal antibodies are used in mice.

PubMed

Bernardo, Lidice; Amash, Alaa; Marjoram, Danielle; Lazarus, Alan H

2016-08-25

Although the prevention of hemolytic disease of the fetus and newborn is highly effective using polyclonal anti-D, a recombinant alternative is long overdue. Unfortunately, anti-D monoclonal antibodies have been, at best, disappointing. To determine the primary attribute defining an optimal antibody, we assessed suppression of murine red blood cell (RBC) immunization by single-monoclonal antibodies vs defined blends of subtype-matched antibodies. Allogeneic RBCs expressing the HOD antigen (hen egg lysozyme [HEL]-ovalbumin-human transmembrane Duffy(b)) were transfused into naïve mice alone or together with selected combinations of HEL-specific antibodies, and the resulting suppressive effect was assessed by evaluating the antibody response. Polyclonal HEL antibodies dramatically inhibited the antibody response to the HOD antigen, whereas single-monoclonal HEL antibodies were less effective despite the use of saturating doses. A blend of monoclonal HEL-specific antibodies reactive with different HEL epitopes significantly increased the suppressive effect, whereas a blend of monoclonal antibodies that block each other's binding to the HEL protein did not increase suppression. In conclusion, these data show that polyclonal antibodies are superior to monoclonal antibodies at suppressing the immune response to the HOD cells, a feature that can be completely recapitulated using monoclonal antibodies to different epitopes. © 2016 by The American Society of Hematology.

Studying red blood cell agglutination by measuring membrane viscosity with optical tweezers

NASA Astrophysics Data System (ADS)

Fernandes, Heloise P.; Fontes, Adriana; de Thomaz, André A.; Barbosa, Luiz C.; Barjas-Castro, Maria L.; Cesar, Carlos L.

2007-09-01

The red blood cell (RBC) viscoelastic membrane contains proteins and glycoproteins embedded in a fluid lipid bilayer that are responsible for cell agglutination. Manipulating RBCs rouleaux with a double optical tweezers, we observed that the cells slide easily one over the others but are strongly connected by their edges. An explanation for this behavior could be the fact that when the cells slide one over the others, proteins are dragged through the membrane. It confers to the movement a viscous characteristic that is dependent of the velocity between the RBCs and justifies why is so easy to slide them apart. Therefore, in a first step of this work, by measuring the force as a function of the relative velocity between two cells, we confirmed this assumption and used this viscous characteristic of the RBC rouleaux to determine the apparent membrane viscosity of the cell. As this behavior is related to the proteins interactions, we can use the apparent membrane viscosity to obtain a better understanding about cell agglutination. Methods related to cell agglutination induced by antigen-antibody interactions are the basis of most of tests used in transfusion centers. Then, in a second step of this work, we measured the apparent membrane viscosity using antibodies. We observed that this methodology is sensitive to different kinds of bindings between RBCs. Better comprehension of the forces and bindings between RBCs could improve the sensibility and specificity of the hemagglutination reactions and also guides the development of new potentiator substances.

An antibody to the lutheran glycoprotein (Lu) recognizing the LU4 blood type variant inhibits cell adhesion to laminin α5.

PubMed

Kikkawa, Yamato; Miwa, Takahiro; Tohara, Yukiko; Hamakubo, Takayuki; Nomizu, Motoyoshi

2011-01-01

The Lutheran blood group glycoprotein (Lu), an Ig superfamily (IgSF) transmembrane receptor, is also known as basal cell adhesion molecule (B-CAM). Lu/B-CAM is a specific receptor for laminin α5, a major component of basement membranes in various tissues. Previous reports have shown that Lu/B-CAM binding to laminin α5 contributes to sickle cell vaso-occlusion. However, as there are no useful tools such as function-blocking antibodies or drugs, it is unclear how epithelial and sickled red blood cells adhere to laminin α5 via Lu/B-CAM. In this study, we discovered a function-blocking antibody that inhibits Lu binding to laminin α5 using a unique binding assay on tissue sections. To characterize the function-blocking antibody, we identified the site on Lu/B-CAM recognized by this antibody. The extracellular domain of Lu/B-CAM contains five IgSF domains, D1-D2-D3-D4-D5. The antibody epitope was localized to D2, but not to the D3 domain containing the major part of the laminin α5 binding site. Furthermore, mutagenesis studies showed that Arg(175), the LU4 blood group antigenic site, was crucial for forming the epitope and the antibody bound sufficiently close to sterically hinder the interaction with α5. Cell adhesion assay using the antibody also showed that Lu/B-CAM serves as a secondary receptor for the adhesion of carcinoma cells to laminin α5. This function-blocking antibody against Lu/B-CAM should be useful for not only investigating cell adhesion to laminin α5 but also for developing drugs to inhibit sickle cell vaso-occlusion.

Microfluidic antibody arrays for simultaneous cell separation and stimulus.

PubMed

Liu, Yan; Germain, Todd; Pappas, Dimitri

2014-12-01

A microfluidic chip containing stamped antibody arrays was developed for simultaneous cell separation and drug testing. Poly(dimethyl siloxane) (PDMS) stamping was used to deposit antibodies in a microfluidic channel, forming discrete cell-capture regions on the surface. Cell mixtures were then introduced, resulting in the separation of cells when specific antibodies were used. Anti-CD19 antibody regions resulted in 94 % capture purity for CD19+ Ramos cells. An antibody that captures multiple cell types, for example anti-CD71, can also be used to capture several cell types simultaneously. Cells could also be loaded onto the arrays with spatial control using laminar streams. Both Ramos B cells and HuT 78 T cells were isolated in the chip and exposed to staurosporine in the same channel. Both cell lines had similar responses to the drug, with 2-10 % of cells remaining viable after 20 h of drug treatment, depending on cell type. The chip can also be used to analyze the efficacy of antibody therapy against cancer cells. Anti-CD95 was deposited on the surface and used for simultaneous cell capture and apoptosis induction via the extrinsic pathway. Cells captured on anti-CD95 surfaces had significant viability loss (15 % viability after 24 h) when compared with a control anti-CD71 antibody (81 % viability after 24 h). This chip can be used for a variety of cell separation and/or drug testing studies, enabling researchers to isolate cells and test them against different anti-cancer compounds and to follow cell response using fluorescence or other readout methods.

Study of blood group B antigen with a specific monoclonal antibody (anti-B, b-183).

PubMed Central

Rouger, P; Edelman, L; Doinel, C; Reviron, J; Salmon, C; Bach, J F

1983-01-01

A murine anti-B monoclonal antibody was obtained by the hybridoma technique. This antibody called anti-B (b-183) is of IgM nature; it is capable of agglutinating normal B, B3, Bx, cis AB and some acquired B red cells. Its association constant is 1.1 X 10(8) l/mol, and appears high compared to those of the monoclonal anti-A. This monoclonal anti-B was used to determine the number of B sites on B3 and Bx red cells. PMID:6840810

Frequency of Red Cell Alloimmunization and Autoimmunization in Thalassemia Patients: A Report from Eastern India.

PubMed

Datta, Suvro Sankha; Mukherjee, Somnath; Talukder, Biplabendu; Bhattacharya, Prasun; Mukherjee, Krishnendu

2015-01-01

Introduction. Red blood cell (RBC) alloimmunization and autoimmunization remain a major problem in transfusion dependent thalassemic patients. There is a paucity of data on the incidence of RBC alloimmunization and autoimmunization in thalassemic patients from eastern part of India, as pretransfusion antibody screening is not routinely performed. Aims. To assess the incidence of RBC alloimmunization and autoimmunization in transfusion dependent thalassemic patients in eastern India. Materials and Methods. Total 500 thalassemia cases were evaluated. The antibody screening and identification were performed with commercially available panel cells (Diapanel, Bio-rad, Switzerland) by column agglutination method. To detect autoantibodies, autocontrol and direct antiglobulin tests were carried out using polyspecific coombs (IgG + C3d) gel cards in all patients. Results. A total of 28 patients developed RBC alloimmunization (5.6%) and 5 patients had autoantibodies (1%). Alloantibody against c had the highest incidence (28.57%) followed by E (21.42%). Five out of 28 (17.85%) patients had developed antibodies against both c and E. Conclusion. Data from this study demonstrate that the RBC alloantibody and autoantibody development rates are significant in our region. Thus, pretransfusion antibody screening needs to be initiated in eastern India in order to ensure safe transfusion practice.

Frequency of Red Cell Alloimmunization and Autoimmunization in Thalassemia Patients: A Report from Eastern India

PubMed Central

Datta, Suvro Sankha; Talukder, Biplabendu; Bhattacharya, Prasun; Mukherjee, Krishnendu

2015-01-01

Introduction. Red blood cell (RBC) alloimmunization and autoimmunization remain a major problem in transfusion dependent thalassemic patients. There is a paucity of data on the incidence of RBC alloimmunization and autoimmunization in thalassemic patients from eastern part of India, as pretransfusion antibody screening is not routinely performed. Aims. To assess the incidence of RBC alloimmunization and autoimmunization in transfusion dependent thalassemic patients in eastern India. Materials and Methods. Total 500 thalassemia cases were evaluated. The antibody screening and identification were performed with commercially available panel cells (Diapanel, Bio-rad, Switzerland) by column agglutination method. To detect autoantibodies, autocontrol and direct antiglobulin tests were carried out using polyspecific coombs (IgG + C3d) gel cards in all patients. Results. A total of 28 patients developed RBC alloimmunization (5.6%) and 5 patients had autoantibodies (1%). Alloantibody against c had the highest incidence (28.57%) followed by E (21.42%). Five out of 28 (17.85%) patients had developed antibodies against both c and E. Conclusion. Data from this study demonstrate that the RBC alloantibody and autoantibody development rates are significant in our region. Thus, pretransfusion antibody screening needs to be initiated in eastern India in order to ensure safe transfusion practice. PMID:26425124

Red cell alloimmunization is associated with development of autoantibodies and increased red cell transfusion requirements in myelodysplastic syndrome

PubMed Central

Singhal, Deepak; Kutyna, Monika M.; Chhetri, Rakchha; Wee, Li Yan A.; Hague, Sophia; Nath, Lakshmi; Nath, Shriram V.; Sinha, Romi; Wickham, Nicholas; Lewis, Ian D.; Ross, David M.; Bardy, Peter G.; To, Luen Bik; Reynolds, John; Wood, Erica M.; Roxby, David J.; Hiwase, Devendra K.

2017-01-01

Up to 90% of patients with a myelodysplastic syndrome require red blood cell transfusion; nevertheless, comprehensive data on red cell alloimmunization in such patients are limited. This study evaluates the incidence and clinical impact of red cell alloimmunization in a large cohort of patients with myelodysplastic syndrome registered in the statewide South Australian-MDS registry. The median age of the 817 patients studied was 73 years, and 66% were male. The cumulative incidence of alloimmunization was 11%. Disease-modifying therapy was associated with a lower risk of alloimmunization while alloimmunization was significantly higher in patients with a revised International Prognostic Scoring System classification of Very Low, Low or Intermediate risk compared to those with a High or Very High risk (P=0.03). Alloantibodies were most commonly directed against antigens in the Rh (54%) and Kell (24%) systems. Multiple alloantibodies were present in 49% of alloimmunized patients. Although 73% of alloimmunized patients developed alloantibodies during the period in which they received their first 20 red cell units, the total number of units transfused was significantly higher in alloimmunized patients than in non-alloimmunized patients (90±100 versus 30±52; P<0.0001). In individual patients, red cell transfusion intensity increased significantly following alloimmunization (2.8±1.3 versus 4.1±2.0; P<0.0001). A significantly higher proportion of alloimmunized patients than non-alloimmunized patients had detectable autoantibodies (65% versus 18%; P<0.0001) and the majority of autoantibodies were detected within a short period of alloimmunization. In conclusion, this study characterizes alloimmunization in a large cohort of patients with myelodysplastic syndrome and demonstrates a signficant increase in red cell transfusion requirements following alloimmunization, most probably due to development of additional alloantibodies and autoantibodies, resulting in subclinical

Rosetting of Plasmodium falciparum-infected red blood cells with uninfected red blood cells enhances microvascular obstruction under flow conditions.

PubMed

Kaul, D K; Roth, E F; Nagel, R L; Howard, R J; Handunnetti, S M

1991-08-01

The occurrence of rosetting of Plasmodium falciparum-infected human red blood cells (IRBC) with uninfected red blood cells (RBC) and its potential pathophysiologic consequences were investigated under flow conditions using the perfused rat mesocecum vasculature. Perfusion experiments were performed using two knobby (K+) lines of P falciparum, ie, rosetting positive (K+R+) and rosetting negative (K+R-). The infusion of K+R+ IRBC resulted in higher peripheral resistance (PRU) than K+R- IRBC (P less than .0012). Video microscopy showed that under conditions of flow, in addition to cytoadherence of K+R+ IRBC to the venular endothelium, rosette formation was also restricted to venules, especially in the areas of slow flow. Rosettes were absent in arterioles and were presumably dissociated by higher wall shear rates. The presence of rosettes in the venules must therefore reflect their rapid reformation after disruption. Cytoadherence of K+R+ IRBC was characterized by formation of focal clusters along the venular wall. In addition, large aggregates of RBC were frequently observed at venular junctions, probably as a result of interaction between flowing rosettes, free IRBC, and uninfected RBC. In contrast, the infusion of K+R+ IRBC resulted in diffuse cytoadherence of these cells exclusively to the venular endothelium but not in rosetting or large aggregate formation. The cytoadherence of K+R+ IRBC showed strong inverse correlation with the venular diameter (r = -.856, P less than .00001). Incubation of K+R+ IRBC with heparin and with monoclonal antibodies to glycoprotein IV/CD36 abolished the rosette formation and resulted in decreased PRU and microvascular blockage. These findings demonstrate that rosetting of K+R+ IRBC with uninfected RBC enhances vasocclusion, suggesting an important in vivo role for rosetting in the microvascular sequestration of P falciparum-infected RBC.

21 CFR 660.30 - Reagent Red Blood Cells.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall be...

21 CFR 660.30 - Reagent Red Blood Cells.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall be...

21 CFR 660.30 - Reagent Red Blood Cells.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall be...

21 CFR 660.30 - Reagent Red Blood Cells.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall be...

Subpopulations of Ig-secreting cells induced by peroxidase immunization: discrimination according to antibody storage and secretion.

PubMed Central

Bernard, J; Jeannesson, P; Thiernesse, N; Zagury, D; Ternynck, T; Avrameas, S

1979-01-01

Mice were injected in their hind footpads with peroxidase (PO) emulsified in Freund's complete adjuvant. The development of cells secreting anti-peroxidase antibody (Ab) and cells secreting immunoglobulins (Ig) were detected in the draining popliteal lymph nodes in the subsequent 35 days, using local haemolysis plaque assay with sheep red cell blood cells coated with either PO or anti-mouse Ig antibody. Plaque-forming cells (PFC) were isolated from the centre of plaques by micromanipulation and after appropriate treatment, were examined by electron microscopy for their intracellular Ab content and in corporation of [3H]-thymidine. Four subpopulations of Ig secreting cells were distinguished: (1) cells secreting Ig without Ab function and not containing intracellular Ab detectable between days 5 and 20; (2) cells secreting Ig without Ab function but containing Ab appearing on day 6 and present throughout the immune response; (3) cells secreting Ab and containing Ab; (4) cells secreting Ab, but without detectable intracellular Ab. These last subpopulations appeared on day 7 and were found in all subsequent assays. The analysis of the kinetics of these subpopulations suggest that cells secreting Ig without Ab function might be precursors of Ab secreting cells. Images Figure 2a Figure 2b Figure 4 PMID:374259

Separation of breast cancer cells from peripherally circulating blood using antibodies fixed in microchannels

NASA Astrophysics Data System (ADS)

Feng, Juan; Soper, Steven A.; McCarley, Robin L.; Murphy, Michael C.

2004-07-01

Bio-Micro Electro Mechanical System (Bio-MEMS) technology was applied to the problem of early breast cancer detection and diagnosis. A micro-device is being developed to identify and specifically collect tumor cells of low abundance (1 tumor cell among 107 normal blood cells) from circulating whole blood. By immobilizing anti-EpCAM (Epithelial Cell Adhesion Molecule) antibodies on polymer micro-channel walls by chemically modifying the surface of the PMMA, breast cancer cells from the MCF-7 cell line, which over-express EpCAM, were selected from a sample volume by the strong binding affinity between the antibody and antigen. To validate the capture of the breast cancer cells, three fluorochrome markers, each identified by a separate color, were used to reliably identify the cancer cells. The cancer cells were defined by DAPI+ (blue), CD45- and the FITC-cell membrane linker+ (green). White blood cells, which may interfere in the detection of the cancer cells, were identified by DAPI+ (blue), CD45+ (red), and the FITC-cell membrane linker+ (green). EpCAM/anti-EpCAM binding models from the literature were used to estimate an optimal velocity, 2mm/sec, for maximizing the number of cells binding and the critical binding force. At higher velocities, shear forces (> 0.48 dyne) will break existing bonds and prevent the formation of new ones. This detection micro-device can be assembled with other lab-on-a-chip components for follow-up gene and protein analysis.

Proteins improving recombinant antibody production in mammalian cells.

PubMed

Nishimiya, Daisuke

2014-02-01

Mammalian cells have been successfully used for the industrial manufacture of antibodies due to their ability to synthesize antibodies correctly. Nascent polypeptides must be subjected to protein folding and assembly in the ER and the Golgi to be secreted as mature proteins. If these reactions do not proceed appropriately, unfolded or misfolded proteins are degraded by the ER-associated degradation (ERAD) pathway. The accumulation of unfolded proteins or intracellular antibody crystals accompanied by this failure triggers the unfolded protein response (UPR), which can considerably attenuate the levels of translation, folding, assembly, and secretion, resulting in reduction of antibody productivity. Accumulating studies by omics-based analysis of recombinant mammalian cells suggest that not only protein secretion processes including protein folding and assembly but also translation are likely to be the rate-limiting factors for increasing antibody production. Here, this review describes the mechanism of antibody folding and assembly and recent advantages which could improve recombinant antibody production in mammalian cells by utilizing proteins such as ER chaperones or UPR-related proteins.

Comparison of Antibody-Dependent Cell-Mediated Cytotoxicity and Virus Neutralization by HIV-1 Env-Specific Monoclonal Antibodies

PubMed Central

von Bredow, Benjamin; Arias, Juan F.; Heyer, Lisa N.; Moldt, Brian; Le, Khoa; Robinson, James E.; Burton, Dennis R.

2016-01-01

ABSTRACT Although antibodies to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been studied extensively for their ability to block viral infectivity, little data are currently available on nonneutralizing functions of these antibodies, such as their ability to eliminate virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 Env-specific antibodies of diverse specificities, including potent broadly neutralizing and nonneutralizing antibodies, were therefore tested for ADCC against cells infected with a lab-adapted HIV-1 isolate (HIV-1NL4-3), a primary HIV-1 isolate (HIV-1JR-FL), and a simian-human immunodeficiency virus (SHIV) adapted for pathogenic infection of rhesus macaques (SHIVAD8-EO). In accordance with the sensitivity of these viruses to neutralization, HIV-1NL4-3-infected cells were considerably more sensitive to ADCC, both in terms of the number of antibodies and magnitude of responses, than cells infected with HIV-1JR-FL or SHIVAD8-EO. ADCC activity generally correlated with antibody binding to Env on the surfaces of virus-infected cells and with viral neutralization; however, neutralization was not always predictive of ADCC, as instances of ADCC in the absence of detectable neutralization, and vice versa, were observed. These results reveal incomplete overlap in the specificities of antibodies that mediate these antiviral activities and provide insights into the relationship between ADCC and neutralization important for the development of antibody-based vaccines and therapies for combating HIV-1 infection. IMPORTANCE This study provides fundamental insights into the relationship between antibody-dependent cell-mediated cytotoxicity (ADCC) and virus neutralization that may help to guide the development of antibody-based vaccines and immunotherapies for the prevention and treatment of HIV-1 infection. PMID:27122574

Comparison of Antibody-Dependent Cell-Mediated Cytotoxicity and Virus Neutralization by HIV-1 Env-Specific Monoclonal Antibodies.

PubMed

von Bredow, Benjamin; Arias, Juan F; Heyer, Lisa N; Moldt, Brian; Le, Khoa; Robinson, James E; Zolla-Pazner, Susan; Burton, Dennis R; Evans, David T

2016-07-01

Although antibodies to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been studied extensively for their ability to block viral infectivity, little data are currently available on nonneutralizing functions of these antibodies, such as their ability to eliminate virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 Env-specific antibodies of diverse specificities, including potent broadly neutralizing and nonneutralizing antibodies, were therefore tested for ADCC against cells infected with a lab-adapted HIV-1 isolate (HIV-1NL4-3), a primary HIV-1 isolate (HIV-1JR-FL), and a simian-human immunodeficiency virus (SHIV) adapted for pathogenic infection of rhesus macaques (SHIVAD8-EO). In accordance with the sensitivity of these viruses to neutralization, HIV-1NL4-3-infected cells were considerably more sensitive to ADCC, both in terms of the number of antibodies and magnitude of responses, than cells infected with HIV-1JR-FL or SHIVAD8-EO ADCC activity generally correlated with antibody binding to Env on the surfaces of virus-infected cells and with viral neutralization; however, neutralization was not always predictive of ADCC, as instances of ADCC in the absence of detectable neutralization, and vice versa, were observed. These results reveal incomplete overlap in the specificities of antibodies that mediate these antiviral activities and provide insights into the relationship between ADCC and neutralization important for the development of antibody-based vaccines and therapies for combating HIV-1 infection. This study provides fundamental insights into the relationship between antibody-dependent cell-mediated cytotoxicity (ADCC) and virus neutralization that may help to guide the development of antibody-based vaccines and immunotherapies for the prevention and treatment of HIV-1 infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Mycoplasma infection of cell lines can simulate the expression of Fc receptors by binding of the carbohydrate moiety of antibodies.

PubMed

Lemke, H; Krausse, R; Lorenzen, J; Havsteen, B

1985-05-01

During the production of Fc receptor (FcR)-bearing hybridomas it was observed with a particular monoclonal anti-sheep red blood cell antibody (anti-SRBC 1/5, IgG1) that the contamination with Mycoplasma arginini of in vitro cultured cell lines leads to an apparent FcR activity. This property did not correspond with the serological typing since other antibodies of the same isotype could not support FcR rosette formation. Another mycoplasma strain M. orale lacked this property. Analysis of the binding reaction revealed that M. arginini contains a lectin which binds the carbohydrate moiety of the anti-SRBC 1/5 antibody, i.e. anti-SRBC 1/5 synthesized under the influence of tunicamycin or deglycosylated by NaIO4 oxidation did not support rosette formation. These data suggest that binding of antibodies to certain mycoplasma strains may be a pathogenic factor during mycoplasma infections by masking the microorganisms with the host's own defense molecules. The experiments with M. arginini-infected cell lines gain immunological importance since we obtained identical results with staphylococcal protein A, as another bacteriological FcR, and cell lines expressing intrinsic membrane FcR. Although it is an open question whether the glycoconjugates are directly bound by the FcR or else by influencing the three-dimensional structure of the antibodies, it seems possible that FcR in general may be lectins.

Pathogenesis and mechanisms of antibody-mediated hemolysis

PubMed Central

Flegel, Willy A

2015-01-01

Background The clinical consequences of antibodies to red blood cells (RBC) have been studied for a century. Most clinically relevant antibodies can be detected by sensitive in vitro assays. Several mechanisms of antibody-mediated hemolysis are well understood. Such hemolysis following transfusion is reliably avoided in a donor/recipient pair, if one individual is negative for the cognate antigen to which the other has the antibody. Study design and results Mechanisms of antibody-mediated hemolysis were reviewed based on a presentation at the Strategies to Address Hemolytic Complications of Immune Globulin Infusions Workshop addressing intravenous immunoglobulin (IVIG) and ABO antibodies. The presented topics included the rates of intravascular and extravascular hemolysis; IgM and IgG isoagglutinins; auto- and alloantibodies; antibody specificity; A, B, A,B and A1 antigens; A1 versus A2 phenotypes; monocytes/macrophages, other immune cells and complement; monocyte monolayer assay (MMA); antibody-dependent cell-mediated cytotoxicity (ADCC); and transfusion reactions due to ABO and other antibodies. Conclusion Several clinically relevant questions remained unresolved, and diagnostic tools were lacking to routinely and reliably predict the clinical consequences of RBC antibodies. Most hemolytic transfusion reactions associated with IVIG were due to ABO antibodies. Reducing the titers of such antibodies in IVIG may lower the frequency of this kind of adverse event. The only way to stop these events is to have no anti-A or anti-B antibodies in the IVIG products. PMID:26174897

Natural Killer Cell Mediated Antibody-Dependent Cellular Cytotoxicity in Tumor Immunotherapy with Therapeutic Antibodies

PubMed Central

Seidel, Ursula J. E.; Schlegel, Patrick; Lang, Peter

2013-01-01

In the last decade several therapeutic antibodies have been Federal Drug Administration (FDA) and European Medicines Agency (EMEA) approved. Although their mechanisms of action in vivo is not fully elucidated, antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is presumed to be a key effector function. A substantial role of ADCC has been demonstrated in vitro and in mouse tumor models. However, a direct in vivo effect of ADCC in tumor reactivity in humans remains to be shown. Several studies revealed a predictive value of FcγRIIIa-V158F polymorphism in monoclonal antibody treatment, indicating a potential effect of ADCC on outcome for certain indications. Furthermore, the use of therapeutic antibodies after allogeneic hematopoietic stem cell transplantation is an interesting option. Studying the role of the FcγRIIIa-V158F polymorphism and the influence of Killer-cell Immunoglobuline-like Receptor (KIR) receptor ligand incompatibility on ADCC in this approach may contribute to future transplantation strategies. Despite the success of approved second-generation antibodies in the treatment of several malignancies, efforts are made to further augment ADCC in vivo by antibody engineering. Here, we review currently used therapeutic antibodies for which ADCC has been suggested as effector function. PMID:23543707

Red cell substitutes.

PubMed

Winslow, Robert M

2007-01-01

Oxygen-carrying plasma expanders (blood substitutes) have been sought for over a century. Development of current products is a result of evolution in the understanding of proteins in general, of hemoglobin in particular, and of how cell-free hemoglobin interacts with the control of local blood flow to ensure adequate tissue oxygenation. Hemoglobin-based products are considered in four "generations" corresponding to major improvements. First-generation products consisted of hemoglobin, freed of red cell membranes (stroma-free hemoglobin [SFH]), which was renal toxic and vasoactive. Second-generation products were polymerized with aldehyde reagents to reduce or eliminate the renal toxicity, but the products were heterogeneous and still vasoactive. Third-generation products employed more specific intramolecular crosslinking to eliminate polymerization and promote homogeneity, but they also remained vasoactive. Fourth-generation products are based on a new understanding of the way in which microvascular blood flow is controlled and the influence of O(2) delivery to vascular walls. After more than a century of research, one of these new solutions should find use as an alternative to red cells for transfusion in certain clinical settings.

Blood bank issues associated with red cell exchanges in sickle cell disease.

PubMed

Sarode, Ravindra; Altuntas, Fevzi

2006-12-01

Sickle cell disease (SCD) patients are prone to develop complications that include stroke, acute chest syndrome, and other crises. Some of these complications require chronic transfusion therapy or red cell exchange (RCE), either for therapeutic or prophylactic reasons. Due to a discrepancy of red cell antigens between African Americans and Caucasians (majority blood donors), the incidence of alloantibody formation is very high, which makes it difficult to find compatible red cell units, especially for urgent RCE. Some of the above conditions require immediate oxygen delivery to the tissues. Thus, SCD patients undergoing RCE should receive red blood cells with special attributes that include matching for Rh and Kell blood group antigens; RBCs should be fresh in order to provide (1) immediate oxygen delivery and (2) longer surviving cells to reduce the interval between RCE. Also, these units should be pre-storage leukoreduced to prevent febrile non-hemolytic reactions and screened for sickle cell traits to avoid transfusing red cells containing HbS. This requires a concerted effort between the apheresis unit, the local blood bank, and the central blood supplier.

Destruction of newly released red blood cells in space flight

NASA Technical Reports Server (NTRS)

Alfrey, C. P.; Udden, M. M.; Huntoon, C. L.; Driscoll, T.

1996-01-01

Space flight results in a rapid change in total blood volume, plasma volume, and red blood cell mass because the space to contain blood is decreased. The plasma volume and total blood volume decreases during the first hours in space and remain at a decreased level for the remainder of the flight. During the first several hours following return to earth, plasma volume and total blood volume increase to preflight levels. During the first few days in space recently produced red blood cells disappear from the blood resulting in a decrease in red blood cell mass of 10-15%. Red cells 12 d old or older survive normally and production of new cells continues at near preflight levels. After the first few days in space, the red cell mass is stable at the decreased level. Following return to earth the hemoglobin and red blood cell mass concentrations decrease reflecting the increase in plasma volume. The erythropoietin levels increase responding to "postflight anemia"; red cell production increases, and the red cell mass is restored to preflight levels after several weeks.

Antibody-Dependent Cellular Cytotoxicity against Reactivated HIV-1-Infected Cells

PubMed Central

Lee, Wen Shi; Richard, Jonathan; Lichtfuss, Marit; Smith, Amos B.; Park, Jongwoo; Courter, Joel R.; Melillo, Bruno N.; Sodroski, Joseph G.; Kaufmann, Daniel E.; Parsons, Matthew S.

2015-01-01

ABSTRACT Lifelong antiretroviral therapy (ART) for HIV-1 does not diminish the established latent reservoir. A possible cure approach is to reactivate the quiescent genome from latency and utilize immune responses to eliminate cells harboring reactivated HIV-1. It is not known whether antibodies within HIV-1-infected individuals can recognize and eliminate cells reactivated from latency through antibody-dependent cellular cytotoxicity (ADCC). We found that reactivation of HIV-1 expression in the latently infected ACH-2 cell line elicited antibody-mediated NK cell activation but did not result in antibody-mediated killing. The lack of CD4 expression on these HIV-1 envelope (Env)-expressing cells likely resulted in poor recognition of CD4-induced antibody epitopes on Env. To examine this further, cultured primary CD4+ T cells from HIV-1+ subjects were used as targets for ADCC. These ex vivo-expanded primary cells were modestly susceptible to ADCC mediated by autologous or heterologous HIV-1+ serum antibodies. Importantly, ADCC mediated against these primary cells could be enhanced following incubation with a CD4-mimetic compound (JP-III-48) that exposes CD4-induced antibody epitopes on Env. Our studies suggest that with sufficient reactivation and expression of appropriate Env epitopes, primary HIV-1-infected cells can be targets for ADCC mediated by autologous serum antibodies and innate effector cells. The results of this study suggest that further investigation into the potential of ADCC to eliminate reactivated latently infected cells is warranted. IMPORTANCE An HIV-1 cure remains elusive due to the persistence of long-lived latently infected cells. An HIV-1 cure strategy, termed “shock and kill,” aims to reactivate HIV-1 expression in latently infected cells and subsequently eliminate the reactivated cells through immune-mediated killing. While recent research efforts have focused on reversing HIV-1 latency, it remains unclear whether preexisting immune

The specificity of the cross-reacting antibodies in blood group O sera which produce mixed agglutination

PubMed Central

Franks, D.; Liske, Rosemary

1968-01-01

γM cross-reacting antibodies in group O sera produce mixed agglutination with secretor buccal cells, but not with non-secretor cells. The mixed agglutination with sera which also contain immune anti-B is produced only with A buccal cells and B indicator red cells, and not with B buccal cells, and is inhibited by B secretor saliva or galactose, but not by A secretor saliva. Mixed agglutination with sera containing immune anti-A is produced only with B buccal cells and A indicator red cells, and is inhibited by A secretor saliva or 2-acetamido-2-deoxygalactose (N-acetyl-D-galactosamine), but not by B secretor saliva. If two sera, one containing immune anti-A and the other containing immune anti-B, are mixed together in equal volumes, mixed agglutination is no longer produced with either A buccal cells (and B indicator red cells) or B buccal cells (and A indicator red cells). These observations are thought to be most readily explicable by the hypothesis that the combining site on the cross-reacting antibody is smaller than on specific anti-A or anti-B, and that it reacts with that part of the antigenic determinant which is common to both A and B antigens. As a consequence of the restricted size of the combining site, it is suggested that cross-reacting antibody will have a lower affinity for A or B antigens than specific anti-A or anti-B does, and competes unsuccessfully with specific anti-A or anti-B for combination with antigen on buccal cells. PMID:5638582

Predictors of Red Cell Alloimmunization in Kurdish Multi Transfused Patients with Hemoglobinopathies in Iraq.

PubMed

Al-Mousawi, Muqdad M N; Al-Allawi, Nasir A S; Alnaqshabandi, Rubad

2015-01-01

Hemoglobinopathies are significant health problems in Iraq, including its Northern Kurdistan region. One of the essential components of management of these disorders is regular lifelong blood transfusions. The latter is associated with several complications including red cell alloimmunization. No study has looked at the frequency of alloimmunization and its associations in the country. To address the latter issue, 401 multi transfused patients [311 with β-thalassemia (β-thal) syndrome and 90 with sickle cell disease], registered at a large thalassemia care center in Iraqi Kurdistan had their records reviewed, and their sera tested for atypical antibodies using screening and extended red cell panels. Red cell alloimmunization was detected in 18 patients (4.5%) with a total of 20 alloantibodies, while no autoantibodies were detected. The most frequent alloantibody was anti-E, followed by anti-D, anti-K, anti-C(w), anti-C, anti-c and anti-Le(a). Ethnicity was an important predictor of alloimmunization, while age at start of transfusion (>2 vs. ≤2 years) (p = 0.005), Rhesus D (RhD) negative status (p = 0.0017) and history of previous transfusion reactions (p = 0.007) showed a statistically significant higher rate of alloimmunization. However, patients' age, gender, number of units transfused, underlying diagnosis and splenectomy were not significantly associated with alloimmunization. Based on our observations, measures to reduce alloimmunization rates may include extended matching for Rhesus and Kell antigens and early initiation of blood transfusions.

B Cells and B Cell Blasts Withstand Cryopreservation While Retaining Their Functionality for Producing Antibody.

PubMed

Fecher, Philipp; Caspell, Richard; Naeem, Villian; Karulin, Alexey Y; Kuerten, Stefanie; Lehmann, Paul V

2018-05-31

In individuals who have once developed humoral immunity to an infectious/foreign antigen, the antibodies present in their body can mediate instant protection when the antigen re-enters. Such antigen-specific antibodies can be readily detected in the serum. Long term humoral immunity is, however, also critically dependent on the ability of memory B cells to engage in a secondary antibody response upon re-exposure to the antigen. Antibody molecules in the body are short lived, having a half-life of weeks, while memory B cells have a life span of decades. Therefore, the presence of serum antibodies is not always a reliable indicator of B cell memory and comprehensive monitoring of humoral immunity requires that both serum antibodies and memory B cells be assessed. The prevailing view is that resting memory B cells and B cell blasts in peripheral blood mononuclear cells (PBMC) cannot be cryopreserved without losing their antibody secreting function, and regulated high throughput immune monitoring of B cell immunity is therefore confined to-and largely limited by-the need to test freshly isolated PBMC. Using optimized protocols for freezing and thawing of PBMC, and four color ImmunoSpot ® analysis for the simultaneous detection of all immunoglobulin classes/subclasses we show here that both resting memory B cells and B cell blasts retain their ability to secrete antibody after thawing, and thus demonstrate the feasibility of B cell immune monitoring using cryopreserved PBMC.

Adhesion between peptides/antibodies and breast cancer cells

NASA Astrophysics Data System (ADS)

Meng, J.; Paetzell, E.; Bogorad, A.; Soboyejo, W. O.

2010-06-01

Atomic force microscopy (AFM) techniques were used to measure the adhesion forces between the receptors on breast cancer cells specific to human luteinizing hormone-releasing hormone (LHRH) peptides and antibodies specific to the EphA2 receptor. The adhesion forces between LHRH-coated AFM tips and human MDA-MB-231 cells (breast cancer cells) were shown to be about five times greater than those between LHRH-coated AFM tips and normal Hs578Bst breast cells. Similarly, those between EphA2 antibody-coated AFM tips and breast cancer cells were over five times greater than those between EphA2 antibody-coated AFM tips and normal breast cells. The results suggest that AFM can be used for the detection of breast cancer cells in biopsies. The implications of the results are also discussed for the early detection and localized treatment of cancer.

Effects of helicopter transport on red blood cell components.

PubMed

Otani, Taiichi; Oki, Ken-ichi; Akino, Mitsuaki; Tamura, Satoru; Naito, Yuki; Homma, Chihiro; Ikeda, Hisami; Sumita, Shinzou

2012-01-01

There are no reported studies on whether a helicopter flight affects the quality and shelf-life of red blood cells stored in mannitol-adenine-phosphate. Seven days after donation, five aliquots of red blood cells from five donors were packed into an SS-BOX-110 container which can maintain the temperature inside the container between 2 °C and 6 °C with two frozen coolants. The temperature of an included dummy blood bag was monitored. After the box had been transported in a helicopter for 4 hours, the red blood cells were stored again and their quality evaluated at day 7 (just after the flight), 14, 21 and 42 after donation. Red blood cell quality was evaluated by measuring adenosine triphosphate, 2,3-diphosphoglycerate, and supernatant potassium, as well as haematocrit, intracellular pH, glucose, supernatant haemoglobin, and haemolysis rate at the various time points. During the experiment the recorded temperature remained between 2 and 6 °C. All data from the red blood cells that had undergone helicopter transportation were the same as those from a control group of red blood cell samples 7 (just after the flight), 14, 21, and 42 days after the donation. Only supernatant Hb and haemolysis rate 42 days after the donation were slightly increased in the helicopter-transported group of red blood cell samples. All other parameters at 42 days after donation were the same in the two groups of red blood cells. These results suggest that red blood cells stored in mannitol-adenine-phosphate are not significantly affected by helicopter transportation. The differences in haemolysis by the end of storage were small and probably not of clinical significance.

Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity

PubMed Central

Calow, Jenny; Bockau, Ulrike; Struwe, Weston B.; Nowaczyk, Marc M.; Loser, Karin; Crispin, Max

2016-01-01

ABSTRACT Antibody glycosylation is a key parameter in the optimization of antibody therapeutics. Here, we describe the production of the anti-cancer monoclonal antibody rituximab in the unicellular ciliate, Tetrahymena thermophila. The resulting antibody demonstrated enhanced antibody-dependent cell-mediated cytotoxicity, which we attribute to unusual N-linked glycosylation. Detailed chromatographic and mass spectrometric analysis revealed afucosylated, oligomannose-type glycans, which, as a whole, displayed isomeric structures that deviate from the typical human counterparts, but whose branches were equivalent to fragments of metabolic intermediates observed in human glycoproteins. From the analysis of deposited crystal structures, we predict that the ciliate glycans adopt protein-carbohydrate interactions with the Fc domain that closely mimic those of native complex-type glycans. In addition, terminal glucose structures were identified that match biosynthetic precursors of human glycosylation. Our results suggest that ciliate-based expression systems offer a route to large-scale production of monoclonal antibodies exhibiting glycosylation that imparts enhanced cell killing activity. PMID:27594301

Evaluation of a malaria antibody ELISA and its value in reducing potential wastage of red cell donations from blood donors exposed to malaria, with a note on a case of transfusion-transmitted malaria.

PubMed

Chiodini, P L; Hartley, S; Hewitt, P E; Barbara, J A; Lalloo, K; Bligh, J; Voller, A

1997-01-01

Blood donations are often wasted for lack of a satisfactory procedure to evaluate donors potentially exposed to malaria. We evaluated a commercial ELISA for the detection of antibodies to malaria and compared it with an immunofluorescent antibody test (IFAT). When 5,311 sera from routine non-exposed donors were tested, 24 (0.45%) were positive by the ELISA, using a Plasmodium falciparum antigen. Seventeen were subjected to confirmatory testing but none were positive by IFAT. Of 1,000 donors potentially exposed in endemic areas 15 (1.5%) were repeatably reactive by ELISA. 10 of these were tested by IFAT and 2 were positive. When 150 patients attending the Hospital for Tropical Diseases in London with acute malaria were tested, 73% of those infected with P. falciparum were repeatably reactive for malarial antibodies by ELISA and 56% with Plasmodium vivax. Of 88 stored clinical sera tested by both IFAT and ELISA 56 were positive by IFAT and of these 52 (93 degrees/0) were positive by ELISA. The ELISA is sufficiently sensitive and specific to screen at-risk donors. Its use could safely retrieve 40,000 red cell units currently discarded each year in Great Britain.

Salivary Prostaglandin E2: Role in Tick-Induced Allergy to Red Meat.

PubMed

Cabezas-Cruz, Alejandro; Mateos-Hernández, Lourdes; Chmelař, Jindrǐch; Villar, Margarita; de la Fuente, José

2017-07-01

Tick-induced allergy to red meat is associated with anti-α-Gal IgE antibody levels. We propose that tick salivary prostaglandin E2 triggers antibody class switching in mature B cells, increasing the levels of anti-α-Gal IgE antibodies. Immune tolerance to α-Gal in blood type B individuals might reduce the risk to this allergy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Viscoelastic Transient of Confined Red Blood Cells

PubMed Central

Prado, Gaël; Farutin, Alexander; Misbah, Chaouqi; Bureau, Lionel

2015-01-01

The unique ability of a red blood cell to flow through extremely small microcapillaries depends on the viscoelastic properties of its membrane. Here, we study in vitro the response time upon flow startup exhibited by red blood cells confined into microchannels. We show that the characteristic transient time depends on the imposed flow strength, and that such a dependence gives access to both the effective viscosity and the elastic modulus controlling the temporal response of red cells. A simple theoretical analysis of our experimental data, validated by numerical simulations, further allows us to compute an estimate for the two-dimensional membrane viscosity of red blood cells, ηmem2D ∼ 10−7 N⋅s⋅m−1. By comparing our results with those from previous studies, we discuss and clarify the origin of the discrepancies found in the literature regarding the determination of ηmem2D, and reconcile seemingly conflicting conclusions from previous works. PMID:25954871

Red colored IgG4 caused by vitamin B12 from cell culture media combined with disulfide reduction at harvest

PubMed Central

Derfus, Gayle E; Dizon-Maspat, Jemelle; Broddrick, Jared T; Velayo, Arleene C; Toschi, Josh D; Santuray, Rodell T; Hsu, Stephen K; Winter, Charles M; Krishnan, Rajesh; Amanullah, Ashraf

2014-01-01

While many antibody therapeutics are formulated at low concentration (~10–20 mg/mL) for intravenous administration, high concentration (> 100 mg/mL) formulations may be required for subcutaneous delivery in certain clinical indications. For such high concentration formulations, product color is more apparent due to the higher molecular density across a given path-length. Color is therefore a product quality attribute that must be well-understood and controlled, to demonstrate process consistency and enable clinical trial blinding. Upon concentration of an IgG4 product at the 2000 L manufacturing scale, variability in product color, ranging from yellow to red, was observed. A small-scale experimental model was developed to assess the effect of processing conditions (medium composition and harvest conditions) on final bulk drug substance (BDS) color. The model was used to demonstrate that, for two distinct IgG4 products, red coloration occurred only in the presence of disulfide reduction-mediated antibody dissociation. The red color-causing component was identified as vitamin B12, in the hydroxocobalamin form, and the extent of red color was correlated with the cobalt (vitamin B12) concentration in the final pools. The intensity of redness in the final BDS was modulated by changing the concentration of vitamin B12 in the cell culture media. PMID:24552690

Human anti-CD30 recombinant antibodies by guided phage antibody selection using cell panning

PubMed Central

Klimka, A; Matthey, B; Roovers, R C; Barth, S; Arends, J-W; Engert, A; Hoogenboom, H R

2000-01-01

In various clinical studies, Hodgkin’s patients have been treated with anti-CD30 immunotherapeutic agents and have shown promising responses. One of the problems that appeared from these studies is the development of an immune response against the non-human therapeutics, which limits repeated administration and reduces efficacy. We have set out to make a recombinant, human anti-CD30 single-chain variable fragment (scFv) antibody, which may serve as a targeting moiety with reduced immunogenicity and more rapid tumour penetration in similar clinical applications. Rather than selecting a naive phage antibody library on recombinant CD30 antigen, we used guided selection of a murine antibody in combination with panning on the CD30-positive cell line L540. The murine monoclonal antibody Ki-4 was chosen as starting antibody, because it inhibits the shedding of the extracellular part of the CD30 antigen. This makes the antibody better suited for CD30-targeting than most other anti-CD30 antibodies. We have previously isolated the murine Ki-4 scFv by selecting a mini-library of hybridoma-derived phage scFv-antibodies via panning on L540 cells. Here, we report that phage display technology was successfully used to obtain a human Ki-4 scFv version by guided selection. The murine variable heavy (VH) and light (VL) chain genes of the Ki-4 scFv were sequentially replaced by human V gene repertoires, while retaining only the major determinant for epitope-specificity: the heavy-chain complementarity determining region 3 (CDR3) of murine Ki-4. After two rounds of chain shuffling and selection by panning on L540 cells, a fully human anti-CD30 scFv was selected. It competes with the parental monoclonal antibody Ki-4 for binding to CD30, inhibits the shedding of the extracellular part of the CD30 receptor from L540 cells and is thus a promising candidate for the generation of anti-CD30 immunotherapeutics. © 2000 Cancer Research Campaign PMID:10901379

Antibody responses of red wolves to canine distemper virus and canine parvovirus vaccination.

PubMed

Harrenstien, L A; Munson, L; Ramsay, E C; Lucash, C F; Kania, S A; Potgieter, L N

1997-07-01

Twenty captive red wolves (Canis rufus), including 16 intended for release into Great Smoky Mountains National Park, Cades Cove, Tennessee (USA), and four housed at Knoxville Zoological Gardens, Inc., Knoxville, Tennessee, were evaluated for immunologic response to vaccination between June 1994 and April 1995. Wolves were vaccinated with modified-live (MLV) canine distemper virus (CDV) and canine parvovirus type-2 (CPV2). Sera were collected, and immunofluorescent staining was performed for determination of immunoglobulin titers (CDV IgM, CDV IgG, and CPV2 IgG). A capture enzyme-linked immunosorbent assay was performed for validation purposes, to confirm the reactivity of our standard diagnostic reagents with red wolf serum. All wolves produced a measurable antibody response to CDV and CPV2 vaccination. Titers against CDV and CPV2 varied widely among individual wolves, but between-litter differences in mean titers were not significant. No consistent response between the degree of response to CDV versus CPV2 vaccination was observed in individual wolves. No differences were seen between IgG responses of pups vaccinated with univalent vaccines given concurrently or during alternating weeks. Pups had an IgG response to CDV and CPV2 vaccination as early as 9 wk of age. Mean post-vaccination IgG titers against CDV were at or above the level normally measured in vaccinated domestic dogs. Mean post-vaccination IgG titers against CPV2 were below the level normally measured in domestic dogs. Adult previously-vaccinated wolves had measurable CDV and CPV2 IgG titers more than 1 yr after vaccination, but did not have significant IgG titer increases after revaccination. We conclude that red wolves are capable of producing an antibody response after vaccination with commercial canine products but that their response to CPV2 vaccination was minimal. This response can be assayed using tests developed for domestic dogs.

CD8+ T Cells and IFN-γ Mediate the Time-Dependent Accumulation of Infected Red Blood Cells in Deep Organs during Experimental Cerebral Malaria

PubMed Central

Claser, Carla; Malleret, Benoît; Gun, Sin Yee; Wong, Alicia Yoke Wei; Chang, Zi Wei; Teo, Pearline; See, Peter Chi Ee; Howland, Shanshan Wu; Ginhoux, Florent; Rénia, Laurent

2011-01-01

Background Infection with Plasmodium berghei ANKA (PbA) in susceptible mice induces a syndrome called experimental cerebral malaria (ECM) with severe pathologies occurring in various mouse organs. Immune mediators such as T cells or cytokines have been implicated in the pathogenesis of ECM. Red blood cells infected with PbA parasites have been shown to accumulate in the brain and other tissues during infection. This accumulation is thought to be involved in PbA–induced pathologies, which mechanisms are poorly understood. Methods and Findings Using transgenic PbA parasites expressing the luciferase protein, we have assessed by real-time in vivo imaging the dynamic and temporal contribution of different immune factors in infected red blood cell (IRBC) accumulation and distribution in different organs during PbA infection. Using deficient mice or depleting antibodies, we observed that CD8+ T cells and IFN-γ drive the rapid increase in total parasite biomass and accumulation of IRBC in the brain and in different organs 6–12 days post-infection, at a time when mice develop ECM. Other cells types like CD4+ T cells, monocytes or neutrophils or cytokines such as IL-12 and TNF-α did not influence the early increase of total parasite biomass and IRBC accumulation in different organs. Conclusions CD8+ T cells and IFN-γ are the major immune mediators controlling the time-dependent accumulation of P. berghei-infected red blood cells in tissues. PMID:21494565

Effects of helicopter transport on red blood cell components

PubMed Central

Otani, Taiichi; Oki, Ken-ichi; Akino, Mitsuaki; Tamura, Satoru; Naito, Yuki; Homma, Chihiro; Ikeda, Hisami; Sumita, Shinzou

2012-01-01

Background There are no reported studies on whether a helicopter flight affects the quality and shelf-life of red blood cells stored in mannitol-adenine-phosphate. Materials and methods Seven days after donation, five aliquots of red blood cells from five donors were packed into an SS-BOX-110 container which can maintain the temperature inside the container between 2 °C and 6 °C with two frozen coolants. The temperature of an included dummy blood bag was monitored. After the box had been transported in a helicopter for 4 hours, the red blood cells were stored again and their quality evaluated at day 7 (just after the flight), 14, 21 and 42 after donation. Red blood cell quality was evaluated by measuring adenosine triphosphate, 2,3-diphosphoglycerate, and supernatant potassium, as well as haematocrit, intracellular pH, glucose, supernatant haemoglobin, and haemolysis rate at the various time points. Results During the experiment the recorded temperature remained between 2 and 6 °C. All data from the red blood cells that had undergone helicopter transportation were the same as those from a control group of red blood cell samples 7 (just after the flight), 14, 21, and 42 days after the donation. Only supernatant Hb and haemolysis rate 42 days after the donation were slightly increased in the helicopter-transported group of red blood cell samples. All other parameters at 42 days after donation were the same in the two groups of red blood cells. Discussion These results suggest that red blood cells stored in mannitol-adenine-phosphate are not significantly affected by helicopter transportation. The differences in haemolysis by the end of storage were small and probably not of clinical significance. PMID:22153688

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in...

Limited value of testing for intrinsic factor antibodies with negative gastric parietal cell antibodies in pernicious anaemia.

PubMed

Khan, S; Del-Duca, C; Fenton, E; Holding, S; Hirst, J; Doré, P C; Sewell, W A C

2009-05-01

The appropriate testing strategy for diagnosing pernicious anaemia using gastric parietal cell (GPC) and/or intrinsic factor antibodies (IFA) is controversial. Intrinsic factor antibodies are found in only about 70% of cases. Indirect immunofluorescence screening for gastric parietal cell antibodies is more sensitive, labour intensive, and less specific. The frequency of antibody positivity (IFA and/or GPC) was retrospectively examined in patients tested for both autoantibodies over a three-year period. It was investigated whether B12 levels were related to antibody status. These findings were validated in a prospective study of IFA in 91 GPC negative patients with low B12 levels. Of 847 samples identified in the retrospective study, 4 (0.47%) were positive for only intrinsic factor antibodies, 731 (86.3%) positive for GPC alone, and 112 (13.2%) for both. Student t test on log-transformed data showed B12 levels had no bearing on autoantibody status. 91 consecutive patients with low B12 levels were tested for both autoantibodies; all were negative for gastric parietal cell antibodies. Only one sample was positive for intrinsic factor antibody using the porcine intrinsic factor assay, but was negative by a human recombinant intrinsic factor-based ELISA. This study provides evidence that testing for gastric parietal cell antibodies is an appropriate screening test for pernicious anaemia, with intrinsic factor antibodies reserved for confirmatory testing or in patients with other autoantibodies that mask the GPC pattern; B12 levels are not related to autoantibody status.

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Red blood cell enzyme assay. 864.7100 Section 864... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects...

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2014 CFR

2014-04-01

... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2013 CFR

2013-04-01

... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...

21 CFR 864.7100 - Red blood cell enzyme assay.

Code of Federal Regulations, 2012 CFR

2012-04-01

... enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in... kinase or 2,3-diphosphoglycerate. A red blood cell enzyme assay is used to determine the enzyme defects... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...

Silane-modified surfaces in specific antibody-mediated cell recognition.

PubMed

Sterzynska, Karolina; Budna, Joanna; Frydrych-Tomczak, Emilia; Hreczycho, Grzegorz; Malinska, Agnieszka; Maciejewski, Hieronim; Zabel, Maciej

2014-01-01

The immobilization of antibodies on various surfaces has been the subject of advanced research in various immunoassay-based diagnostic devices. The physical and chemical stabilities of the immobilized antibodies on a solid surface still cause many problems because upon immobilizing antibody molecules, the antigen-binding ability usually decreases. The silanization of surfaces with organosilanes carrying chemically active groups such as (3-aminopropyl) triethoxysilane (APTES) can accommodate these antigen-binding molecules in an appropriate orientation so that their functionality and binding activity are essentially retained. In this study, n-butyltrimethoxysilane (BMS) and 3-(octafluoropentyloxy)-propyltriethoxysilane (OFPOS) were used as "blocking silanes". The aims of this study were to compare the effectiveness of specific antibody binding of APTES, APTES + BMS and APTES + OFPOS and to characterize the modified surfaces by contact angle measurements and immunofluorescence measurements prior to and after immobilizing proteins. Additionally, we have evaluated the functionality of the immobilized antibodies by their abilities to bind EpCAM-positive human colon adenocarcinoma cell line (LoVo) and EpCAM-negative mouse embryonic fibroblast cell line (3T3). Cell enumeration was conducted on the basis of DAPI-positive signals and recorded using a confocal laser scanning biological microscope. The results of our study showed that the immobilization capability and reactivity of APTES, APTES + BMS and APTES + OFPOS differ. The modification of APTES with unreactive silanes (BMS,OFPOS) is recommended to improve the antibody binding efficiency. However, using OFPOS resulted in more effective antibody and cell binding, and it appears to be the most useful compound in specific antibody-mediated cell recognition.

Trypanosoma cruzi infection induces a massive extrafollicular and follicular splenic B-cell response which is a high source of non-parasite-specific antibodies.

PubMed

Bermejo, Daniela A; Amezcua Vesely, María C; Khan, Mahmood; Acosta Rodríguez, Eva V; Montes, Carolina L; Merino, Maria C; Toellner, Kai Michael; Mohr, Elodie; Taylor, Dale; Cunningham, Adam F; Gruppi, Adriana

2011-01-01

Acute infection with Trypanosoma cruzi, the aetiological agent of Chagas' disease, results in parasitaemia and polyclonal lymphocyte activation. It has been reported that polyclonal B-cell activation is associated with hypergammaglobulinaemia and delayed parasite-specific antibody response. In the present study we analysed the development of a B-cell response within the different microenvironments of the spleen during acute T. cruzi infection. We observed massive germinal centre (GC) and extrafollicular (EF) responses at the peak of infection. However, the EF foci were evident since day 3 post-infection (p.i.), and, early in the infection, they mainly provided IgM. The EF foci response reached its peak at 11 days p.i. and extended from the red pulp into the periarteriolar lymphatic sheath. The GCs were detected from day 8 p.i. At the peak of parasitaemia, CD138(+) B220(+) plasma cells in EF foci, red pulp and T-cell zone expressed IgM and all the IgG isotypes. Instead of the substantial B-cell response, most of the antibodies produced by splenic cells did not target the parasite, and parasite-specific IgG isotypes could be detected in sera only after 18 days p.i. We also observed that the bone marrow of infected mice presented a strong reduction in CD138(+) B220(+) cells compared with that of normal mice. Hence, in acute infection with T. cruzi, the spleen appears to be the most important lymphoid organ that lodges plasma cells and the main producer of antibodies. The development of a B-cell response during T. cruzi infection shows features that are particular to T. cruzi and other protozoan infection but different to other infections or immunization with model antigens.

Interaction between phloretin and the red blood cell membrane

PubMed Central

1976-01-01

Phloretin binding to red blood cell components has been characterized at pH6, where binding and inhibitory potency are maximal. Binding to intact red cells and to purified hemoglobin are nonsaturated processes approximately equal in magnitude, which strongly suggests that most of the red cell binding may be ascribed to hemoglobin. This conclusion is supported by the fact that homoglobin-free red cell ghosts can bind only 10% as much phloretin as an equivalent number of red cells. The permeability of the red cell membrane to phloretin has been determined by a direct measurement at the time-course of the phloretin uptake. At a 2% hematocrit, the half time for phloretin uptake is 8.7s, corresponding to a permeability coefficient of 2 x 10(-4) cm/s. The concentration dependence of the binding to ghosts reveals two saturable components. Phloretin binds with high affinity (K diss = 1.5 muM) to about 2.5 x 10(6) sites per cell; it also binds with lower affinity (Kdiss = 54 muM) to a second (5.5 x 10(7) per cell) set of sites. In sonicated total lipid extracts of red cell ghosts, phloretin binding consists of a single, saturable component. Its affinity and total number of sites are not significantly different from those of the low affinity binding process in ghosts. No high affinity binding of phloretin is exhibited by the red cell lipid extracts. Therefore, the high affinity phloretin binding sites are related to membrane proteins, and the low affinity sites result from phloretin binding to lipid. The identification of these two types of binding sites allows phloretin effects on protein-mediated transport processes to be distinguished from effects on the lipid region of the membrane. PMID:5575

Sickle red cell adhesion: many issues and some answers.

PubMed

Kaul, D K

2008-01-01

Among multiple pathologies associated with sickle cell disease, sickle red cell-endothelial interaction has been implicated as a potential initiating mechanism in vaso-occlusive events that characterize this disease. Vast literature exists on various aspects of sickle red cell adhesion, but many issues remain unresolved, especially pertaining to the role of sickle red cell heterogeneity, the relative role of multiple adhesion mechanisms and targets of antiadhesive therapy. This review briefly analyzes these issues.

A droplet-based heterogeneous immunoassay for screening single cells secreting antigen-specific antibodies.

PubMed

Akbari, Samin; Pirbodaghi, Tohid

2014-09-07

High throughput heterogeneous immunoassays that screen antigen-specific antibody secreting cells are essential to accelerate monoclonal antibody discovery for therapeutic applications. Here, we introduce a heterogeneous single cell immunoassay based on alginate microparticles as permeable cell culture chambers. Using a microfluidic device, we encapsulated single antibody secreting cells in 35-40 μm diameter alginate microbeads. We functionalized the alginate to capture the secreted antibodies inside the microparticles, enabling single cell analysis and preventing the cross-talk between the neighboring encapsulated cells. We demonstrated non-covalent functionalization of alginate microparticles by adding three secondary antibodies to the alginate solution to form high molecular weight complexes that become trapped in the porous nanostructure of alginate and capture the secreted antibodies. We screened anti-TNF-alpha antibody-secreting cells from a mixture of antibody-secreting cells.

The hSK4 (KCNN4) isoform is the Ca2+-activated K+ channel (Gardos channel) in human red blood cells.

PubMed

Hoffman, Joseph F; Joiner, William; Nehrke, Keith; Potapova, Olga; Foye, Kristen; Wickrema, Amittha

2003-06-10

The question is, does the isoform hSK4, also designated KCNN4, represent the small conductance, Ca2+-activated K+ channel (Gardos channel) in human red blood cells? We have analyzed human reticulocyte RNA by RT-PCR, and, of the four isoforms of SK channels known, only SK4 was found. Northern blot analysis of purified and synchronously growing human erythroid progenitor cells, differentiating from erythroblasts to reticulocytes, again showed only the presence of SK4. Western blot analysis, with an anti-SK4 antibody, showed that human erythroid progenitor cells and, importantly, mature human red blood cell ghost membranes, both expressed the SK4 protein. The Gardos channel is known to turn on, given inside Ca2+, in the presence but not the absence of external Ko+ and remains refractory to Ko+ added after exposure to inside Ca2+. Heterologously expressed SK4, but not SK3, also shows this behavior. In inside-out patches of red cell membranes, the open probability (Po) of the Gardos channel is markedly reduced when the temperature is raised from 27 to 37 degrees C. Net K+ efflux of intact red cells is also reduced by increasing temperature, as are the Po values of inside-out patches of Chinese hamster ovary cells expressing SK4 (but not SK3). Thus the envelope of evidence indicates that SK4 is the gene that codes for the Gardos channel in human red blood cells. This channel is important pathophysiologically, because it represents the major pathway for cell shrinkage via KCl and water loss that occurs in sickle cell disease.

Rheologic and hemodynamic characteristics of red cells of mouse, rat and human.

PubMed

Chen, D; Kaul, D K

1994-01-01

The present study compares hematologic, rheologic and hemodynamic characteristics of red cells from mouse, rat and human. Red cells in these species are biconcave discs that show significant differences in diameter and mean corpuscular volume (MCV). However, differences in mean corpuscular hemoglobin concentration (MCHC) are not significant. Viscosity measurement of washed red cell suspensions (in each case the medium osmolarity adjusted to match plasma osmolarity) showed significant interspecies differences at shear rates of 37.5 and 750 sec-1 as follows: Human > rat > mouse. Hemodynamic and microcirculatory behavior of these red cells was investigated in the artificially perfused ex vivo mesocecum vasculature of the rat. Hemodynamic measurements in the whole ex vivo mesocecum preparation revealed maximal increase in the peripheral resistance unit (PRU) for the human red cells followed by the rat and mouse red cells, respectively at a hematocrit (Hct) of 40%. Further, measurements of red cell velocities (Vrbc) in single arterioles of the mesocecum vasculature, during sustained perfusion with washed red cell suspensions, showed that at any given perfusion pressure (Pa), Vrbc for both mouse and rat red cells was higher than that for human red cells, while Vrbc for mouse red cells was higher than that for the rat. These results demonstrate that the microvascular flow behavior of these red cells is likely to be influenced by both physical and rheologic characteristics.

Rapid Isolation of Antibody from a Synthetic Human Antibody Library by Repeated Fluorescence-Activated Cell Sorting (FACS)

PubMed Central

Yim, Sung Sun; Bang, Hyun Bae; Kim, Young Hwan; Lee, Yong Jae; Jeong, Gu Min; Jeong, Ki Jun

2014-01-01

Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS). First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv) was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show KD values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼106). These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required. PMID:25303314

Facts and Fallacies of Kidd Antibodies: Experience in a Tertiary Care Hospital in North India.

PubMed

Makroo, R N; Nayak, Sweta; Chowdhry, Mohit; Karna, Prashant

2017-06-01

We have analyzed the method used in our laboratory to detect the most elusive, clinically significant alloantibody: the Kidd alloantibodies and find the most convenient procedure. A retrospective analysis of the method used in our laboratory for determining Kidd alloantibodies from January 2013 to May 2015 was conducted. The details of the event that sensitized the patient for red cell antibody formation and procedure used to detect the alloantibody were retrieved from the departmental records. Of 405 red cell antibody identification cases, 24 (5.9 %) had Kidd antibody (anti-Jka in 12: 50 % cases; anti-Jkb in 4: 16.7 % cases; multiple antibodies in 8: 32 % cases). Thirteen of 24 patients (54.2 %) had autocontrol positive of which 6 cases needed adsorption procedures whereas antibody/ies could be identified without adsorption procedure in the remaining 7 cases. All the 7 cases had autocontrol of 1+ strength. Of the 11 patients (45.8 %) with autocontrol negative, the antibody was identified using solid phase in 7 cases whereas tube panels were also used in the remaining 4 cases. Kidd alloantibodies though deceptive can be identified by sensitive techniques like the solid phase and simple but laborious techniques using the tube cell panels. Depending upon the reaction strength of the autocontrol, the routine autoadsorption process may be skipped and tube cell enzyme treated cells or solid phase techniques be used to get the results.

Profiling antibody responses by multiparametric analysis of primary B cells

PubMed Central

Story, Craig M.; Papa, Eliseo; Hu, Chih-Chi Andrew; Ronan, Jehnna L.; Herlihy, Kara; Ploegh, Hidde L.; Love, J. Christopher

2008-01-01

Determining the efficacy of a vaccine generally relies on measuring neutralizing antibodies in sera. This measure cannot elucidate the mechanisms responsible for the development of immunological memory at the cellular level, however. Quantitative profiles that detail the cellular origin, extent, and diversity of the humoral (antibody-based) immune response would improve both the assessment and development of vaccines. Here, we describe a novel approach to collect multiparametric datasets that describe the specificity, isotype, and apparent affinity of the antibodies secreted from large numbers of individual primary B cells (≈103-104). The antibody/antigen binding curves obtained by this approach can be used to classify closely related populations of cells using algorithms for data clustering, and the relationships among populations can be visualized graphically using affinity heatmaps. The technique described was used to evaluate the diversity of antigen-specific antibody-secreting cells generated during an in vivo humoral response to a series of immunizations designed to mimic a multipart vaccination. Profiles correlating primary antibody-producing cells with the molecular characteristics of their secreted antibodies should facilitate both the evaluation of candidate vaccines and, broadly, studies on the repertoires of antibodies generated in response to infectious or autoimmune diseases. PMID:19004776

Patient-Derived Antibody Targets Tumor Cells

Cancer.gov

An NCI Cancer Currents blog on an antibody derived from patients that killed tumor cells in cell lines of several cancer types and slowed tumor growth in mouse models of brain and lung cancer without evidence of side effects.

Selecting antagonistic antibodies that control differentiation through inducible expression in embryonic stem cells

PubMed Central

Melidoni, Anna N.; Dyson, Michael R.; Wormald, Sam; McCafferty, John

2013-01-01

Antibodies that modulate receptor function have great untapped potential in the control of stem cell differentiation. In contrast to many natural ligands, antibodies are stable, exquisitely specific, and are unaffected by the regulatory mechanisms that act on natural ligands. Here we describe an innovative system for identifying such antibodies by introducing and expressing antibody gene populations in ES cells. Following induced antibody expression and secretion, changes in differentiation outcomes of individual antibody-expressing ES clones are monitored using lineage-specific gene expression to identify clones that encode and express signal-modifying antibodies. This in-cell expression and reporting system was exemplified by generating blocking antibodies to FGF4 and its receptor FGFR1β, identified through delayed onset of ES cell differentiation. Functionality of the selected antibodies was confirmed by addition of exogenous antibodies to three different ES reporter cell lines, where retained expression of pluripotency markers Oct4, Nanog, and Rex1 was observed. This work demonstrates the potential for discovery and utility of functional antibodies in stem cell differentiation. This work is also unique in constituting an example of ES cells carrying an inducible antibody that causes a functional protein “knock-down” and allows temporal control of stable signaling components at the protein level. PMID:24082130

[Correlation between red blood cell count and liver function status].

PubMed

Xie, Xiaomeng; Wang, Leijie; Yao, Mingjie; Wen, Xiajie; Chen, Xiangmei; You, Hong; Jia, Jidong; Zhao, Jingmin; Lu, Fengmin

2016-02-01

To investigate the changes in red blood cell count in patients with different liver diseases and the correlation between red blood cell count and degree of liver damage. The clinical data of 1427 patients with primary liver cancer, 172 patients with liver cirrhosis, and 185 patients with hepatitis were collected, and the Child-Pugh class was determined for all patients. The differences in red blood cell count between patients with different liver diseases were retrospectively analyzed, and the correlation between red blood cell count and liver function status was investigated. The Mann-Whitney U test, Kruskal-Wallis H test, rank sum test, Spearman rank sum correlation test, and chi-square test were performed for different types of data. Red blood cell count showed significant differences between patients with chronic hepatitis, liver cancer, and liver cirrhosis and was highest in patients with chronic hepatitis and lowest in patients with liver cirrhosis (P < 0.05). In the patients with liver cirrhosis, red blood cell count tended to decrease in patients with a higher Child-Pugh class (P < 0.05). For patients with liver cirrhosis, red blood cell count can reflect the degree of liver damage, which may contribute to an improved liver function prediction model for these patients.

Genomic Typing of Red Cell Antigens

DTIC Science & Technology

2011-09-01

Antigen‐Matched  Red  Cells  for  Sickle  Cell  Anemia   Patients  Using  Molecular Typing to Augment Testing: Meghan Delaney, Prashant Gaur, Askale...Antigen‐Matched Red Cells for Sickle Cell  Anemia  Patients  Using Molecular Typing to Augment Testing: AABB (poster) 2009.  Background: Patients with sickle...not used. Delivery of a  healthy female  neonate  was uneventful. The serologic studies showed the mother and baby’s  phenotypes as O and AB

Accelerated removal of antibody-coated red blood cells from the circulation is accurately tracked by a biotin label

PubMed Central

Mock, Donald M.; Lankford, Gary L.; Matthews, Nell I.; Burmeister, Leon F.; Kahn, Daniel; Widness, John A.; Strauss, Ronald G.

2013-01-01

BACKGROUND Safe, accurate methods to reliably measure circulating red blood cell (RBC) kinetics are critical tools to investigate pathophysiology and therapy of anemia, including hemolytic anemias. This study documents the ability of a method using biotin-labeled RBCs (BioRBCs) to measure RBC survival (RCS) shortened by coating with a highly purified monomeric immunoglobulin G antibody to D antigen. STUDY DESIGN AND METHODS Autologous RBCs from 10 healthy D+ subjects were labeled with either biotin or 51Cr (reference method), coated (opsonized) either lightly (n = 4) or heavily (n = 6) with anti-D, and transfused. RCS was determined for BioRBCs and for 51Cr independently as assessed by three variables: 1) posttransfusion recovery at 24 hours (PTR24) for short-term RCS; 2) time to 50% decrease of the label (T50), and 3) mean potential life span (MPL) for long-term RCS. RESULTS BioRBCs tracked both normal and shortened RCS accurately relative to 51Cr. For lightly coated RBCs, mean PTR24, T50, and MPL results were not different between BioRBCs and 51Cr. For heavily coated RBCs, both short-term and long-term RCS were shortened by approximately 17 and 50%, respectively. Mean PTR24 by BioRBCs (84 ± 18%) was not different from 51Cr (81 ± 10%); mean T50 by BioRBCs (23 ± 17 days) was not different from 51Cr (22 ± 18 days). CONCLUSION RCS shortened by coating with anti-D can be accurately measured by BioRBCs. We speculate that BioRBCs will be useful for studying RCS in conditions involving accelerated removal of RBCs including allo- and autoimmune hemolytic anemias. PMID:22023312

Red blood cell-derived microparticles: An overview.

PubMed

Westerman, Maxwell; Porter, John B

2016-07-01

The red blood cell (RBC) is historically the original parent cell of microparticles (MPs). In this overview, we describe the discovery and the early history of red cell-derived microparticles (RMPs) and present an overview of the evolution of RMP. We report the formation, characteristics, effects of RMP and factors which may affect RMP evaluation. The review examines RMP derived from both normal and pathologic RBC. The pathologic RBC studies include sickle cell anemia (SCA), sickle cell trait (STr), thalassemia intermedia (TI), hereditary spherocytosis (HS), hereditary elliptocytosis (HE), hereditary stomatocytosis (HSt) and glucose-6-phosphate dehydrogenase deficiency (G6PD). Copyright © 2016 Elsevier Inc. All rights reserved.

In vivo regeneration of red cell 2,3-diphosphoglycerate following transfusion of DPG-depleted AS-1, AS-3 and CPDA-1 red cells.

PubMed

Heaton, A; Keegan, T; Holme, S

1989-01-01

Regeneration of 2,3-diphosphoglycerate (DPG) was determined following transfusion of DPG-depleted group O red cells into group A recipients. Blood from five donors was stored in the adenine-containing solutions CPDA-1, AS-1 or AS-3 for 35 d at 4 degrees C. Post-transfusion red cell DPG and ATP were measured in separated group O red cells over a 7 d period. The studies confirmed rapid in vivo DPG regeneration with greater than or equal to 50% of the maximum level being achieved within 7 h. An average of 95% of the recipients' pre-transfusion DPG level was achieved by 72 h and by 7 d mean (+/- SEM) DPG levels relative to recipient's pre-transfusion DPG averaged 84% (+/- 13%), 92% (+/- 17%) and 84% (+/- 21%) for CPDA-1, AS-1 and AS-3 red cells, respectively. Results were comparable to those previously reported for blood stored in ACD for 15-20 d (Valeri & Hirsch, 1969; Beutler & Wood, 1969). The immediate regeneration rate, V, closely approximated first order regeneration kinetics with AS-3 red cells exhibiting double the rate of CPDA-1 red cells (P less than 0.001). AS-1 red cells exhibited an intermediate rate of regeneration which was not significantly different compared to either CPDA-1 or AS-3 (P greater than 0.05). V exhibited a significant (P less than 0.05) positive correlation with ATP levels 5-7 h post-infusion. ATP regeneration of the infused cells was rapid with a mean increase of 1.2 mumol/g Hb above post-storage levels being achieved 1 h following transfusion.

Antibody-targeted interleukin 2 stimulates T-cell killing of autologous tumor cells.

PubMed Central

Gillies, S D; Reilly, E B; Lo, K M; Reisfeld, R A

1992-01-01

A genetically engineered fusion protein consisting of a chimeric anti-ganglioside GD2 antibody (ch14.18) and interleukin 2 (IL2) was tested for its ability to enhance the killing of autologous GD2-expressing melanoma target cells by a tumor-infiltrating lymphocyte line (660 TIL). The fusion of IL2 to the carboxyl terminus of the immunoglobulin heavy chain did not reduce IL2 activity as measured in a standard proliferation assay using either mouse or human T-cell lines. Antigen-binding activity was greater than that of the native chimeric antibody. The ability of resting 660 TIL cells to kill their autologous GD2-positive target cells was enhanced if the target cells were first coated with the fusion protein. This stimulation of killing was greater than that of uncoated cells in the presence of equivalent or higher concentrations of free IL2. Such antibody-cytokine fusion proteins may prove useful in targeting the biological effect of IL2 and other cytokines to tumor cells and in this way stimulate their immune destruction. Images PMID:1741398

Cryopreservation of red blood cells.

PubMed

Lagerberg, Johan W

2015-01-01

Cryopreservation of red blood cell concentrates (RBCs) is an important method for maintaining an inventory of rare RBC units and managing special transfusion circumstances. The permeating additive glycerol is used as a cryoprotectant to protect RBCs against freezing damage. The use of thawed RBCs was hampered a 24-h outdating period due to potential bacterial contamination when a functionally open system was used for addition and removal of the glycerol. With the introduction of a functionally closed system for the glycerolization and deglycerolization of RBC units, extended post-thaw storage became possible. Here, we describe the cryopreservation of red blood cells according to the high-glycerol method, using a functionally closed processing system.

Red cell metabolism studies on Skylab

NASA Technical Reports Server (NTRS)

Mengel, C. E.

1974-01-01

On the basis of these background data, metabolic studies were performed on humans involved in space flight. These studies included the Skylab experiences. The primary purpose of the investigations was to study red cells for: (1) evidences of lipid peroxidation, or (2) changes at various points in the glycolytic pathway. The Skylab missions were an opportunity to study blood samples before, during, and after flight and to compare results with simultaneous controls. No direct evidence that lipid peroxidation had occurred in the red blood cells was apparent in the studies.

Red blood cells aligning inside innovative liquid crystal cell

NASA Astrophysics Data System (ADS)

Likhomanova, S. V.; Kamanin, A. A.; Kamanina, N. V.

2017-11-01

Investigation results of red blood cells (human erythrocytes) aligning and fixing inside the liquid crystal (LC) cell have been presented in the present paper. LC cells have been modified through the improved nanostructured relief and LC sensitized with intermolecular charge transfer complex COANP-C70.

New Developments in Red Blood Cell Preservation Using Liquid and Freezing Procedures.

DTIC Science & Technology

1982-04-02

restore or improve the red cell 2,3 DPG and ATP levels . Biochemically modified red blood cells may be cryopreserved for indefinite storage, or they may...salvage outdated red blood cells. However,,-ndated red blood cells are also being biochemically modified to increase’the 2,3 DPG levels to 2 to 3...restore or improve the edcell 2,3 DPG and ATP levels . Biochemically modified red blood cells iay-be cryopreserved for indefinite storage. or-thy my be

Effect of reconstructive vascular surgery on red cell deformability--preliminary results.

PubMed Central

Irwin, S T; Rocks, M J; McGuigan, J A; Patterson, C C; Morris, T C; O'Reilly, M J

1983-01-01

Using a simple filtration method, red cell deformability was measured in healthy control subjects and in patients with peripheral vascular disease. Impaired red cell deformability was demonstrated in patients with rest pain or gangrene and in patients with intermittent claudication. An improvement in red cell deformability was demonstrated after successful reconstructive vascular surgery in both patient groups. An improvement in red cell deformability was demonstrated in patients undergoing major limb amputation. PMID:6619311

Quantification of the fraction poorly deformable red blood cells using ektacytometry.

PubMed

Streekstra, G J; Dobbe, J G G; Hoekstra, A G

2010-06-21

We describe a method to obtain the fraction of poorly deformable red blood cells in a blood sample from the intensity pattern in an ektacytometer. In an ektacytometer red blood cells are transformed into ellipsoids by a shear flow between two transparent cylinders. The intensity pattern, due to a laser beam that is sent through the suspension, is projected on a screen. When measuring a healthy red blood cell population iso-intensity curves are ellipses with an axial ratio equal to that of the average red blood cell. In contrast poorly deformable cells result in circular iso-intensity curves. In this study we show that for mixtures of deformable and poorly deformable red blood cells, iso-intensity curves in the composite intensity pattern are neither elliptical nor circular but obtain cross-like shapes. We propose a method to obtain the fraction of poorly deformable red blood cells from those intensity patterns. Experiments with mixtures of poorly deformable and deformable red blood cells validate the method and demonstrate its accuracy. In a clinical setting our approach is potentially of great value for the detection of the fraction of sickle cells in blood samples of patients with sickle cell disease or to find a measure for the parasitemia in patients infected with malaria.

Antibody-Dependent Cell-Mediated Cytotoxicity Effector-Enhanced EphA2 Agonist Monoclonal Antibody Demonstrates Potent Activity against Human Tumors1

PubMed Central

Bruckheimer, Elizabeth M; Fazenbaker, Christine A; Gallagher, Sandra; Mulgrew, Kathy; Fuhrmann, Stacy; Coffman, Karen T; Walsh, William; Ready, Shannon; Cook, Kim; Damschroder, Melissa; Kinch, Michael; Kiener, Peter A; Woods, Rob; Gao, Changshou; Dall'Acqua, William; Wu, Herren; Coats, Steven

2009-01-01

EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated ∼80% tumor cell killing. In a dose-dependent manner, natural killer (NK) cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID) mice (which have functional NK cells and monocytes) and SCID nonobese diabetic (NOD) mice (which largely lack functional NK cells and monocytes). Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells. PMID:19484140

Droplet based microfluidics for highthroughput screening of antibody secreting cells

NASA Astrophysics Data System (ADS)

Cai, Liheng; Heyman, John; Mazutis, Linas; Ung, Lloyd; Guerra, Rodrigo; Aubrecht, Donald; Weitz, David

2014-03-01

We present a droplet based microfluidic platform that allows highthroughput screening of antibody secreting cells. We coencapsulate single cells, fluorescent probes, and detection beads into emulsion droplets with diameter of 40 micron. The beads capture antibodies secreted by cells, resulting in a pronounced fluorescent signal that activates dielectrophoresis sorting at rate about 500 droplets per second. Moreover, we demonstrate that Reverse Transcription Polymerase Chain Reaction (RT-PCR) can be successfully applied to the cell encapsulated in a single sorted droplet. Our work highlights the potential of droplet based microfluidics as a platform to generate recombinant antibodies.

A monoclonal antibody that recognizes B cells and B cell precursors in mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coffman, R.L.; Weissman, I.L.

1981-02-01

The monoclonal antibody, RA3-2C2, appears to be specific for cells within the B cell lineage. This antibody does not recognize thymocytes, peripheral T cells, or nonlymphoid hematopoietic cells in the spleen or bone marrow. Nor does it recognize the pluripotent hematopoietic stem cells, the spleen colony-forming unit, All sIg+ B cells and most plasma cells are RA3-2C2+. In addition, approximately 20% of nucleated bone marrow cells are RA3-2C2+ but sIg-. This population contains B cell precursors that can give rise to sIg+ cells within 2 d in vitro.

Neuronal uptake of anti-Hu antibody, but not anti-Ri antibody, leads to cell death in brain slice cultures.

PubMed

Greenlee, John E; Clawson, Susan A; Hill, Kenneth E; Wood, Blair; Clardy, Stacey L; Tsunoda, Ikuo; Jaskowski, Troy D; Carlson, Noel G

2014-09-17

Anti-Hu and anti-Ri antibodies are paraneoplastic immunoglobulin (Ig)G autoantibodies which recognize cytoplasmic and nuclear antigens present in all neurons. Although both antibodies produce similar immunohistological labeling, they recognize different neuronal proteins. Both antibodies are associated with syndromes of central nervous system dysfunction. However, the neurological deficits associated with anti-Hu antibody are associated with neuronal death and are usually irreversible, whereas neurological deficits in patients with anti-Ri antibody may diminish following tumor removal or immunosuppression. To study the effect of anti-Hu and anti-Ri antibodies on neurons, we incubated rat hippocampal and cerebellar slice cultures with anti-Hu or anti-Ri sera from multiple patients. Cultures were evaluated in real time for neuronal antibody uptake and during prolonged incubation for neuronal death. To test the specificity of anti-Hu antibody cytotoxic effect, anti-Hu serum IgG was incubated with rat brain slice cultures prior to and after adsorption with its target Hu antigen, HuD. We demonstrated that: 1) both anti-Hu and anti-Ri antibodies were rapidly taken up by neurons throughout both cerebellum and hippocampus; 2) antibody uptake occurred in living neurons and was not an artifact of antibody diffusion into dead cells; 3) intracellular binding of anti-Hu antibody produced neuronal cell death, whereas uptake of anti-Ri antibody did not affect cell viability during the period of study; and 4) adsorption of anti-Hu antisera against HuD greatly reduced intraneuronal IgG accumulation and abolished cytotoxicity, confirming specificity of antibody-mediated neuronal death. Both anti-Hu and anti-Ri antibodies were readily taken up by viable neurons in slice cultures, but the two antibodies differed markedly in terms of their effects on neuronal viability. The ability of anti-Hu antibodies to cause neuronal death could account for the irreversible nature of paraneoplastic

An Alternative Chemical Redox Method for the Production of Bispecific Antibodies: Implication in Rapid Detection of Food Borne Pathogens

PubMed Central

Owais, Mohammad; Kazmi, Shadab; Tufail, Saba; Zubair, Swaleha

2014-01-01

Bi-functional antibodies with the ability to bind two unrelated epitopes have remarkable potential in diagnostic and bio-sensing applications. In the present study, bispecific antibodies that recognize human red blood cell (RBC) and the food borne pathogen Listeria monocytogenes (L. monocytogenes) were engineered. The procedure involves initial reduction of a mixture of anti-RBC and anti-Listeria antibodies followed by gradual re-oxidation of the reduced disulphides. This facilitates association of the separated antibody chains and formation of hybrid immunoglobulins with affinity for the L. monocytogenes and human RBC. The bispecific antibodies caused the agglutination of the RBCs only in the presence of L. monocytogenes cells. The agglutination process necessitated the specific presence of L. monocytogenes and the red colored clumps formed were readily visible with naked eyes. The RBC agglutination assay described here provides a remarkably simple approach for the rapid and highly specific screening of various pathogens in their biological niches. PMID:24637674

The restoration in vivo of 2,3-diphosphoglycerate (2,3-DPG) in stored red cells, after transfusion. The levels of red cells 2,3-DPG.

PubMed

Stan, Ana; Zsigmond, Eva

2009-01-01

Since the main reason for transfusing preserved red cells is to increase the oxygen carrying capacity of the recipient, the circulating preserved red cells should have at the time of transfusion normal oxygen uptake and normal oxyhemoglobin dissociation characteristics. We evaluated the effectiveness of transfused red cells, through periodical determination of erythrocyte components, during 72 hours after transfusions of large quantities (3,000 mL) of blood. Three patients with massive hemorrhages, two after amputation and one after nephrectomy were given each 3,000 mL preserved blood (in ACD, 10 days, at 4 degrees C). Red cell 2,3-DPG and serum inorganic phosphorus were determined prior to transfusion and after, periodically, for three days. Red cell 2,3-DPG was determined by Krimsky's method and inorganic phosphorus by Kuttner and Lichtenstein's method. The in vivo restoration of 2,3-DPG--of transfused red cells is shown as a percentage of recipient's final 2,3-DPG level, and was calculated in each of the three patients. The level of erythrocyte 2,3-DPG was greater than 60% of the final level within 24 hours, after the end of transfusion. The in vivo rates of restoration of 2,3-DPG in transfused red cells for periods of 0-6, 6-24, 24-48 and 48-72 hours are 0.251, 0.238, 0.133, 0.120 mM/L cells/hour. The therapeutic significance of the increased oxygen affinity of stored blood becomes very important in clinical conditions, when large volumes of red cells are urgently needed. After massive transfusions, the restoration of 2,3-DPG in red cells produces a decrease of serum inorganic phosphorus through its consumption. The stored blood with low values of erythrocyte 2,3-DPG can be used without hesitation when correcting a chronic anemia for instance, but in acute situation, when the organism needs restoration of the oxygen releasing capacity within minutes, the resynthesis is obviously insufficient. In such situations, fresh blood or blood with a near normal 2,3-DPG

A novel bispecific antibody, S-Fab, induces potent cancer cell killing.

PubMed

Li, Li; He, Ping; Zhou, Changhua; Jing, Li; Dong, Bin; Chen, Siqi; Zhang, Ning; Liu, Yawei; Miao, Ji; Wang, Zhong; Li, Qing

2015-01-01

Bispecific antibodies that engage immune cells to kill cancer cells have been actively studied in cancer immunotherapy. In this study, we present a novel bispecific format, S-Fab, fabricated by linking a single-domain anti-carcinoembryonic antigen VHH to a conventional anti-CD3 Fab. In contrast to most bispecific antibodies, the S-Fab bispecific antibody can be efficiently expressed and purified from bacteria. The purified S-Fab is stable in serum and is able to recruit T cells to drive potent cancer cell killing. In xenograft models, the S-Fab antibody suppresses tumor growth in the presence of human immune cells. Our study suggested that the bispecific S-Fab format can be applied to a wide range of immunotherapies.

Red Blood Cell Count Automation Using Microscopic Hyperspectral Imaging Technology.

PubMed

Li, Qingli; Zhou, Mei; Liu, Hongying; Wang, Yiting; Guo, Fangmin

2015-12-01

Red blood cell counts have been proven to be one of the most frequently performed blood tests and are valuable for early diagnosis of some diseases. This paper describes an automated red blood cell counting method based on microscopic hyperspectral imaging technology. Unlike the light microscopy-based red blood count methods, a combined spatial and spectral algorithm is proposed to identify red blood cells by integrating active contour models and automated two-dimensional k-means with spectral angle mapper algorithm. Experimental results show that the proposed algorithm has better performance than spatial based algorithm because the new algorithm can jointly use the spatial and spectral information of blood cells.

IGE AND IGGA ANTIBODY-MEDIATED RELEASE OF HISTAMINE FROM RAT PERITONEAL CELLS

PubMed Central

Bach, Michael K.; Bloch, Kurt J.; Austen, K. Frank

1971-01-01

IgGa, in contrast to IgE, antibodies mediated the antigen-induced release of histamine from rat peritoneal mast cells without a requirement for a latent period and without the capacity to bind firmly to the target cell. Nonetheless, IgGa anti-DNP antibody interfered with the capacity of rat anti-N. brasiliensis antiserum rich in IgE antibodies to prepare the target cells for histamine release by worm antigen. Further, interaction of IgE antibody-prepared cells with IgGa anti-DNP antibody and DNP-BSA at 0°C so as to achieve sterile activation, or at 30°C to permit histamine release, inactivated such cells as determined by the subsequent failure to release histamine upon challenge with worm antigen. Thus, although IgE and IgGa antibodies are immunochemically distinct homologous immunoglobulins and exhibit different functional characteristics, their interaction at the target cell involves a common receptor and at least one common point in the pathway to the release of pharmacologic agents from the cell. PMID:4101607

Blood volume and red cell life span (M113), part C

NASA Technical Reports Server (NTRS)

Johnson, P. C., Jr.

1973-01-01

Prechamber, in-chamber, and postchamber blood samples taken from Skylab simulation crewmembers did not indicate significant shortening of the red cell life span during the mission. This does not suggest that the space simulation environment could not be associated with red cell enzyme changes. It does show that any changes in enzymes were not sufficiently great to significantly shorten red cell survival. There was no evidence of bone marrow erythropoetic suppression nor was there any evidence of increased red cell destruction.

Prolonged red cell storage before transfusion increases extravascular hemolysis

PubMed Central

Rapido, Francesca; Brittenham, Gary M.; Bandyopadhyay, Sheila; La Carpia, Francesca; L’Acqua, Camilla; McMahon, Donald J.; Rebbaa, Abdelhadi; Wojczyk, Boguslaw S.; Netterwald, Jane; Wang, Hangli; Schwartz, Joseph; Eisenberger, Andrew; Soffing, Mark; Yeh, Randy; Divgi, Chaitanya; Ginzburg, Yelena Z.; Shaz, Beth H.; Sheth, Sujit; Francis, Richard O.; Spitalnik, Steven L.; Hod, Eldad A.

2016-01-01

BACKGROUND. Some countries have limited the maximum allowable storage duration for red cells to 5 weeks before transfusion. In the US, red blood cells can be stored for up to 6 weeks, but randomized trials have not assessed the effects of this final week of storage on clinical outcomes. METHODS. Sixty healthy adult volunteers were randomized to a single standard, autologous, leukoreduced, packed red cell transfusion after 1, 2, 3, 4, 5, or 6 weeks of storage (n = 10 per group). 51-Chromium posttransfusion red cell recovery studies were performed and laboratory parameters measured before and at defined times after transfusion. RESULTS. Extravascular hemolysis after transfusion progressively increased with increasing storage time (P < 0.001 for linear trend in the AUC of serum indirect bilirubin and iron levels). Longer storage duration was associated with decreasing posttransfusion red cell recovery (P = 0.002), decreasing elevations in hematocrit (P = 0.02), and increasing serum ferritin (P < 0.0001). After 6 weeks of refrigerated storage, transfusion was followed by increases in AUC for serum iron (P < 0.01), transferrin saturation (P < 0.001), and nontransferrin-bound iron (P < 0.001) as compared with transfusion after 1 to 5 weeks of storage. CONCLUSIONS. After 6 weeks of refrigerated storage, transfusion of autologous red cells to healthy human volunteers increased extravascular hemolysis, saturated serum transferrin, and produced circulating nontransferrin-bound iron. These outcomes, associated with increased risks of harm, provide evidence that the maximal allowable red cell storage duration should be reduced to the minimum sustainable by the blood supply, with 35 days as an attainable goal. REGISTRATION. ClinicalTrials.gov NCT02087514. FUNDING. NIH grant HL115557 and UL1 TR000040. PMID:27941245

Experimental prestorage filtration removes antibodies and decreases lipids in RBC supernatants mitigating TRALI in vivo

PubMed Central

Kelher, Marguerite R.; Khan, Samina Y.; LaSarre, Monica; West, F. Bernadette; Land, Kevin J.; Mish, Barbara; Ceriano, Linda; Sowemimo-Coker, Samuel

2014-01-01

Transfusion-related acute lung injury (TRALI) remains a significant cause of transfusion-related mortality with red cell transfusion. We hypothesize that prestorage filtration may reduce proinflammatory activity in the red blood cell (RBC) supernatant and prevent TRALI. Filters were manufactured for both small volumes and RBC units. Plasma containing antibodies to human lymphocyte antigen (HLA)-A2 or human neutrophil antigen (HNA)-3a was filtered, and immunoglobulins and specific HNA-3a and HLA-2a neutrophil (PMN) priming activity were measured. Antibodies to OX27 were added to plasma, and filtration was evaluated in a 2-event animal model of TRALI. RBC units from 31 donors known to have antibodies against HLA antigens and from 16 antibody-negative controls were filtered. Furthermore, 4 RBC units were drawn and underwent standard leukoreduction. Immunoglobulins, HLA antibodies, PMN priming activity, and the ability to induce TRALI in an animal model were measured. Small-volume filtration of plasma removed >96% of IgG, antibodies to HLA-A2 and HNA-3a, and their respective priming activity, as well as mitigating antibody-mediated in vivo TRALI. In RBC units, experimental filtration removed antibodies to HLA antigens and inhibited the accumulation of lipid priming activity and lipid-mediated TRALI. We conclude that filtration removes proinflammatory activity and the ability to induce TRALI from RBCs and may represent a TRALI mitigation step. PMID:24747436

Stabilization of red blood cells by the plasticizer, diethylhexylphthalate.

PubMed

Horowitz, B; Stryker, M H; Waldman, A A; Woods, K R; Gass, J D; Drago, J

1985-01-01

The red blood cells of blood stored in containers made of polyvinylchloride (PVC) film are osmotically more stable and lose on average about 1/3 less hemoglobin than when blood is stored in another plastic [poly-(ethylene-co-ethyl acrylate); EEA]. The stability of uniform volumes of stored red blood cells varies directly with PVC surface area, whereas changes in EEA surface area have comparatively little or no effect. PVC contains high concentrations of the plasticizer, diethylhexylphthalate (DEHP), known to migrate into blood and to have a high potential for toxicity. To determine if DEHP could be the red cell stabilizing agent in PVC, whole blood was stored in containers made from EEA into which was incorporated varying amounts of DEHP. Incorporation of DEHP into EEA significantly reduced erythrocyte osmotic fragility (p = 0.01). The degree of reduced fragility correlated with the level of DEHP in the cell phase implicating DEHP in PVC containers as the stabilizing agent for red cells.

Anti-MUC1 antibody inhibits EGF receptor signaling in cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hisatsune, Akinori, E-mail: hisatsun@kumamoto-u.ac.jp; Nakayama, Hideki; Kawasaki, Mitsuru

2011-02-18

Research highlights: {yields} We identified changes in the expression and function of EGFR by anti-MUC1 antibody. {yields} An anti-MUC1 antibody GP1.4 decreased EGFR from cell surface by internalization. {yields} GP1.4 specifically inhibited ERK signaling triggered EGF-EGFR signaling pathway. {yields} Internalization of EGFR was dependent on the presence of MUC1 on cell surface. {yields} GP1.4 significantly inhibited EGF-dependent cancer cell proliferation and migration. -- Abstract: MUC1 is a type I transmembrane glycoprotein aberrantly overexpressed in various cancer cells. High expression of MUC1 is closely associated with cancer progression and metastasis, leading to poor prognosis. We previously reported that MUC1 is internalizedmore » by the binding of the anti-MUC1 antibody, from the cell surface to the intracellular region via the macropinocytotic pathway. Since MUC1 is closely associated with ErbBs, such as EGF receptor (EGFR) in cancer cells, we examined the effect of the anti-MUC1 antibody on EGFR trafficking. Our results show that: (1) anti-MUC1 antibody GP1.4, but not another anti-MUC1 antibody C595, triggered the internalization of EGFR in pancreatic cancer cells; (2) internalization of EGFR by GP1.4 resulted in the inhibition of ERK phosphorylation by EGF stimulation, in a MUC1 dependent manner; (3) inhibition of ERK phosphorylation by GP1.4 resulted in the suppression of proliferation and migration of pancreatic cancer cells. We conclude that the internalization of EGFR by anti-MUC1 antibody GP1.4 inhibits the progression of cancer cells via the inhibition of EGFR signaling.« less

Studying red blood cell agglutination by measuring electrical and mechanical properties with a double optical tweezers

NASA Astrophysics Data System (ADS)

Fontes, Adriana; Fernandes, Heloise P.; de Thomaz, André A.; Barbosa, Luiz C.; Barjas-Castro, Maria L.; Cesar, Carlos L.

2007-07-01

The red blood cell (RBC) viscoelastic membrane contains proteins and glycolproteins embedded in, or attached, to a fluid lipid bilayer and are negatively charged, which creates a repulsive electric (zeta) potential between the cells and prevents their aggregation in the blood stream. The basis of the immunohematologic tests is the interaction between antigens and antibodies that causes hemagglutination. The identification of antibodies and antigens is of fundamental importance for the transfusional routine. This agglutination is induced by decreasing the zeta-potential through the introduction of artificial potential substances. This report proposes the use of the optical tweezers to measure the membrane viscosity, the cell adhesion, the zeta-potential and the size of the double layer of charges (CLC) formed around the cell in an electrolytic solution. The adhesion was quantified by slowly displacing two RBCs apart until the disagglutination. The CLC was measured using the force on the bead attached to a single RBC in response to an applied voltage. The zeta-potential was obtained by measuring the terminal velocity after releasing the RBC from the optical trap at the last applied voltage. For the membrane viscosity experiment, we trapped a bead attached to RBCs and measured the force to slide one RBC over the other as a function of the relative velocity. After we tested the methodology, we performed measurements using antibody and potential substances. We observed that this experiment can provide information about cell agglutination that helps to improve the tests usually performed in blood banks. We also believe that this methodology can be applied for measurements of zeta-potentials in other kind of samples.

Nanotube antibody biosensor arrays for the detection of circulating breast cancer cells

NASA Astrophysics Data System (ADS)

Shao, Ning; Wickstrom, Eric; Panchapakesan, Balaji

2008-11-01

Recent reports have shown that nanoscale electronic devices can be used to detect a change in electrical properties when receptor proteins bind to their corresponding antibodies functionalized on the surface of the device, in extracts from as few as ten lysed tumor cells. We hypothesized that nanotube-antibody devices could sensitively and specifically detect entire live cancer cells. We report for the first time a single nanotube field effect transistor array, functionalized with IGF1R-specific and Her2-specific antibodies, which exhibits highly sensitive and selective sensing of live, intact MCF7 and BT474 human breast cancer cells in human blood. Those two cell lines both overexpress IGF1R and Her2, at different levels. Single or small bundle of nanotube devices that were functionalized with IGF1R-specific or Her2-specific antibodies showed 60% decreases in conductivity upon interaction with BT474 or MCF7 breast cancer cells in two µl drops of blood. Control experiments with non-specific antibodies or with MCF10A control breast cells produced a less than 5% decrease in electrical conductivity, illustrating the high sensitivity for whole cell binding by these single nanotube-antibody devices. We postulate that the free energy change due to multiple simultaneous cell-antibody binding events exerted stress along the nanotube surface, decreasing its electrical conductivity due to an increase in band gap. Because the free energy change upon cell-antibody binding, the stress exerted on the nanotube, and the change in conductivity are specific to a specific antigen-antibody interaction; these properties might be used as a fingerprint for the molecular sensing of circulating cancer cells. From optical microscopy observations during sensing, it appears that the binding of a single cell to a single nanotube field effect transistor produced the change in electrical conductivity. Thus we report a nanoscale oncometer with single cell sensitivity with a diameter 1000 times

Macrophage-mediated trogocytosis leads to death of antibody-opsonized tumor cells

PubMed Central

Velmurugan, Ramraj; Challa, Dilip K.; Ram, Sripad; Ober, Raimund J.; Ward, E. Sally

2016-01-01

Understanding the complex behavior of effector cells such as monocytes or macrophages in regulating cancerous growth is of central importance for cancer immunotherapy. Earlier studies using CD20-specific antibodies have demonstrated that the Fcγ receptor (FcγR)-mediated transfer of the targeted receptors from tumor cells to these effector cells through trogocytosis can enable escape from antibody therapy, leading to the viewpoint that this process is pro-tumorigenic. In the current study we demonstrate that persistent trogocytic attack results in the killing of HER2-overexpressing breast cancer cells. Further, antibody engineering to increase FcγR interactions enhances this tumoricidal activity. These studies extend the complex repertoire of activities of macrophages to trogocytic-mediated cell death of HER2-overexpressing target cells and have implications for the development of effective antibody-based therapies. PMID:27226489

Energy of adhesion of human T cells to adsorption layers of monoclonal antibodies measured by a film trapping technique.

PubMed Central

Ivanov, I B; Hadjiiski, A; Denkov, N D; Gurkov, T D; Kralchevsky, P A; Koyasu, S

1998-01-01

A novel method for studying the interaction of biological cells with interfaces (e.g., adsorption monolayers of antibodies) is developed. The method is called the film trapping technique because the cell is trapped within an aqueous film of equilibrium thickness smaller than the cell diameter. A liquid film of uneven thickness is formed around the trapped cell. When observed in reflected monochromatic light, this film exhibits an interference pattern of concentric bright and dark fringes. From the radii of the fringes one can restore the shape of interfaces and the cell. Furthermore, one can calculate the adhesive energy between the cell membrane and the aqueous film surface (which is covered by a layer of adsorbed proteins and/or specific ligands), as well as the disjoining pressure, representing the force of interaction per unit area of the latter film. The method is applied to two human T cell lines: Jurkat and its T cell receptor negative (TCR-) derivative. The interaction of these cells with monolayers of three different monoclonal antibodies adsorbed at a water-air interface is studied. The results show that the adhesive energy is considerable (above 0.5 mJ/m2) when the adsorption monolayer contains antibodies acting as specific ligands for the receptors expressed on the cell surface. In contrast, the adhesive energy is close to zero in the absence of such a specific ligand-receptor interaction. In principle, the method can be applied to the study of the interaction of a variety of biological cells (B cells, natural killer cells, red blood cells, etc.) with adsorption monolayers of various biologically active molecules. In particular, film trapping provides a tool for the gentle micromanipulation of cells and for monitoring of processes (say the activation of a T lymphocyte) occurring at the single-cell level. PMID:9649417

Inhibitory Effect of Individual or Combinations of Broadly Neutralizing Antibodies and Antiviral Reagents against Cell-Free and Cell-to-Cell HIV-1 Transmission

PubMed Central

Kolodkin-Gal, Dror; Eslamizar, Leila; Owuor, Joshua O.; Mazzola, Emanuele; Gonzalez, Ana M.; Korioth-Schmitz, Birgit; Gelman, Rebecca S.; Montefiori, David C.; Haynes, Barton F.; Schmitz, Joern E.

2015-01-01

ABSTRACT To date, most therapeutic and vaccine candidates for human immunodeficiency virus type 1 (HIV-1) are evaluated preclinically for efficacy against cell-free viral challenges. However, cell-associated HIV-1 is suggested to be a major contributor to sexual transmission by mucosal routes. To determine if neutralizing antibodies or inhibitors block cell-free and cell-associated virus transmission of diverse HIV-1 strains with different efficiencies, we tested 12 different antibodies and five inhibitors against four green fluorescent protein (GFP)-labeled HIV-1 envelope (Env) variants from transmitted/founder (T/F) or chronic infection isolates. We evaluated antibody/inhibitor-mediated virus neutralization using either TZM-bl target cells, in which infectivity was determined by virus-driven luciferase expression, or A3R5 lymphoblastoid target cells, in which infectivity was evaluated by GFP expression. In both the TZM-bl and A3R5 assays, cell-free virus or infected CD4+ lymphocytes were used as targets for neutralization. We further hypothesized that the combined use of specific neutralizing antibodies targeting HIV-1 Env would more effectively prevent cell-associated virus transmission than the use of individual antibodies. The tested antibody combinations included two gp120-directed antibodies, VRC01 and PG9, or VRC01 with the gp41-directed antibody 10E8. Our results demonstrated that cell-associated virus was less sensitive to neutralizing antibodies and inhibitors, particularly using the A3R5 neutralization assay, and the potencies of these neutralizing agents differed among Env variants. A combination of different neutralizing antibodies that target specific sites on gp120 led to a significant reduction in cell-associated virus transmission. These assays will help identify ideal combinations of broadly neutralizing antibodies to use for passive preventive antibody administration and further characterize targets for the most effective neutralizing antibodies

Surface design of antibody-immobilized thermoresponsive cell culture dishes for recovering intact cells by low-temperature treatment.

PubMed

Kobayashi, Jun; Hayashi, Masaki; Ohno, Takahiro; Nishi, Masanori; Arisaka, Yoshinori; Matsubara, Yoshinori; Kakidachi, Hiroshi; Akiyama, Yoshikatsu; Yamato, Masayuki; Horii, Akihiro; Okano, Teruo

2014-11-01

Antibody-immobilized thermoresponsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) [poly(IPAAm-co-CIPAAm)]-grafted cell culture surfaces were designed to enhance both the initial adhesion of weakly adhering cells and the ability of cells to detach in response to low temperature through the regulation of affinity binding between immobilized antibodies and antigens on the cellular surface. Ty-82 cells and neonatal normal human dermal fibroblasts (NHDFs), which express CD90 on the cell surface, adhered to anti-CD90 antibody-immobilized thermoresponsive surfaces at 37°C, a condition at which the grafted thermoresponsive polymer chains shrank. Adherent Ty-82 cells were detached from the surfaces by lowering the temperature to 20°C and applying external forces, such as pipetting, whereas cultured NHDF sheets spontaneously detached themselves from the surface in response to reduced temperature alone. When the temperature was decreased to 20°C, the swelling of grafted thermoresponsive polymer chains weakened the affinity binding between immobilized antibody and antigen on the cells due to the increasing steric hindrance of the polymer chains around the antigen-recognition site of the immobilized antibodies. No contamination was detected on cells harvested from covalently immobilized antibodies on the culture surfaces by low-temperature treatment, whereas a carryover of the antibody and avidin from the avidin-biotin binding surface was observed. Furthermore, the initial adhesion of adipose tissue-derived cells, which adhere weakly to PIPAAm-grafted surfaces, was enhanced on the antibody-immobilized thermoresponsive surfaces. © 2013 Wiley Periodicals, Inc.

RED BLOOD CELL PRESERVATION.

DTIC Science & Technology

red cells were assayed by ion exchange chromatography. The O-day 2,3- diphosphoglycerate concentrations of the ACD-AP bloods were below the normal...adenosine or guanosine. After a small initial increase, the ADP levels remained fairly constant. The AMP values increased as the ATP decreased and in

Hyperemic peripheral red marrow in a patient with sickle cell anemia demonstrated on Tc-99m labeled red blood cell venography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heiden, R.A.; Locko, R.C.; Stent, T.R.

1991-03-01

A 25-year-old gravid woman, homozygous for sickle cell anemia, with a history of recent deep venous thrombosis, was examined using Tc-99m labeled red blood cell venography for recurrent thrombosis. Although negative for thrombus, the study presented an unusual incidental finding: the patient's peripheral bone marrow was hyperemic in a distribution consistent with peripheral red bone marrow expansion. Such a pattern has not been documented before using this technique. This report supports other literature that has demonstrated hyperemia of peripheral red bone marrow in other hemolytic anemias. This finding may ultimately define an additional role of scintigraphy in assessing the pathophysiologicmore » status of the sickle cell patient.« less

Patient-Specific B-Cell Antibody Factories to Treat Metastatic Disease

DTIC Science & Technology

2013-08-01

Immortalization of these selected clones using Epstein - Barr viral transformation provides a method to maintain these antibody producing cell lines as a...2013 Please see next page. None provided. Patient-Specific B-Cell Antibody Factories to Treat Metastatic Disease Kevin Claffey University of

Surrogate target cells expressing surface anti-idiotype antibody for the clinical evaluation of an internalizing CD22-specific antibody.

PubMed

Leung, Shui-On; Gao, Kai; Wang, Guang Yu; Cheung, Benny Ka-Wa; Lee, Kwan-Yeung; Zhao, Qi; Cheung, Wing-Tai; Wang, Jun Zhi

2015-01-01

SM03, a chimeric antibody that targets the B-cell restricted antigen CD22, is currently being clinically evaluated for the treatment of lymphomas and other autoimmune diseases in China. SM03 binding to surface CD22 leads to rapid internalization, making the development of an appropriate cell-based bioassay for monitoring changes in SM03 bioactivities during production, purification, storage, and clinical trials difficult. We report herein the development of an anti-idiotype antibody against SM03. Apart from its being used as a surrogate antigen for monitoring SM03 binding affinities, the anti-idiotype antibody was engineered to express as fusion proteins on cell surfaces in a non-internalizing manner, and the engineered cells were used as novel "surrogate target cells" for SM03. SM03-induced complement-mediated cytotoxicity (CMC) against these "surrogate target cells" proved to be an effective bioassay for monitoring changes in Fc functions, including those resulting from minor structural modifications borne within the Fc-appended carbohydrates. The approach can be generally applied for antibodies that target rapidly internalizing or non-surface bound antigens. The combined use of the anti-idiotype antibody and the surrogate target cells could help evaluate clinical parameters associated with safety and efficacies, and possibly the mechanisms of action of SM03.

Stably Fluorescent Cell Line of Human Ovarian Epithelial Cancer Cells SK-OV-3ip-red.

PubMed

Konovalova, E V; Shulga, A A; Chumakov, S P; Khodarovich, Yu M; Woo, Eui-Jeon; Deev, S M

2017-11-01

Stable red fluorescing line of human ovarian epithelial cancer cells SK-OV-3ip-red was generated expressing gene coding for protein TurboFP635 (Katushka) fluorescing in the far-red spectrum region with excitation and emission peaks at 588 and 635 nm, respectively. Fluorescence of SK-OV-3ip-red line remained high during long-term cell culturing and after cryogenic freezing. The obtained cell line SK-OV-3ip-red can serve a basis for a model of a scattered tumor with numerous/extended metastases and used both for testing anticancer drugs inhibiting metastasis growth and for non-invasive monitoring of the growth dynamics with high precision.

Polymer-mediated immunocamouflage of red blood cells: effects of polymer size on antigenic and immunogenic recognition of allogeneic donor blood cells.

PubMed

Wang, DunCheng; Kyluik, Dana L; Murad, Kari L; Toyofuku, Wendy M; Scott, Mark D

2011-07-01

Developing a practical means of reducing alloimmunization in chronically transfused patients would be of significant clinical benefit. Immunocamouflaging red blood cells (RBCs) by membrane grafting of methoxypoly(ethylene glycol) (mPEG) may reduce the risk of allo-immunization. The results of this study showed that antibody recognition of non-ABO antigens was significantly reduced in an mPEG-dose- and polymer size-dependent manner, with higher molecular weight mPEGs providing better immunoprotection. Furthermore, in vivo immunogenicity was significantly reduced in mice serially transfused with mPEG-modified xenogeneic (sheep; sRBCs), allogeneic (C57Bl/6), or syngeneic (Balb/c) RBCs. Following a primary transfusion of sRBCs, mice receiving mPEG-sRBCs showed a >90% reduction in anti-sRBC IgG antibody levels. After two transfusions, mice receiving mPEG-sRBCs showed reductions of >80% in anti-sRBC IgG levels. Importantly, mPEG-modified autologous cells did not induce neoantigens or an immune (IgG or IgM) response. These data suggest that the global immunocamouflage of RBCs by polymer grafting may provide a safe and cost-effective means of reducing the risk of alloimmunization.

Monoclonal Antibody and an Antibody-Toxin Conjugate to a Cell Surface Proteoglycan of Melanoma Cells Suppress in vivo Tumor Growth

NASA Astrophysics Data System (ADS)

Bumol, T. F.; Wang, Q. C.; Reisfeld, R. A.; Kaplan, N. O.

1983-01-01

A monoclonal antibody directed against a cell surface chondroitin sulfate proteoglycan of human melanoma cells, 9.2.27, and its diphtheria toxin A chain (DTA) conjugate were investigated for their effects on in vitro protein synthesis and in vivo tumor growth of human melanoma cells. The 9.2.27 IgG and its DTA conjugate display similar serological activities against melanoma target cells but only the conjugate can induce consistent in vitro inhibition of protein synthesis and toxicity in M21 melanoma cells. However, both 9.2.27 IgG and its DTA conjugate effect significant suppression of M21 tumor growth in vivo in an immunotherapy model of a rapidly growing tumor in athymic nu/nu mice, suggesting that other host mechanisms may mediate monoclonal antibody-induced tumor suppression.

Trispecific antibodies for CD16A-directed NK cell engagement and dual-targeting of tumor cells.

PubMed

Gantke, Thorsten; Weichel, Michael; Herbrecht, Carmen; Reusch, Uwe; Ellwanger, Kristina; Fucek, Ivica; Eser, Markus; Müller, Thomas; Griep, Remko; Molkenthin, Vera; Zhukovsky, Eugene A; Treder, Martin

2017-09-01

Bispecific antibodies that redirect the lytic activity of cytotoxic immune effector cells, such as T- and NK cells, onto tumor cells have emerged as a highly attractive and clinically validated treatment modality for hematological malignancies. Advancement of this therapeutic concept into solid tumor indications, however, is hampered by the scarcity of targetable antigens that are surface-expressed on tumor cells but demonstrate only limited expression on healthy tissues. To overcome this limitation, the concept of dual-targeting, i.e. the simultaneous targeting of two tumor-expressed surface antigens with limited co-expression on non-malignant cells, with multispecific antibodies has been proposed to increase tumor selectivity of antibody-induced effector cell cytotoxicity. Here, a novel CD16A (FcγRIIIa)-directed trispecific, tetravalent antibody format, termed aTriFlex, is described, that is capable of redirecting NK cell cytotoxicity to two surface-expressed antigens. Using a BCMA/CD200-based in vitro model system, the potential use of aTriFlex antibodies for dual-targeting and selective induction of NK cell-mediated target cell lysis was investigated. Bivalent bispecific target cell binding was found to result in significant avidity gains and up to 17-fold increased in vitro potency. These data suggest trispecific aTriFlex antibodies may support dual-targeting strategies to redirect NK cell cytotoxicity with increased selectivity to enable targeting of solid tumor antigens. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Agonist antibody that induces human malignant cells to kill one another

PubMed Central

Yea, Kyungmoo; Zhang, Hongkai; Xie, Jia; Jones, Teresa M.; Lin, Chih-Wei; Francesconi, Walter; Berton, Fulvia; Fallahi, Mohammad; Sauer, Karsten; Lerner, Richard A.

2015-01-01

An attractive, but as yet generally unrealized, approach to cancer therapy concerns discovering agents that change the state of differentiation of the cancer cells. Recently, we discovered a phenomenon that we call “receptor pleiotropism” in which agonist antibodies against known receptors induce cell fates that are very different from those induced by the natural agonist to the same receptor. Here, we show that one can take advantage of this phenomenon to convert acute myeloblastic leukemic cells into natural killer cells. Upon induction with the antibody, these leukemic cells enter into a differentiation cascade in which as many as 80% of the starting leukemic cells can be differentiated. The antibody-induced killer cells make large amounts of perforin, IFN-γ, and granzyme B and attack and kill other members of the leukemic cell population. Importantly, induction of killer cells is confined to transformed cells, in that normal bone marrow cells are not induced to form killer cells. Thus, it seems possible to use agonist antibodies to change the differentiation state of cancer cells into those that attack and kill other members of the malignant clone from which they originate. PMID:26487683

Control of red blood cell mass during spaceflight

NASA Technical Reports Server (NTRS)

Lane, H. W.; Alfrey, C. P.; Driscoll, T. B.; Smith, S. M.; Nyquist, L. E.

1996-01-01

Data are reviewed from twenty-two astronauts from seven space missions in a study of red blood cell mass. The data show that decreased red cell mass in all astronauts exposed to space for more than nine days, although the actual dynamics of mass changes varies with flight duration. Possible mechanisms for these changes, including alterations in erythropoietin levels, are discussed.

Cell-Cell Transmission Enables HIV-1 to Evade Inhibition by Potent CD4bs Directed Antibodies

PubMed Central

Schanz, Merle; Reynell, Lucy; Günthard, Huldrych F.; Rusert, Peter; Trkola, Alexandra

2012-01-01

HIV is known to spread efficiently both in a cell-free state and from cell to cell, however the relative importance of the cell-cell transmission mode in natural infection has not yet been resolved. Likewise to what extent cell-cell transmission is vulnerable to inhibition by neutralizing antibodies and entry inhibitors remains to be determined. Here we report on neutralizing antibody activity during cell-cell transmission using specifically tailored experimental strategies which enable unambiguous discrimination between the two transmission routes. We demonstrate that the activity of neutralizing monoclonal antibodies (mAbs) and entry inhibitors during cell-cell transmission varies depending on their mode of action. While gp41 directed agents remain active, CD4 binding site (CD4bs) directed inhibitors, including the potent neutralizing mAb VRC01, dramatically lose potency during cell-cell transmission. This implies that CD4bs mAbs act preferentially through blocking free virus transmission, while still allowing HIV to spread through cell-cell contacts. Thus providing a plausible explanation for how HIV maintains infectivity and rapidly escapes potent and broadly active CD4bs directed antibody responses in vivo. PMID:22496655

Korean Red Ginseng Extract Enhances the Anticancer Effects of Sorafenib through Abrogation of CREB and c-Jun Activation in Renal Cell Carcinoma.

PubMed

Kim, Chulwon; Lee, Jong Hyun; Baek, Seung Ho; Ko, Jeong-Hyeon; Nam, Dongwoo; Ahn, Kwang Seok

2017-07-01

Although application of sorafenib in the treatment of human renal cell carcinoma (RCC) remains one of the best examples of successful targeted therapy, majority of RCC patients suffer from its side effects as well as develop resistance to this targeted therapy. Thus, there is a need to promote novel alternative therapies for the treatment of RCC. In this study, we investigated whether Korean red ginseng extract (KRGE) could inhibit the proliferation and induce chemosensitization in human renal cancer cells. Also, we used a human phospho-antibody array containing 46 antibodies against signaling molecules to examine a subset of phosphorylation events after KRGE and sorafenib combination treatment. Korean red ginseng extract suppressed the proliferation of two RCC cell lines; activated caspase-3; caused poly(ADP-ribose) polymerase cleavage; abrogated the expression of B-cell lymphoma 2, B-cell lymphoma extra large, survivin, inhibitors of apoptosis proteins-1/2, cyclooxygenase-2, cyclin D1, matrix metallopeptidase-9, and vascular endothelial growth factor; and upregulated pro-apoptotic gene products. Interestingly, KRGE enhanced the cytotoxic and apoptotic effects of sorafenib in RCC cells. The combination treatment of KRGE and sorafenib more clearly suppressed cyclic adenosine monophosphate response element-binding protein and c-Jun phosphorylation and induced phosphorylation of p53 than did the individual treatment regimen. Our results clearly demonstrate that KRGE can enhance the anticancer activity of sorafenib and may have a substantial potential in the treatment of RCC. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Cell-free measurements of brightness of fluorescently labeled antibodies

PubMed Central

Zhou, Haiying; Tourkakis, George; Shi, Dennis; Kim, David M.; Zhang, Hairong; Du, Tommy; Eades, William C.; Berezin, Mikhail Y.

2017-01-01

Validation of imaging contrast agents, such as fluorescently labeled imaging antibodies, has been recognized as a critical challenge in clinical and preclinical studies. As the number of applications for imaging antibodies grows, these materials are increasingly being subjected to careful scrutiny. Antibody fluorescent brightness is one of the key parameters that is of critical importance. Direct measurements of the brightness with common spectroscopy methods are challenging, because the fluorescent properties of the imaging antibodies are highly sensitive to the methods of conjugation, degree of labeling, and contamination with free dyes. Traditional methods rely on cell-based assays that lack reproducibility and accuracy. In this manuscript, we present a novel and general approach for measuring the brightness using antibody-avid polystyrene beads and flow cytometry. As compared to a cell-based method, the described technique is rapid, quantitative, and highly reproducible. The proposed method requires less than ten microgram of sample and is applicable for optimizing synthetic conjugation procedures, testing commercial imaging antibodies, and performing high-throughput validation of conjugation procedures. PMID:28150730

Measuring electrical and mechanical properties of red blood cells with a double optical tweezers

NASA Astrophysics Data System (ADS)

Fontes, Adriana; Fernandes, Heloise P.; Barjas-Castro, Maria L.; de Thomaz, André A.; Pozzo, Liliana d. Y.; Barbosa, Luiz C.; Cesar, Carlos L.

2006-08-01

The fluid lipid bilayer viscoelastic membrane of red blood cells (RBC) contains antigen glycolproteins and proteins which can interact with antibodies to cause cell agglutination. This is the basis of most of the immunohematologic tests in blood banks and the identification of the antibodies against the erythrocyte antigens is of fundamental importance for transfusional routines. The negative charges of the RBCs creates a repulsive electric (zeta) potential between the cells and prevents their aggregation in the blood stream. The first counterions cloud strongly binded moving together with the RBC is called the compact layer. This report proposes the use of a double optical tweezers for a new procedure for measuring: (1) the apparent membrane viscosity, (2) the cell adhesion, (3) the zeta potential and (4) the compact layer's size of the charges formed around the cell in the electrolytic solution. To measure the membrane viscosity we trapped silica beads strongly attached to agglutinated RBCs and measured the force to slide one RBC over the other as a function of the relative velocity. The RBC adhesion was measured by slowly displacing two RBCs apart until the disagglutination happens. The compact layer's size was measured using the force on the silica bead attached to a single RBC in response to an applied voltage and the zeta potential was obtained by measuring the terminal velocity after releasing the RBC from the optical trap at the last applied voltage. We believe that the methodology here proposed can improve the methods of diagnosis in blood banks.

Red blood cell transfusion in newborn infants

PubMed Central

Whyte, Robin K; Jefferies, Ann L

2014-01-01

Red blood cell transfusion is an important and frequent component of neonatal intensive care. The present position statement addresses the methods and indications for red blood cell transfusion of the newborn, based on a review of the current literature. The most frequent indications for blood transfusion in the newborn are the acute treatment of perinatal hemorrhagic shock and the recurrent correction of anemia of prematurity. Perinatal hemorrhagic shock requires immediate treatment with large quantities of red blood cells; the effects of massive transfusion on other blood components must be considered. Some guidelines are now available from clinical trials investigating transfusion in anemia of prematurity; however, considerable uncertainty remains. There is weak evidence that cognitive impairment may be more severe at follow-up in extremely low birth weight infants transfused at lower hemoglobin thresholds; therefore, these thresholds should be maintained by transfusion therapy. Although the risks of transfusion have declined considerably in recent years, they can be minimized further by carefully restricting neonatal blood sampling. PMID:24855419

Sickle red cell dehydration: mechanisms and interventions.

PubMed

Bookchin, Robert M; Lew, Virgilio L

2002-03-01

A critical link between the single molecular defect in sickle cell anemia and the extensive pathology of this disease is the reversible increase in red cell membrane permeability generated by hemoglobin S polymers in the deoxygenated state. This permeability, usually described as P (sickle), triggers a chain of events in which two constitutive transporters of the red cell membrane become activated-the recently cloned intermediate conductance, Ca 2+ -sensitive K channel, and the electroneutral K:Cl cotransporter-leading to sickle cell dehydration. This article reviews knowledge of the dehydration mechanism, stressing the marked heterogeneity of dehydration rates in sickle cell populations, and discusses recent contributions to understanding of the function and regulation of P (sickle), Ca 2+ -sensitive K channel, and K:Cl cotransporter, and of therapies targeted at these transporters.

Distinctions Among Circulating Antibody Secreting Cell Populations, Including B-1 Cells, in Human Adult Peripheral Blood1

PubMed Central

Quách, Tâm D.; Rodríguez-Zhurbenko, Nely; Hopkins, Thomas J.; Guo, Xiaoti; Vázquez, Ana María Hernández; Li, Wentian; Rothstein, Thomas L.

2015-01-01

Human antibody secreting cell (ASC) populations in circulation are not well studied. In addition to B-1 (CD20+CD27+CD38lo/intCD43+) cell and the conventional plasmablast (CD20-CD27hiCD38hi) cell populations, here we identified a novel B cell population termed 20+38hi B cells (CD20+CD27hiCD38hi) that spontaneously secretes antibody. At steady state, 20+38hi B cells are distinct from plasmablasts on the basis of CD20 expression, amount of antibody production, frequency of mutation, and diversity of B cell receptor repertoire. However, cytokine treatment of 20+38hi B cells induces loss of CD20 and acquisition of CD138, suggesting that 20+38hi B cells are precursors to plasmablasts, or pre-plasmablasts. We then evaluated similarities and differences between CD20+CD27+CD38lo/intCD43+ B-1 cells, CD20+CD27hiCD38hi 20+38hi B cells, CD20-CD27hiCD38hi plasmablasts, and CD20+CD27+CD38lo/intCD43- memory B cells. We found that B-1 cells differ from 20+38hi B cells and plasmablasts in numbers of ways, including antigen expression, morphological appearance, transcriptional profiling, antibody skewing, antibody repertoire, and secretory response to stimulation. In terms of gene expression, B-1 cells align more closely with memory B cells than with 20+38hi B cells or plasmablasts, but differ in that memory B cells do not express antibody secretion related genes. We found that, B-1 cell antibodies utilize Vh4-34, which is often associated with autoreactivity, 3 to 6-fold more often than other B cell populations. Along with selective production of IgM anti-PC, this data suggests that human B-1 cells might be preferentially selected for autoreactivity/natural-specificity. In sum, our results indicate that human healthy adult peripheral blood at steady state consists of 3 distinct ASC populations. PMID:26740107

Alloimmunization is associated with older age of transfused red blood cells in sickle cell disease.

PubMed

Desai, Payal C; Deal, Allison M; Pfaff, Emily R; Qaqish, Bahjat; Hebden, Leyna M; Park, Yara A; Ataga, Kenneth I

2015-08-01

Red blood cell (RBC) alloimmunization is a significant clinical complication of sickle cell disease (SCD). It can lead to difficulty with cross-matching for future transfusions and may sometimes trigger life-threatening delayed hemolytic transfusion reactions. We conducted a retrospective study to explore the association of clinical complications and age of RBC with alloimmunization in patients with SCD followed at a single institution from 2005 to 2012. One hundred and sixty six patients with a total of 488 RBC transfusions were evaluated. Nineteen patients (11%) developed new alloantibodies following blood transfusions during the period of review. The median age of RBC units was 20 days (interquartile range: 14-27 days). RBC antibody formation was significantly associated with the age of RBC units (P = 0.002), with a hazard ratio of 3.5 (95% CI: 1.71-7.11) for a RBC unit that was 7 days old and 9.8 (95% CI: 2.66-35.97) for a unit that was 35 days old, 28 days after the blood transfusion. No association was observed between RBC alloimmunization and acute vaso-occlusive complications. Although increased echocardiography-derived tricuspid regurgitant jet velocity (TRV) was associated with the presence of RBC alloantibodies (P = 0.02), TRV was not significantly associated with alloimmunization when adjusted for patient age and number of transfused RBC units. Our study suggests that RBC antibody formation is significantly associated with older age of RBCs at the time of transfusion. Prospective studies in patients with SCD are required to confirm this finding. © 2015 Wiley Periodicals, Inc.

Surface decoration of red blood cells with maleimidophenyl-polyethylene glycol facilitated by thiolation with iminothiolane: an approach to mask A, B, and D antigens to generate universal red blood cells.

PubMed

Nacharaju, Parimala; Boctor, Fouad N; Manjula, Belur N; Acharya, Seetharama A

2005-03-01

The surface decoration of red blood cells (RBCs) by polyethylene glycol (PEG) chains has been an approach developed to camouflage the blood group antigens from their antibodies. A PEGylation protocol, however, that can mask the antigens appropriately to inhibit the agglutination of RBCs with the respective antibodies is not available so far. A new approach for PEGylation of RBC membrane proteins has been designed with thiolation-mediated maleimide chemistry. The accessibility of the surface lysine residues of membrane proteins to bulky PEG reagents was increased by linking an extension arm carrying a thiol group. RBCs have been PEGylated by thiolation-mediated chemistry with maleimidophenyl-PEG (Mal-Phe-PEG) reagents of different chain lengths. Mal-Phe-PEG-5000 chains alone masked the most important antigens of the Rh system (C, c, E, e, and D) from their antibodies. The masking of the A and B antigens needed a combination of Mal-Phe-PEG-5000 and Mal-Phe-PEG-20000 chains to inhibit the agglutination of RBCs completely with anti-A or anti-B. Thiolation-mediated PEGylation of RBCs with Mal-Phe-PEG-5000 and Mal-Phe-PEG-20000 converts Group A Rh(D)+ and B Rh(D)+ RBCs into RBCs with serologic behavior comparable to Group O Rh(D)- RBCs that are considered as universal RBCs for transfusion.

Red Dot Basal Cell Carcinoma: An Unusual Variant of a Common Malignancy.

PubMed

Loh, Tiffany Y; Cohen, Philip R

2016-05-01

Red dot basal cell carcinoma is a distinct but rare subtype of basal cell carcinoma (BCC). It presents as a red macule or papule; therefore, in most cases, it may easily be mistaken for a benign vascular lesion, such as a telangiectasia or angioma.
A red dot BCC in an older woman is described. Clinical and histological differences between red dot BCCs and telangiectasias are described.
A 72-year-old woman initially presented with a painless red macule on her nose. Biopsy of the lesion established the diagnosis of a red dot BCC. Pubmed was searched for the following terms: angioma, basal cell carcinoma, dermoscope, diascopy, red dot, non-melanoma skin cancer, telangiectasia, and vascular. The papers were reviewed for cases of red dot basal cell carcinoma. Clinical and histological characteristics of red dot basal cell carcinoma and telangiectasias were compared.
Red dot BCC is an extremely rare variant of BCC that may be confused with benign vascular lesions. Although BCCs rarely metastasize and are associated with low mortality, they have the potential to become locally invasive and destructive if left untreated. Thus, a high index of suspicion for red dot BCC is necessary.

J Drugs Dermatol. 2016;15(5):645-647.

Reflection coefficients of permeant molecules in human red cell suspensions.

PubMed

Owen, J D; Eyring, E M

1975-08-01

The Staverman reflection coefficient, sigma for several permeant molecules was determined in human red cell suspensions with a Durrum stopped-flow spectrophotometer. This procedure was first used with dog, cat, and beef red cells and with human red cells. The stopped-flow technique used was similar to the rapid-flow method used by those who originally reported sigma measurements in human red cells for molecules which rapidly penetrate the red cell membrane. The sigma values we obtained agreed with those previously reported for most of the slow penetrants, except malonamide, but disagreed with all the sigma values previously reported for the rapid penetrants. We were unable to calculate an "equivalent pore radius" with our sigma data. The advantages of our equipment and our experimental procedure are discussed. Our sigma data suggest that sigma is indirectly proportional to the log of the nonelectrolyte permeability coefficient, omega. Since a similar trend has been previously shown for log omega and molar volume of the permeant molecules, a correlatioo was shown between sigma and molar volume suggesting the membrane acts as a sieve.

Antibody-Directed Cytotoxic Agents: Use of Monoclonal Antibody to Direct the Action of Toxin A Chains to Colorectal Carcinoma Cells

NASA Astrophysics Data System (ADS)

Gilliland, D. Gary; Steplewski, Zenon; Collier, R. John; Mitchell, Kenneth F.; Chang, Tong H.; Koprowski, Hilary

1980-08-01

We have constructed cell-specific cytotoxic agents by covalently coupling the A chain from diphtheria toxin or ricin toxin to monoclonal antibody directed against a colorectal carcinoma tumor-associated antigen. Antibody 1083-17-1A was modified by attachment of 3-(2-pyridyldithio)propionyl or cystaminyl groups and then treated with reduced A chain to give disulfide-linked conjugates that retained the original binding specificity of the antibody moiety. The conjugates showed cytotoxic activity for colorectal carcinoma cells in culture, but were not toxic in the same concentration range for a variety of cell lines that lacked the antigen. Under defined conditions virtually 100% of antigen-bearing cultured cells were killed, whereas cells that lacked the antigen were not affected. Conjugates containing toxin A chains coupled to monoclonal antibodies may be useful in studying functions of various cell surface components and, possibly, as tumor-specific therapeutic agents.

VEGFR2-targeted fusion antibody improved NK cell-mediated immunosurveillance against K562 cells.

PubMed

Ren, Xueyan; Xie, Wei; Wang, Youfu; Xu, Menghuai; Liu, Fang; Tang, Mingying; Li, Chenchen; Wang, Min; Zhang, Juan

2016-08-01

MHC class I polypeptide-related sequence A (MICA), which is normally expressed on cancer cells, activates NK cells via NK group 2-member D pathway. However, some cancer cells escape NK-mediated immune surveillance by shedding membrane MICA causing immune suppression. To address this issue, we designed an antibody-MICA fusion targeting tumor-specific antigen (vascular endothelial growth factor receptor 2, VEGFR2) based on our patented antibody (mAb04) against VEGFR2. In vitro results demonstrate that the fusion antibody retains both the antineoplastic and the immunomodulatory activity of mAb04. Further, we revealed that it enhanced NK-mediated immunosurveillance against K562 cells through increasing degranulation and cytokine production of NK cells. The overall data suggest our new fusion protein provides a promising approach for cancer-targeted immunotherapy and has prospects for potential application of chronic myeloid leukemia.

Drugs for preventing red blood cell dehydration in people with sickle cell disease.

PubMed

Nagalla, Srikanth; Ballas, Samir K

2012-07-11

Sickle cell disease is an inherited disorder of hemoglobin, resulting in abnormal red blood cells. These are rigid and may block blood vessels leading to acute painful crises and other complications. Recent research has focused on therapies to rehydrate the sickled cells by reducing the loss of water and ions from them. Little is known about the effectiveness and safety of such drugs. To assess the relative risks and benefits of drugs to rehydrate sickled red blood cells. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Haemoglobinopathies Trials Register.Last search of the Group's Trials Register: 25 October 2011. Randomized or quasi-randomized controlled trials of drugs to rehydrate sickled red blood cells compared to placebo or an alternative treatment. Both authors independently selected studies for inclusion, assessed study quality and extracted data. Of the 51 studies identified, three met the inclusion criteria. The first study tested the effectiveness of zinc sulphate to prevent sickle cell-related crises in a total of 145 participants and showed a significant reduction in painful crises over one and a half years, mean difference -2.83 (95% confidence interval -3.51 to -2.15). However, analysis was restricted due to limited statistical data. Changes to red cell parameters and blood counts were inconsistent. No serious adverse events were noted in the study.The second study was a Phase II dose-finding study of senicapoc (a Gardos channel blocker) compared to placebo. Compared to the placebo group the high dose senicapoc showed significant improvement in change in hemoglobin level, number and proportion of dense red blood cells, red blood cell count and indices and hematocrit. The results with low-dose senicapoc were similar to the high-dose senicapoc group but of lesser magnitude. There was no difference in the frequency of painful crises between the three groups. A subsequent Phase III study of senicapoc was terminated early since

A Novel Assay for Antibody-Dependent Cell-Mediated Cytotoxicity against HIV-1- or SIV-Infected Cells Reveals Incomplete Overlap with Antibodies Measured by Neutralization and Binding Assays

PubMed Central

Alpert, Michael D.; Heyer, Lisa N.; Williams, David E. J.; Harvey, Jackson D.; Greenough, Thomas; Allhorn, Maria

2012-01-01

The resistance of human immunodeficiency virus type 1 (HIV-1) to antibody-mediated immunity often prevents the detection of antibodies that neutralize primary isolates of HIV-1. However, conventional assays for antibody functions other than neutralization are suboptimal. Current methods for measuring the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC) are limited by the number of natural killer (NK) cells obtainable from individual donors, donor-to-donor variation, and the use of nonphysiological targets. We therefore developed an ADCC assay based on NK cell lines that express human or macaque CD16 and a CD4+ T-cell line that expresses luciferase from a Tat-inducible promoter upon HIV-1 or simian immunodeficiency virus (SIV) infection. NK cells and virus-infected targets are mixed in the presence of serial plasma dilutions, and ADCC is measured as the dose-dependent loss of luciferase activity. Using this approach, ADCC titers were measured in plasma samples from HIV-infected human donors and SIV-infected macaques. For the same plasma samples paired with the same test viruses, this assay was approximately 2 orders of magnitude more sensitive than optimized assays for neutralizing antibodies—frequently allowing the measurement of ADCC in the absence of detectable neutralization. Although ADCC correlated with other measures of Env-specific antibodies, neutralizing and gp120 binding titers did not consistently predict ADCC activity. Hence, this assay affords a sensitive method for measuring antibodies capable of directing ADCC against HIV- or SIV-infected cells expressing native conformations of the viral envelope glycoprotein and reveals incomplete overlap of the antibodies that direct ADCC and those measured in neutralization and binding assays. PMID:22933282

The absolute counting of red cell-derived microparticles with red cell bead by flow rate based assay.

PubMed

Nantakomol, Duangdao; Imwong, Malika; Soontarawirat, Ingfar; Kotjanya, Duangporn; Khakhai, Chulalak; Ohashi, Jun; Nuchnoi, Pornlada

2009-05-01

Activation of red blood cell is associated with the formation of red cell-derived microparticles (RMPs). Analysis of circulating RMPs is becoming more refined and clinically useful. A quantitative Trucount tube method is the conventional method uses for quantitating RMPs. In this study, we validated a quantitative method called "flow rate based assay using red cell bead (FCB)" to measure circulating RMPs in the peripheral blood of healthy subjects. Citrated blood samples collected from 30 cases of healthy subjects were determined the RMPs count by using double labeling of annexin V-FITC and anti-glycophorin A-PE. The absolute RMPs numbers were measured by FCB, and the results were compared with the Trucount or with flow rate based calibration (FR). Statistical correlation and agreement were analyzed using linear regression and Bland-Altman analysis. There was no significant difference in the absolute number of RMPs quantitated by FCB when compared with those two reference methods including the Trucount tube and FR method. The absolute RMPs count obtained from FCB method was highly correlated with those obtained from Trucount tube (r(2) = 0.98, mean bias 4 cell/microl, limit of agreement [LOA] -20.3 to 28.3 cell/microl), and FR method (r(2) = 1, mean bias 10.3 cell/microl, and LOA -5.5 to 26.2 cell/microl). This study demonstrates that FCB is suitable and more affordable for RMPs quantitation in the clinical samples. This method is a low cost and interchangeable to latex bead-based method for generating the absolute counts in the resource-limited areas. (c) 2008 Clinical Cytometry Society.

Antibodies and Selection of Monoclonal Antibodies.

PubMed

Hanack, Katja; Messerschmidt, Katrin; Listek, Martin

Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology.

The hSK4 (KCNN4) isoform is the Ca2+-activated K+ channel (Gardos channel) in human red blood cells

PubMed Central

Hoffman, Joseph F.; Joiner, William; Nehrke, Keith; Potapova, Olga; Foye, Kristen; Wickrema, Amittha

2003-01-01

The question is, does the isoform hSK4, also designated KCNN4, represent the small conductance, Ca2+-activated K+ channel (Gardos channel) in human red blood cells? We have analyzed human reticulocyte RNA by RT-PCR, and, of the four isoforms of SK channels known, only SK4 was found. Northern blot analysis of purified and synchronously growing human erythroid progenitor cells, differentiating from erythroblasts to reticulocytes, again showed only the presence of SK4. Western blot analysis, with an anti-SK4 antibody, showed that human erythroid progenitor cells and, importantly, mature human red blood cell ghost membranes, both expressed the SK4 protein. The Gardos channel is known to turn on, given inside Ca2+, in the presence but not the absence of external \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{K}}_{{\\mathrm{o}}}^{+}\\end{equation*}\\end{document} and remains refractory to \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{K}}_{{\\mathrm{o}}}^{+}\\end{equation*}\\end{document} added after exposure to inside Ca2+. Heterologously expressed SK4, but not SK3, also shows this behavior. In inside–out patches of red cell membranes, the open probability (Po) of the Gardos channel is markedly reduced when the temperature is raised from 27 to 37°C. Net K+ efflux of intact red cells is also reduced by increasing temperature, as are the Po values of inside–out patches of Chinese hamster ovary cells expressing SK4 (but not SK3). Thus the envelope of evidence indicates that SK4 is the gene that codes for the Gardos channel in human red blood cells. This channel is important

Immunoproteomics of Plasmodium falciparum-infected red blood cell membrane fractions

PubMed Central

Cabral, Fernanda J; Vianna, Luciana G; Medeiros, Marcia M; Carlos, Bianca Cechetto; Martha, Rosimeire D; Silva, Nadia Maria; da Silva, Luiz Hildebrando P; Stabeli, Rodrigo G; Wunderlich, Gerhard

2017-01-01

BACKGROUND The surface of infected red blood cells (iRBCs) has been widely investigated because of the molecular complexity and pathogenesis mechanisms involved. Asymptomatic individuals are important in the field because they can perpetuate transmission as natural reservoirs and present a challenge for diagnosing malaria because of their low levels of circulating parasites. Recent studies of iRBC antibody recognition have shown that responses are quantitatively similar in symptomatic and asymptomatic infections, but no studies have characterised the plasmodial proteins targeted by this response. OBJECTIVES Our main objective was to identify Plasmodium falciparum proteins associated with iRBC ghosts recognised by antibodies in the sera of symptomatic and asymptomatic individuals in the Brazilian Amazon. METHODS We collected symptomatic and asymptomatic sera from patients residing in the Brazilian Amazon and P. falciparum iRBC ghosts to identify the proteins involved in natural antibody recognition by 2D-electrophoresis, western blotting, and high- resolution mass spectrometry. FINDINGS 2D gel-based immunoproteome analysis using symptomatic and asymptomatic sera identified 11 proteins with at least one unique peptide, such as chaperones HSP70-1 and HSP70-x, which likely are components of the secretion machinery/PTEX translocon. PfEMP1 is involved in antigenic variation in symptomatic infections and we found putative membrane proteins whose functions are unknown. MAIN FINDINGS Our results suggest a potential role of old and new proteins, such as antigenic variation proteins, iRBC remodelling, and membrane proteins, with no assigned functions related to the immune response against P. falciparum, providing insights into the pathogenesis, erythrocyte remodelling, and secretion machinery important for alternative diagnosis and/or malaria therapy. PMID:29211247

Detection of microparticles from human red blood cells by multiparametric flow cytometry

PubMed Central

Grisendi, Giulia; Finetti, Elena; Manganaro, Daniele; Cordova, Nicoletta; Montagnani, Giuliano; Spano, Carlotta; Prapa, Malvina; Guarneri, Valentina; Otsuru, Satoru; Horwitz, Edwin M.; Mari, Giorgio; Dominici, Massimo

2015-01-01

Background During storage, red blood cells (RBC) undergo chemical and biochemical changes referred to as “storage lesions”. These events determine the loss of RBC integrity, resulting in lysis and release of microparticles. There is growing evidence of the clinical importance of microparticles and their role in blood transfusion-related side effects and pathogen transmission. Flow cytometry is currently one of the most common techniques used to quantify and characterise microparticles. Here we propose multiparametric staining to monitor and quantify the dynamic release of microparticles by stored human RBC. Material and methods RBC units (n=10) were stored under blood bank conditions for up to 42 days. Samples were tested at different time points to detect microparticles and determine the haemolysis rate (HR%). Microparticles were identified by flow cytometry combining carboxyfluorescein diacetate succinimidyl ester (CFSE) dye, annexin V and anti-glycophorin A antibody. Results We demonstrated that CFSE can be successfully used to label closed vesicles with an intact membrane. The combination of CFSE and glycophorin A antibody was effective for monitoring and quantifying the dynamic release of microparticles from RBC during storage. Double staining with CFSE/glycophorin A was a more precise approach, increasing vesicle detection up to 4.7-fold vs the use of glycophorin A/annexin V alone. Moreover, at all the time points tested, we found a robust correlation (R=0.625; p=0.0001) between HR% and number of microparticles detected. Discussion Multiparametric staining, based on a combination of CFSE, glycophorin A antibody and annexin V, was able to detect, characterise and monitor the release of microparticles from RBC units during storage, providing a sensitive approach to labelling and identifying microparticles for transfusion medicine and, more broadly, for cell-based therapies. PMID:25369588

Image classification of unlabeled malaria parasites in red blood cells.

PubMed

Zheng Zhang; Ong, L L Sharon; Kong Fang; Matthew, Athul; Dauwels, Justin; Ming Dao; Asada, Harry

2016-08-01

This paper presents a method to detect unlabeled malaria parasites in red blood cells. The current "gold standard" for malaria diagnosis is microscopic examination of thick blood smear, a time consuming process requiring extensive training. Our goal is to develop an automate process to identify malaria infected red blood cells. Major issues in automated analysis of microscopy images of unstained blood smears include overlapping cells and oddly shaped cells. Our approach creates robust templates to detect infected and uninfected red cells. Histogram of Oriented Gradients (HOGs) features are extracted from templates and used to train a classifier offline. Next, the ViolaJones object detection framework is applied to detect infected and uninfected red cells and the image background. Results show our approach out-performs classification approaches with PCA features by 50% and cell detection algorithms applying Hough transforms by 24%. Majority of related work are designed to automatically detect stained parasites in blood smears where the cells are fixed. Although it is more challenging to design algorithms for unstained parasites, our methods will allow analysis of parasite progression in live cells under different drug treatments.

[Establishment and identification of mouse lymphoma cell line EL4 expressing red fluorescent protein].

PubMed

Li, Yan-Jie; Cao, Jiang; Chen, Chong; Wang, Dong-Yang; Zeng, Ling-Yu; Pan, Xiu-Ying; Xu, Kai-Lin

2010-02-01

This study was purposed to construct a lentiviral vector encoding red fluorescent protein (DsRed) and transfect DsRed into EL4 cells for establishing mouse leukemia/lymphoma model expressing DsRed. The bicistronic SIN lentiviral transfer plasmid containing the genes encoding neo and internal ribosomal entry site-red fluorescent protein (IRES-DsRed) was constructed. Human embryonic kidney 293FT cells were co-transfected with the three plasmids by liposome method. The viral particles were collected and used to transfect EL4 cells, then the cells were selected by G418. The results showed that the plasmid pXZ208-neo-IRES-DsRed was constructed successfully, and the viral titer reached to 10(6) U/ml. EL4 cells were transfected by the viral solution efficiently. The transfected EL4 cells expressing DsRed survived in the final concentration 600 microg/ml of G418. The expression of DsRed in the transfected EL4 cells was demonstrated by fluorescence microscopy and flow cytometry. In conclusion, the EL4/DsRed cell line was established successfully.

Measuring skewness of red blood cell deformability distribution by laser ektacytometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nikitin, S Yu; Priezzhev, A V; Lugovtsov, A E

An algorithm is proposed for measuring the parameters of red blood cell deformability distribution based on laser diffractometry of red blood cells in shear flow (ektacytometry). The algorithm is tested on specially prepared samples of rat blood. In these experiments we succeeded in measuring the mean deformability, deformability variance and skewness of red blood cell deformability distribution with errors of 10%, 15% and 35%, respectively. (laser biophotonics)

A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells

NASA Astrophysics Data System (ADS)

Colcher, D.; Horan Hand, P.; Nuti, M.; Schlom, J.

1981-05-01

Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts of apparently normal human liver. As a result of further screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a ``pancarcinoma'' reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.

Development and characterization of a monoclonal antibody to human embryonal carcinoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khazaeli, M.B.; Beierwaltes, W.H.; Pitt, G.S.

1987-06-01

A monoclonal anti-testicular carcinoma antibody was obtained via the somatic cell fusion technique by immunization of BALB/c mice with freshly prepared single cell suspension from a patient with testicular embryonal carcinoma with choriocarcinoma components. The hybridoma supernates were screened against the testicular carcinoma cells used in the immunization as well as normal mononuclear white blood cells isolated from the same patient. An antibody (5F9) was selected which bound to fresh tumor cells from two patients with embryonal testicular carcinoma and failed to bind to fresh tumor cells from 24 patients (2 seminoma, 2 melanoma, 3 neck, 2 esophageal, 1 ovarian,more » 3 colon, 1 prostate, 2 breast, 1 liposarcoma, 3 endometrial, 1 kidney, 1 adrenal, 1 larynx and 1 bladder tumors) or cell suspensions prepared from normal liver, lung, spleen, ovary, testes, kidney, red blood cells or white blood cells. The antibody was tested for its binding to several well established cancer cell lines, and was found to bind to the BeWo human choriocarcinoma and two human embryonal carcinoma cell lines. The antibody did not react with 22 other cell lines or with hCG. The antibody was labeled with /sup 131/I and injected into nude mice bearing BeWo tumors and evaluated for tumor localization by performing whole body scans with a gamma camera 5 days later. Six mice injected with the antibody showed positive tumor localization without the need for background subtraction while six mice injected with MOPC-21, a murine myeloma immunoglobulin, demonstrated much less tumor localization. Tissue distribution studies performed after scanning showed specific tumor localization (8:1 tumor: muscle) for the monoclonal antibody and no specific localization for MOPC-21.« less

Comparative study on antibody immobilization strategies for efficient circulating tumor cell capture.

PubMed

Ates, Hatice Ceren; Ozgur, Ebru; Kulah, Haluk

2018-03-23

Methods for isolation and quantification of circulating tumor cells (CTCs) are attracting more attention every day, as the data for their unprecedented clinical utility continue to grow. However, the challenge is that CTCs are extremely rare (as low as 1 in a billion of blood cells) and a highly sensitive and specific technology is required to isolate CTCs from blood cells. Methods utilizing microfluidic systems for immunoaffinity-based CTC capture are preferred, especially when purity is the prime requirement. However, antibody immobilization strategy significantly affects the efficiency of such systems. In this study, two covalent and two bioaffinity antibody immobilization methods were assessed with respect to their CTC capture efficiency and selectivity, using an anti-epithelial cell adhesion molecule (EpCAM) as the capture antibody. Surface functionalization was realized on plain SiO 2 surfaces, as well as in microfluidic channels. Surfaces functionalized with different antibody immobilization methods are physically and chemically characterized at each step of functionalization. MCF-7 breast cancer and CCRF-CEM acute lymphoblastic leukemia cell lines were used as EpCAM positive and negative cell models, respectively, to assess CTC capture efficiency and selectivity. Comparisons reveal that bioaffinity based antibody immobilization involving streptavidin attachment with glutaraldehyde linker gave the highest cell capture efficiency. On the other hand, a covalent antibody immobilization method involving direct antibody binding by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)-N-hydroxysuccinimide (NHS) reaction was found to be more time and cost efficient with a similar cell capture efficiency. All methods provided very high selectivity for CTCs with EpCAM expression. It was also demonstrated that antibody immobilization via EDC-NHS reaction in a microfluidic channel leads to high capture efficiency and selectivity.

Automated microscopy system for detection and genetic characterization of fetal nucleated red blood cells on slides

NASA Astrophysics Data System (ADS)

Ravkin, Ilya; Temov, Vladimir

1998-04-01

The detection and genetic analysis of fetal cells in maternal blood will permit noninvasive prenatal screening for genetic defects. Applied Imaging has developed and is currently evaluating a system for semiautomatic detection of fetal nucleated red blood cells on slides and acquisition of their DNA probe FISH images. The specimens are blood smears from pregnant women (9 - 16 weeks gestation) enriched for nucleated red blood cells (NRBC). The cells are identified by using labeled monoclonal antibodies directed to different types of hemoglobin chains (gamma, epsilon); the nuclei are stained with DAPI. The Applied Imaging system has been implemented with both Olympus BX and Nikon Eclipse series microscopes which were equipped with transmission and fluorescence optics. The system includes the following motorized components: stage, focus, transmission, and fluorescence filter wheels. A video camera with light integration (COHU 4910) permits low light imaging. The software capabilities include scanning, relocation, autofocusing, feature extraction, facilities for operator review, and data analysis. Detection of fetal NRBCs is achieved by employing a combination of brightfield and fluorescence images of nuclear and cytoplasmic markers. The brightfield and fluorescence images are all obtained with a single multi-bandpass dichroic mirror. A Z-stack of DNA probe FISH images is acquired by moving focus and switching excitation filters. This stack is combined to produce an enhanced image for presentation and spot counting.

Method for determining properties of red blood cells

DOEpatents

Gourley, Paul L.

2001-01-01

A method for quantifying the concentration of hemoglobin in a cell, and indicia of anemia, comprises determining the wavelength of the longitudinal mode of a liquid in a laser microcavity; determining the wavelength of the fundamental transverse mode of a red blood cell in the liquid in the laser microcavity; and determining if the cell is anemic from the difference between the wavelength of the longitudinal mode and the fundamental transverse mode. In addition to measuring hemoglobin, the invention includes a method using intracavity laser spectroscopy to measure the change in spectra as a function of time for measuring the influx of water into a red blood cell and the cell's subsequent rupture.

Naive T-cell receptor transgenic T cells help memory B cells produce antibody

PubMed Central

Duffy, Darragh; Yang, Chun-Ping; Heath, Andrew; Garside, Paul; Bell, Eric B

2006-01-01

Injection of the same antigen following primary immunization induces a classic secondary response characterized by a large quantity of high-affinity antibody of an immunoglobulin G class produced more rapidly than in the initial response – the products of memory B cells are qualitatively distinct from that of the original naive B lymphocytes. Very little is known of the help provided by the CD4 T cells that stimulate memory B cells. Using antigen-specific T-cell receptor transgenic CD4 T cells (DO11.10) as a source of help, we found that naive transgenic T cells stimulated memory B cells almost as well (in terms of quantity and speed) as transgenic T cells that had been recently primed. There was a direct correlation between serum antibody levels and the number of naive transgenic T cells transferred. Using T cells from transgenic interleukin-2-deficient mice we showed that interleukin-2 was not required for a secondary response, although it was necessary for a primary response. The results suggested that the signals delivered by CD4 T cells and required by memory B cells for their activation were common to both antigen-primed and naive CD4 T cells. PMID:17067314

Hemoglobin Aggregation in Single Red Blood Cells of Sickle Cell Anemia

NASA Astrophysics Data System (ADS)

Nishio, Izumi; Tanaka, Toyoichi; Sun, Shao-Tang; Imanishi, Yuri; Tsuyoshi Ohnishi, S.

1983-06-01

A laser light scattering technique was used to observe the extent of hemoglobin aggregation in solitary red blood cells of sickle cell anemia. Hemoglobin aggregation was confirmed in deoxygenated cells. The light scattering technique can also be applied to cytoplasmic studies of any biological cell.

Red cell density is sex and race dependent in the adult.

PubMed

Blumenfeld, N; Fabry, M E; Thysen, B; Nagel, R L

1988-09-01

Using a highly sensitive method for the determination of red cell densities (Percoll-Stractan continuous isopyknic gradients), we find that, in adults, this parameter varies with sex and race. Whites have red cell densities (expressed as mean corpuscular hemoglobin concentration [MCHC]) that are, on the average, 0.7 gm/dl higher than those in blacks (the difference of the means has p less than 2 x 10(-7]. White men have, on the average, 0.6 gm/dl higher MCHC than white women (the difference of the means has p less than 6 x 10(-5]. We find a strong correlation between all red cell densities and intracellular K+ and a slightly weaker correlation between red cell density and intracellular Na+ + K+. Men have an average intraerythrocytic K+ that is approximately 4.5 mmol/L of red cells less than that of women among whites as well as blacks (p less than 10(-5) and p less than 9 x 10(-4), respectively). Blacks have significantly higher plasma ferritin levels than do whites (in addition to the sex difference). Future work will have to dissect the possible causes of these differences, which include the high incidence of deletional alpha-thalassemia (-a/aa) among blacks, menstruation, hormonal effects, and the red cell transport and volume regulation differences between sexes and races. Whatever the cause of the sex and racial differences reported here, they are bound to affect the pathophysiologic expression of genetic red cell diseases that are particularly sensitive to the MCHC, such as the sickle cell syndromes.

From Red Cells to Soft Porous Lubrication

NASA Astrophysics Data System (ADS)

Wu, Qianhong; Gacka, Thomas; Nathan, Rungun; Crawford, Robert; Vucbmss Team

2014-11-01

Biological scientists have wondered, since the motion of red cells was first observed in capillaries, how the highly flexible red cell can move with so little friction in tightly fitting microvessels without being damaged or undergoing hemolysis. Theoretical studies (Feng and Weinbaum, 2000, JFM; Wu et al., 2004, PRL) attributed this frictionless motion to the dramatically enhanced hydrodynamic lifting force generated inside the soft, porous, endothelial surface layer (ESL) covering the inner surfaces of our capillaries, as a red blood cell glides over it. Herein we report the first experimental examination of this concept. The results conclusively demonstrate that significant fraction of the overall lifting force generated in a soft porous layer as a planing surface glides over it, is contributed by the pore fluid pressure, and thus frictional loss is reduced significantly. Moreover, the experimental predictions showed excellent agreement with the experimental data. This finding has the potential of dramatically changing existing lubrication approaches, and can result in substantial savings in energy consumption and thus reduction in greenhouse gas emissions.

Avoiding Anemia: Boost Your Red Blood Cells

MedlinePlus

... Issues Subscribe January 2014 Print this issue Avoiding Anemia Boost Your Red Blood Cells En español Send ... Disease When Blood Cells Bend Wise Choices Preventing Anemia To prevent or treat iron-deficiency anemia: Eat ...

Drugs for preventing red blood cell dehydration in people with sickle cell disease.

PubMed

Nagalla, Srikanth; Ballas, Samir K

2016-03-04

Sickle cell disease is an inherited disorder of hemoglobin, resulting in abnormal red blood cells. These are rigid and may block blood vessels leading to acute painful crises and other complications. Recent research has focused on therapies to rehydrate the sickled cells by reducing the loss of water and ions from them. Little is known about the effectiveness and safety of such drugs. This is an updated version of a previously published review. To assess the relative risks and benefits of drugs to rehydrate sickled red blood cells. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Haemoglobinopathies Trials Register.Last search of the Group's Trials Register: 28 November 2015. Randomized or quasi-randomized controlled trials of drugs to rehydrate sickled red blood cells compared to placebo or an alternative treatment. Both authors independently selected studies for inclusion, assessed study quality and extracted data. Of the 51 studies identified, three met the inclusion criteria. The first study tested the effectiveness of zinc sulphate to prevent sickle cell-related crises in a total of 145 participants and showed a significant reduction in painful crises over one and a half years, mean difference -2.83 (95% confidence interval -3.51 to -2.15). However, analysis was restricted due to limited statistical data. Changes to red cell parameters and blood counts were inconsistent. No serious adverse events were noted in the study.The second study was a Phase II dose-finding study of senicapoc (a Gardos channel blocker) compared to placebo. Compared to the placebo group the high dose senicapoc showed significant improvement in change in hemoglobin level, number and proportion of dense red blood cells, red blood cell count and indices and hematocrit. The results with low-dose senicapoc were similar to the high-dose senicapoc group but of lesser magnitude. There was no difference in the frequency of painful crises between the three groups. A

Immunization with SV40-transformed cells yields mainly MHC-restricted monoclonal antibodies

PubMed Central

1986-01-01

Recognition of antigens on cell surfaces only in the context of the MHC- encoded alloantigens of the presenting cell (self + X) has classically been considered the province of T cells. However, evidence from several sources has indicated that B cells and antibodies can exhibit self + X- restricted recognition as well. This report concerns the mAb response to SV40-transformed H-2b fibroblast cell lines. The specificities of the antibodies obtained have been analyzed for binding to a panel of SV40-transformed H-2-syngeneic, H-2-allogeneic, and H-2b mutant fibroblast cell lines, as well as cell lines not bearing cell surface SV40 transformation-associated antigens. A large proportion of primary C57BL/6 (71%) and BALB/c (68%) splenic B cells responding to in vitro stimulation with SV40-transformed H-2b cells recognize cell surface antigens associated with SV40 transformation only when coexpressed with MHC antigens of the immunizing cell, particularly the Kb molecule, on transformed cells. To extensively define the nature of antigen recognition by these antibodies, we have generated and characterized nine hybridoma antibodies specific for SV40-transformed H-2-syngeneic cell lines. Seven of these hybridoma antibodies recognize SV40- associated transformation antigens in the context of H-2b molecules. Six of these are restricted by the Kb molecule and discriminate among a panel of SV40-transformed Kb mutant cell lines, thus confirming the participation of class I MHC-encoded molecules in the recognition by B cells of cell surface antigens. PMID:3014034

Elimination of human T cell leukemia virus type-1-infected cells by neutralizing and antibody-dependent cellular cytotoxicity-inducing antibodies against human t cell leukemia virus type-1 envelope gp46.

PubMed

Tanaka, Yuetsu; Takahashi, Yoshiaki; Tanaka, Reiko; Kodama, Akira; Fujii, Hideki; Hasegawa, Atsuhiko; Kannagi, Mari; Ansari, Aftab A; Saito, Mineki

2014-06-01

Human T cell leukemia virus type-1 (HTLV-1) is prevalent worldwide with foci of high prevalence. However, to date no effective vaccine or drug against HTLV-1 infection has been developed. In efforts to define the role of antibodies in the control of HTLV-1 infection, we capitalized on the use of our previously defined anti-gp46 neutralizing monoclonal antibody (mAb) (clone LAT-27) and high titers of human anti-HTLV-1 IgG purified from HAM/TSP patients (HAM-IgG). LAT-27 and HAM-IgG completely blocked syncytium formation and T cell immortalization mediated by HTLV-1 in vitro. The addition of these antibodies to cultures of CD8(+) T cell-depleted peripheral blood mononuclear cells (PBMCs) from HAM/TSP patients at the initiation of culture not only decreased the numbers of Tax-expressing cells and the production of HTLV-1 p24 but also inhibited the spontaneous immortalization of T cells. Coculture of in vitro-HTLV-1-immortalized T cell lines with autologous PBMCs in the presence of LAT-27 or HAM-IgG, but not an F(ab')2 fragment of LAT-27 or nonneutralizing anti-gp46 mAbs, resulted in depletion of HTLV-1-infected cells. A 24-h (51)Cr release assay showed the presence of significant antibody-dependent cellular cytotoxicity (ADCC) activity in LAT-27 and HAM-IgG, but not F(ab')2 of LAT-27, resulting in the depletion of HTLV-1-infected T cells by autologous PBMCs. The depletion of natural killer (NK) cells from the effector PBMCs reduced this ADCC activity. Altogether, the present data demonstrate that the neutralizing and ADCC-inducing activities of anti-HTLV-1 antibodies are capable of reducing infection and eliminating HTLV-1-infected cells in the presence of autologous PBMCs.

A Quantitative Method for Comparing the Brightness of Antibody-dye Reagents and Estimating Antibodies Bound per Cell.

PubMed

Kantor, Aaron B; Moore, Wayne A; Meehan, Stephen; Parks, David R

2016-07-01

We present a quantitative method for comparing the brightness of antibody-dye reagents and estimating antibodies bound per cell. The method is based on complementary binding of test and fill reagents to antibody capture microspheres. Several aliquots of antibody capture beads are stained with varying amounts of the test conjugate. The remaining binding sites on the beads are then filled with a second conjugate containing a different fluorophore. Finally, the fluorescence of the test conjugate compared to the fill conjugate is used to measure the relative brightness of the test conjugate. The fundamental assumption of the test-fill method is that if it takes X molecules of one test antibody to lower the fill signal by Y units, it will take the same X molecules of any other test antibody to give the same effect. We apply a quadratic fit to evaluate the test-fill signal relationship across different amounts of test reagent. If the fit is close to linear, we consider the test reagent to be suitable for quantitative evaluation of antibody binding. To calibrate the antibodies bound per bead, a PE conjugate with 1 PE molecule per antibody is used as a test reagent and the fluorescence scale is calibrated with Quantibrite PE beads. When the fluorescence per antibody molecule has been determined for a particular conjugate, that conjugate can be used for measurement of antibodies bound per cell. This provides comparisons of the brightness of different conjugates when conducted on an instrument whose statistical photoelectron (Spe) scales are known. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

[Studies on red orpiment induction of NB4 and HL-60 cell apoptosis].

PubMed

Bai, Y; Huang, S

1998-09-01

To study the possible mechanism of red orpiment, which is main component of composite indigo naturalis tablets, in the treatment of acute promyelocytic leukemia(APL). The effect of red orpiment on induction of APL cell line NB4 and HL-60 apoptosis were studied by cell morphology, DNA gel electrophoresis and flow cytometry assay. Red orpiment induced NB4 and HL-60 cell apoptosis. When treated with different concentration of red orpiment(25-200 micrograms/ml) for 16 hours, both NB4 and HL-60 cells showed typical apoptosis features. If decreased the concentration of red orpiment to 12.5 micrograms/ml, the NB4 cell still showed apoptosis features while the HL-60 cell did not when cultured for 72 hours. Arsenic disulfide(As2S2) had the same effect as red orpiment did under the same experiment condition. It is the main component, As2S2 of the red orpiment that can induces NB4 and HL-60 cell apoptosis. and the red orpiment is responsible for the high CR rate of APL induced by the composite indigo naturalis tablets.

Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells

PubMed Central

2010-01-01

Trastuzumab (Herceptin®), a humanized IgG1 antibody raised against the human epidermal growth factor receptor 2 (HER2/neu), is the main antibody in clinical use against breast cancer. Pre-clinical evidence and clinical studies indicate that trastuzumab employs several anti-tumour mechanisms that most likely contribute to enhanced survival of patients with HER2/neu-positive breast carcinomas. New strategies are aimed at improving antibody-based therapeutics like trastuzumab, e.g. by enhancing antibody-mediated effector function mechanisms. Based on our previous findings that a chimaeric ovarian tumour antigen-specific IgE antibody showed greater efficacy in tumour cell killing, compared to the corresponding IgG1 antibody, we have produced an IgE homologue of trastuzumab. Trastuzumab IgE was engineered with the same light- and heavy-chain variable-regions as trastuzumab, but with an epsilon in place of the gamma-1 heavy-chain constant region. We describe the physical characterisation and ligand binding properties of the trastuzumab IgE and elucidate its potential anti-tumour activities in functional assays. Both trastuzumab and trastuzumab IgE can activate monocytic cells to kill tumour cells, but they operate by different mechanisms: trastuzumab functions in antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE functions in antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab IgE, incubated with mast cells and HER2/neu-expressing tumour cells, triggers mast cell degranulation, recruiting against cancer cells a potent immune response, characteristic of allergic reactions. Finally, in viability assays both antibodies mediate comparable levels of tumour cell growth arrest. These functional characteristics of trastuzumab IgE, some distinct from those of trastuzumab, indicate its potential to complement or improve upon the existing clinical benefits of trastuzumab. PMID:18941743

Phosphate Ion Exchange Resin Used in the Liquid Preservation of Baboon Red Blood Cells.

DTIC Science & Technology

1982-06-08

absence of resin. The addition of a phosphate anion exchange resin to the CPD anticoagulant provided better maintenance of red cell 23 DPG and P50 levels ...than red blood cells S.prepared from blood without resin. Red blood cell ATP levels and 24-hour post- transfusion survival values were similar whether or...coagulant provided better maintenance of red cell 2,3 DPG and P50 levels during storage of whole blood at 4 C, and red blood cells prepared from whole

Biosignatures of Kerala red rain cells: Implications in understanding their origin

NASA Astrophysics Data System (ADS)

Gangappa, R.; Thomas, M.; Hogg, S.

2013-09-01

The red rain that fell over Kerala, southern India (2001-2012) was characterised by the red pigmented particles. Earlier proposal claiming that these are known algal bloom blown from trees (Sampath et al, 2001; DiGregorio, 2007) has been studied by us and disproved. Also, further investigation reporting their extraordinary properties including a suggestion that they lack DNA (Louis and Kumar 2003; 2006; 2008) has been invalidated (Gangappa and Hogg, 2013). However, their claim regarding the growth and replication of these cells at 300ºC needs more investigation if it is to gain acceptance. Current study provide evidences regarding the biological properties of Kerala red rain cells to gain insights into environmental conditions from which they may have originated. Combined with various research strategies and high resolution instruments, we have demonstrated the following interesting properties of Kerala red rain cells: (1) unusually thick external envelope enclosing the central core; (2)stability of red pigment at temperatures about 100ºC and pH variations; (3) absence of eukaryotic ultrastructures; (4) possible replication at 121ºC with nanostructures (possible daughter cells) having similar morphological features inside the large mother cells at such high temperature. They contain high percentage of carbon, iron, silicon and aluminum and often enclosed in a silicon rich biofilms. Further investigation shows that the positive detection of DNA in these cells was possible only after the complete removal of red pigment, thereby providing an explanation for the negative outcome of earlier studies in this regard. Moreover, evidences are shown to support that these cells contain high amounts of UV absorbing compounds, porphyrin complexes and possible scytonemin. Kerala red rain cells may prove to be polyextermophiles belonging to prokaryotes and may have possibly originated from the environment containing above mentioned chemical elements, high energy UV exposure and

Red Dot Basal Cell Carcinoma: Report of Cases and Review of This Unique Presentation of Basal Cell Carcinoma.

PubMed

Cohen, Philip R

2017-03-22

Red dot basal cell carcinoma is a unique variant of basal cell carcinoma. Including the three patients described in this report, red dot basal cell carcinoma has only been described in seven individuals. This paper describes the features of two males and one female with red dot basal cell carcinoma and reviews the characteristics of other patients with this clinical subtype of basal cell carcinoma. A 70-year-old male developed a pearly-colored papule with a red dot in the center on his nasal tip. A 71-year-old male developed a red dot surrounded by a flesh-colored papule on his left nostril. Lastly, a 74-year-old female developed a red dot within an area of erythema on her left mid back. Biopsy of the lesions all showed nodular and/or superficial basal cell carcinoma. Correlation of the clinical presentation and pathology established the diagnosis of red dot basal cell carcinoma. The tumors were treated by excision using the Mohs surgical technique. Pubmed was searched with the keyword: basal, cell, cancer, carcinoma, dot, red, and skin. The papers generated by the search and their references were reviewed. Red dot basal cell carcinoma has been described in three females and two males; the gender was not reported in two patients. The tumor was located on the nose (five patients), back (one patient) and thigh (one patient). Cancer presented as a solitary small red macule or papule; often, the carcinoma was surrounded by erythema or a flesh-colored papule. Although basal cell carcinomas usually do not blanch after a glass microscope slide is pressed against them, the red dot basal cell carcinoma blanched after diascopy in two of the patients, resulting in a delay of diagnosis in one of these individuals. Dermoscopy may be a useful non-invasive modality for evaluating skin lesions when the diagnosis of red dot basal cell carcinoma is considered. Mohs surgery is the treatment of choice; in some of the patients, the ratio of the area of the postoperative wound to that

Red Dot Basal Cell Carcinoma: Report of Cases and Review of This Unique Presentation of Basal Cell Carcinoma

PubMed Central

2017-01-01

Red dot basal cell carcinoma is a unique variant of basal cell carcinoma. Including the three patients described in this report, red dot basal cell carcinoma has only been described in seven individuals. This paper describes the features of two males and one female with red dot basal cell carcinoma and reviews the characteristics of other patients with this clinical subtype of basal cell carcinoma. A 70-year-old male developed a pearly-colored papule with a red dot in the center on his nasal tip. A 71-year-old male developed a red dot surrounded by a flesh-colored papule on his left nostril. Lastly, a 74-year-old female developed a red dot within an area of erythema on her left mid back. Biopsy of the lesions all showed nodular and/or superficial basal cell carcinoma. Correlation of the clinical presentation and pathology established the diagnosis of red dot basal cell carcinoma. The tumors were treated by excision using the Mohs surgical technique. Pubmed was searched with the keyword: basal, cell, cancer, carcinoma, dot, red, and skin. The papers generated by the search and their references were reviewed. Red dot basal cell carcinoma has been described in three females and two males; the gender was not reported in two patients. The tumor was located on the nose (five patients), back (one patient) and thigh (one patient). Cancer presented as a solitary small red macule or papule; often, the carcinoma was surrounded by erythema or a flesh-colored papule. Although basal cell carcinomas usually do not blanch after a glass microscope slide is pressed against them, the red dot basal cell carcinoma blanched after diascopy in two of the patients, resulting in a delay of diagnosis in one of these individuals. Dermoscopy may be a useful non-invasive modality for evaluating skin lesions when the diagnosis of red dot basal cell carcinoma is considered. Mohs surgery is the treatment of choice; in some of the patients, the ratio of the area of the postoperative wound to that

Circulating red cell-derived microparticles in human malaria.

PubMed

Nantakomol, Duangdao; Dondorp, Arjen M; Krudsood, Srivicha; Udomsangpetch, Rachanee; Pattanapanyasat, Kovit; Combes, Valery; Grau, Georges E; White, Nicholas J; Viriyavejakul, Parnpen; Day, Nicholas P J; Chotivanich, Kesinee

2011-03-01

In patients with falciparum malaria, plasma concentrations of cell-derived microparticles correlate with disease severity. Using flow cytometry, we quantified red blood cell-derived microparticles (RMPs) in patients with malaria and identified the source and the factors associated with production. RMP concentrations were increased in patients with Plasmodium falciparum (n = 29; median, 457 RMPs/μL [range, 13-4,342 RMPs/μL]), Plasmodium vivax (n = 5; median, 409 RMPs/μL [range, 281-503/μL]), and Plasmodium malariae (n = 2; median, 163 RMPs/μL [range, 127-200 RMPs/μL]) compared with those in healthy subjects (n = 11; median, 8 RMPs/μL [range, 3-166 RMPs/μL]; P = .01). RMP concentrations were highest in patients with severe falciparum malaria (P = .01). Parasitized red cells produced >10 times more RMPs than did unparasitized cells, but the overall majority of RMPs still derived from uninfected red blood cells (URBCs). In cultures, RMP production increased as the parasites matured. Hemin and parasite products induced RMP production in URBCs, which was inhibited by N-acetylcysteine, suggesting heme-mediated oxidative stress as a pathway for the generation of RMPs.

Risk of pure red cell aplasia in patients with hepatitis C receiving antiviral therapy and an erythropoiesis-stimulating agent.

PubMed

Rossert, Jerome; Yue, Susan; Smirnakis, Karen; Mytych, Daniel T; Johnson, Larry; Kouchakji, Elias; Casadevall, Nicole

2014-02-01

Antibody-mediated pure red cell aplasia (PRCA) has been primarily observed in patients with chronic kidney disease treated with an erythropoiesis-stimulating agent (ESA); only a few anecdotal cases have been reported in other patient populations. We searched the Amgen Global Safety Adverse Event Database and identified 14 patients with hepatitis C who developed severe anemia, anti-erythropoietin antibodies, and bone marrow biopsy-proven PRCA, while receiving interferon therapy (with or without ribavirin) and an ESA. During the follow-up period and after ESA treatment stopped, 11 patients no longer required transfusions and 3 did. Analysis of antibody isotypes showed that, contrary to reports of patients with chronic kidney disease, immunoglobulin G1 was the predominant isotype rather than immunoglobulin G4 (immunoglobulin G4 was detected in only 1 of 6 patients). Epitope mapping showed the anti-erythropoietin antibodies bound domains required for receptor binding. Therefore, the potential benefits of ESA therapy must be weighed against the risk for PRCA in patients with hepatitis C who are receiving treatment with interferon and ribavirin. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Phosphatidylserine exposure on stored red blood cells as a parameter for donor-dependent variation in product quality.

PubMed

Dinkla, Sip; Peppelman, Malou; Van Der Raadt, Jori; Atsma, Femke; Novotný, Vera M J; Van Kraaij, Marian G J; Joosten, Irma; Bosman, Giel J C G M

2014-04-01

Exposure of phosphatidylserine on the outside of red blood cells contributes to recognition and removal of old and damaged cells. The fraction of phosphatidylserine-exposing red blood cells varies between donors, and increases in red blood cell concentrates during storage. The susceptibility of red blood cells to stress-induced phosphatidylserine exposure increases with storage. Phosphatidylserine exposure may, therefore, constitute a link between donor variation and the quality of red blood cell concentrates. In order to examine the relationship between storage parameters and donor characteristics, the percentage of phosphatidylserine-exposing red blood cells was measured in red blood cell concentrates during storage and in fresh red blood cells from blood bank donors. The percentage of phosphatidylserine-exposing red blood cells was compared with red blood cell susceptibility to osmotic stress-induced phosphatidylserine exposure in vitro, with the regular red blood cell concentrate quality parameters, and with the donor characteristics age, body mass index, haemoglobin level, gender and blood group. Phosphatidylserine exposure varies between donors, both on red blood cells freshly isolated from the blood, and on red blood cells in red blood cell concentrates. Phosphatidylserine exposure increases with storage time, and is correlated with stress-induced phosphatidylserine exposure. Increased phosphatidylserine exposure during storage was found to be associated with haemolysis and vesicle concentration in red blood cell concentrates. The percentage of phosphatidylserine-exposing red blood cells showed a positive correlation with the plasma haemoglobin concentration of the donor. The fraction of phosphatidylserine-exposing red blood cells is a parameter of red blood cell integrity in red blood cell concentrates and may be an indicator of red blood cell survival after transfusion. Measurement of phosphatidylserine exposure may be useful in the selection of donors and

Density increment and decreased survival of rat red blood cells induced by cadmium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kunimoto, M.; Miura, T.

1986-01-01

Male Wistar rats were injected with CdCl/sub 2/ subcutaneously to examine in vivo effects of Cd on density and survival of red blood cells. During the 7 days after administration of 1.0 mg Cd/kg, the following sequence of events occurred: (1) a progressive increase in the amount of more dense red blood cells concomitant with a decrease in that of light red blood cells from the first to the third day; (2) an increase in the spleen weight at the third day; (3) a decrease in the hematocrit value and an increase in the amount of light red blood cellsmore » at the fifth day; and (4) a recovery of the hematocrit value at the seventh day. Five days after administration, the hematocrit value decreased in a dose-dependent mode and the decrease was significant at the 1% level at 1.0 and 1.5 mg Cd/kg. A highly significant splenomegaly was also observed at 0.5 to 1.5 mg Cd/kg. In order to label red blood cells in vivo, (/sup 3/H) diisopropylfluorophosphate ((/sup 3/H)DFP) was injected into rats. At Day 11, Cd at either 0.5 or 1.0 mg/kg was administered to (/sup 3/H)DFP-prelabeled animals. Cd administration accelerated /sup 3/H-labeled red cell clearance from the blood. Six days after Cd administration, the radioactivity of red blood cells was 76 and 68% of the control at 0.5 and 1.0 mg Cd/kg, respectively. In vitro treatment of rat red density and accelerated in vivo clearance of red blood cells from the recipient circulation. These results show that Cd at low dose can cause anemia by increasing red cell density and by accelerating red cell sequestration, presumably in the spleen.« less

Red cell distribution width does not predict stroke severity or functional outcome.

PubMed

Ntaios, George; Gurer, Ozgur; Faouzi, Mohamed; Aubert, Carole; Michel, Patrik

2012-01-01

Red cell distribution width was recently identified as a predictor of cardiovascular and all-cause mortality in patients with previous stroke. Red cell distribution width is also higher in patients with stroke compared with those without. However, there are no data on the association of red cell distribution width, assessed during the acute phase of ischemic stroke, with stroke severity and functional outcome. In the present study, we sought to investigate this relationship and ascertain the main determinants of red cell distribution width in this population. We used data from the Acute Stroke Registry and Analysis of Lausanne for patients between January 2003 and December 2008. Red cell distribution width was generated at admission by the Sysmex XE-2100 automated cell counter from ethylene diamine tetraacetic acid blood samples stored at room temperature until measurement. An χ(2) -test was performed to compare frequencies of categorical variables between different red cell distribution width quartiles, and one-way analysis of variance for continuous variables. The effect of red cell distribution width on severity and functional outcome was investigated in univariate and multivariate robust regression analysis. Level of significance was set at 95%. There were 1504 patients (72±15·76 years, 43·9% females) included in the analysis. Red cell distribution width was significantly associated to NIHSS (β-value=0·24, P=0·01) and functional outcome (odds ratio=10·73 for poor outcome, P<0·001) at univariate analysis but not multivariate. Prehospital Rankin score (β=0·19, P<0·001), serum creatinine (β=0·008, P<0·001), hemoglobin (β=-0·009, P<0·001), mean platelet volume (β=0·09, P<0·05), age (β=0·02, P<0·001), low ejection fraction (β=0·66, P<0·001) and antihypertensive treatment (β=0·32, P<0·001) were independent determinants of red cell distribution width. Red cell distribution width, assessed during the early phase of acute ischemic stroke

Using a combined computational-experimental approach to predict antibody-specific B cell epitopes.

PubMed

Sela-Culang, Inbal; Benhnia, Mohammed Rafii-El-Idrissi; Matho, Michael H; Kaever, Thomas; Maybeno, Matt; Schlossman, Andrew; Nimrod, Guy; Li, Sheng; Xiang, Yan; Zajonc, Dirk; Crotty, Shane; Ofran, Yanay; Peters, Bjoern

2014-04-08

Antibody epitope mapping is crucial for understanding B cell-mediated immunity and required for characterizing therapeutic antibodies. In contrast to T cell epitope mapping, no computational tools are in widespread use for prediction of B cell epitopes. Here, we show that, utilizing the sequence of an antibody, it is possible to identify discontinuous epitopes on its cognate antigen. The predictions are based on residue-pairing preferences and other interface characteristics. We combined these antibody-specific predictions with results of cross-blocking experiments that identify groups of antibodies with overlapping epitopes to improve the predictions. We validate the high performance of this approach by mapping the epitopes of a set of antibodies against the previously uncharacterized D8 antigen, using complementary techniques to reduce method-specific biases (X-ray crystallography, peptide ELISA, deuterium exchange, and site-directed mutagenesis). These results suggest that antibody-specific computational predictions and simple cross-blocking experiments allow for accurate prediction of residues in conformational B cell epitopes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Recombinant blood group proteins for use in antibody screening and identification tests.

PubMed

Seltsam, Axel; Blasczyk, Rainer

2009-11-01

The present review elucidates the potentials of recombinant blood group proteins (BGPs) for red blood cell (RBC) antibody detection and identification in pretransfusion testing and the achievements in this field so far. Many BGPs have been eukaryotically and prokaryotically expressed in sufficient quantity and quality for RBC antibody testing. Recombinant BGPs can be incorporated in soluble protein reagents or solid-phase assays such as ELISA, color-coded microsphere and protein microarray chip-based techniques. Because novel recombinant protein-based assays use single antigens, a positive reaction of a serum with the recombinant protein directly indicates the presence and specificity of the target antibody. Inversely, conventional RBC-based assays use panels of human RBCs carrying a huge number of blood group antigens at the same time and require negative reactions of samples with antigen-negative cells for indirect determination of antibody specificity. Because of their capacity for single-step, direct RBC antibody determination, recombinant protein-based assays may greatly facilitate and accelerate the identification of common and rare RBC antibodies.

Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes.

PubMed

Dørum, Siri; Steinsbø, Øyvind; Bergseng, Elin; Arntzen, Magnus Ø; de Souza, Gustavo A; Sollid, Ludvig M

2016-05-05

This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells.

Gluten-specific antibodies of celiac disease gut plasma cells recognize long proteolytic fragments that typically harbor T-cell epitopes

PubMed Central

Dørum, Siri; Steinsbø, Øyvind; Bergseng, Elin; Arntzen, Magnus Ø.; de Souza, Gustavo A.; Sollid, Ludvig M.

2016-01-01

This study aimed to identify proteolytic fragments of gluten proteins recognized by recombinant IgG1 monoclonal antibodies generated from single IgA plasma cells of celiac disease lesions. Peptides bound by monoclonal antibodies in complex gut-enzyme digests of gluten treated with the deamidating enzyme transglutaminase 2, were identified by mass spectrometry after antibody pull-down with protein G beads. The antibody bound peptides were long deamidated peptide fragments that contained the substrate recognition sequence of transglutaminase 2. Characteristically, the fragments contained epitopes with the sequence QPEQPFP and variants thereof in multiple copies, and they typically also harbored many different gluten T-cell epitopes. In the pull-down setting where antibodies were immobilized on a solid phase, peptide fragments with multivalent display of epitopes were targeted. This scenario resembles the situation of the B-cell receptor on the surface of B cells. Conceivably, B cells of celiac disease patients select gluten epitopes that are repeated multiple times in long peptide fragments generated by gut digestive enzymes. As the fragments also contain many different T-cell epitopes, this will lead to generation of strong antibody responses by effective presentation of several distinct T-cell epitopes and establishment of T-cell help to B cells. PMID:27146306

Selective Blockade of Human Natural Killer Cells by a Monoclonal Antibody

NASA Astrophysics Data System (ADS)

Newman, Walter

1982-06-01

A murine monoclonal antibody, 13.1, which blocks human natural killer (NK) cell-mediated lysis, has been developed. Hybridoma 13.1 was derived by fusion of NS-1 cells with spleen cells from mice immunized with an enriched population of NK cells. Supernatants of growing hybridomas were screened for their ability to block NK cell-mediated lysis of K562 targets. Antibody 13.1 is an IgG1 with a single light chain type and it does not fix complement. The 13.1 antigen is expressed on all peripheral blood mononuclear cells, with an antigen density approximately 1/30th that of HLA antigen heavy chain. Pretreatment and washing experiments revealed that inhibition of cytotoxicity occurred at the effector cell level only. Significant blocking was achieved with nanogram quantities of antibody and was not due to toxic effects on NK cells. Likewise, controls with other antibodies of the same subclass demonstrated that blocking was not a consequence of mere binding to NK cells. When a panel of 17 NK cell-susceptible targets was tested, the lysis of only 5 of these was blocked, namely K562, HL-60, KG-1, Daudi, and HEL, a human erythroleukemic cell line. The lysis of 12 human B cell and T cell line targets was not inhibited. In addition to the demonstration that the 13.1 antigen is a crucial cell surface structure involved in NK lysis, a heterogeneity of target cell recognition has been revealed that argues for the proposition that individual NK cells have multiple recognitive capabilities.

Red Blood Cell Hematocrit Influences Platelet Adhesion Rate in a Microchannel

NASA Astrophysics Data System (ADS)

Spann, Andrew; Campbell, James; Fitzgibbon, Sean; Rodriguez, Armando; Shaqfeh, Eric

2014-11-01

The creation of a blood clot to stop bleeding involves platelets forming a plug at the site of injury. Red blood cells indirectly play a role in ensuring that the distribution of platelets across the height of the channel is not uniform - the contrast in deformability and size between platelets and red blood cells allows the platelets to preferentially marginate close to the walls. We perform 3D boundary integral simulations of a suspension of platelets and red blood cells in a periodic channel with a model that allows for platelet binding at the walls. The relative rate of platelet activity with varying hematocrit (volume fraction of red blood cells) is compared to experiments in which red blood cells and platelets flow through a channel coated with von Willebrand factor. In the simulations as well as the experiments, a decrease in hematocrit of red blood cells is found to reduce the rate at which platelets adhere to the channel wall in a manner that is both qualitatively and quantitatively similar. We conclude with a discussion of the tumbling and wobbling motions of platelets in 3D leading up to the time at which the platelets bind to the wall. Funded by Stanford Army High Performance Computing Research Center, experiments by US Army Institute of Surgical Research.

Role of red cells and plasma composition on blood sessile droplet evaporation

NASA Astrophysics Data System (ADS)

Lanotte, Luca; Laux, Didier; Charlot, Benoît; Abkarian, Manouk

2017-11-01

The morphology of dried blood droplets derives from the deposition of red cells, the main components of their solute phase. Up to now, evaporation-induced convective flows were supposed to be at the base of red cell distribution in blood samples. Here, we present a direct visualization by videomicroscopy of the internal dynamics in desiccating blood droplets, focusing on the role of cell concentration and plasma composition. We show that in diluted suspensions, the convection is promoted by the rich molecular composition of plasma, whereas it is replaced by an outward red blood cell displacement front at higher hematocrits. We also evaluate by ultrasounds the effect of red cell deposition on the temporal evolution of sample rigidity and adhesiveness.

Antibody engineering of a cytotoxic monoclonal antibody 84 against human embryonic stem cells: Investigating the effects of multivalency on cytotoxicity.

PubMed

Klement, Maximilian; Zheng, Jiyun; Liu, Chengcheng; Tan, Heng-Liang; Wong, Victor Vai Tak; Choo, Andre Boon-Hwa; Lee, Dong-Yup; Ow, Dave Siak-Wei

2017-02-10

Antibody fragments have shown targeted specificity to their antigens, but only modest tissue retention times in vivo and in vitro. Multimerization has been used as a protein engineering tool to increase the number of binding units and thereby enhance the efficacy and retention time of antibody fragments. In this work, we explored the effects of valency using a series of self-assembling polypeptides based on the GCN4 leucine zipper multimerization domain fused to a single-chain variable fragment via an antibody upper hinge sequence. Four engineered antibody fragments with a valency from one to four antigen-binding units of a cytotoxic monoclonal antibody 84 against human embryonic stem cells (hESC) were constructed. We hypothesized that higher cytotoxicity would be observed for fragments with increased valency. Flow cytometry analysis revealed that the trimeric and tetrameric engineered antibody fragments resulted in the highest degree of cytotoxicity to the undifferentiated hESC, while the engineered antibody fragments were observed to have improved tissue penetration into cell clusters. Thus, a trade off was made for the trimeric versus tetrameric fragment due to improved tissue penetration. These results have direct implications for antibody-mediated removal of undifferentiated hESC during regenerative medicine and cell therapy. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

DOE Office of Scientific and Technical Information (OSTI.GOV)

Frankfurt, O.S.

A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to themore » drug dose. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.« less

Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides.

PubMed

Hermand, P; Mouro, I; Huet, M; Bloy, C; Suyama, K; Goldstein, J; Cartron, J P; Bailly, P

1993-07-15

Rabbit polyclonal antibodies were raised against synthetic peptides corresponding to hydrophilic regions of the human Rhesus (Rh) IX cDNA-encoded polypeptide predicted to be extracellularly or intracellularly exposed in the topologic model of the Rh blood group protein. Four antibodies encompassing residues 33-45 (MPC1), 224-233 (MPC4), 390-404 (MPC6), and 408-416 (MPC8) were characterized and compared with a polyclonal anti-Rh protein obtained by immunization with purified Rh proteins. All antibodies had specificity for authentic Rh polypeptides and reacted on Western blot with Rh proteins immunoprecipitated with human monoclonal anti-RhD, -c, and -E. MPC1, but not the other antibodies, agglutinated all human erythrocytes except Rhnull and Rhmod cells, which either lack totally or are severely deficient in Rh proteins, respectively. Immunoblotting analysis with membrane proteins from common and rare variants showed that MPC1 and MPC8 reacted in Western blot with 32-Kd Rh polypeptides from all common red blood cells except those from Rhnull and Rhmod, indicating that peptide regions 33-45 and 408-416 may be common to several if not all Rh proteins, whatever the Rh blood group specificity. MPC4 reacted only with membrane preparations from cells carrying the E antigen, whereas MPC6 recognized preferentially the Rh proteins from E and Ee preparations, suggesting that the protein encoded by the RhIXb cDNA carries the E and/or e antigen(s). Immunoadsorption experiments using inside-out or right-side-out sealed vesicules from DccEE red blood cells as competing antigen showed that the MPC6 and MPC8 antibodies bound only to the cytoplasmic side of the erythrocyte membrane, thus providing evidence for the intracellular orientation of the C-terminal 27 residues of the Rh polypeptides. Attempts to transiently or stably express the Rh polypeptides. Attempts to transiently or stably express the Rh cDNA in eukaryotic cells were largely unsuccessful, suggesting that Rh antigen

Early alterations of red blood cell rheology in critically ill patients.

PubMed

Reggiori, Giulia; Occhipinti, Giovanna; De Gasperi, Andrea; Vincent, Jean-Louis; Piagnerelli, Michael

2009-12-01

To investigate red blood cell rheology in a large intensive care unit population on admission, and to assess the possible influence of comorbidities on the rheology. : Prospective study. Medico-surgical intensive care unit with 31 beds. All intensive care unit admissions during a 5-month period and 20 healthy volunteers. Blood sampling. A total of 196 intensive care patients (160 without and 36 with sepsis) and 20 healthy volunteers were studied. Red blood cell rheology (deformability and aggregation) was assessed ex vivo using the laser-assisted optical rotational cell analyzer (LORCA; Mechatronics Instruments BV, AN Zwaag, Netherlands) within the first 24 hrs after intensive care unit admission. Red blood cell deformability was determined by the elongation index in relation to the shear stress (0.3 to 50 Pa) applied on the red blood cell membrane surface. Aggregation was assessed by the aggregation index. Septic patients were more likely to have anemia, coagulation abnormalities, and comorbidities than were nonseptic patients. Red blood cell deformability was significantly altered in septic compared to nonseptic patients and volunteers for the majority of shear stress rates studied. The aggregation index was greater in septic patients than in volunteers (67.9% [54.7-73.5] vs. 61.8% [58.2-68.4]; p < .05). Only sepsis and hematologic disease influenced the elongation index (both p < .01). Other comorbidities, like cancer, diabetes mellitus, cirrhosis, and terminal renal failure, had no effect on the elongation index. Aggregation index was related to the degree of organ failure (Sequential Organ Failure Assessment score), the red blood cell count, and fibrinogen concentrations. Early alterations of red blood cell rheology are common in intensive care unit patients, especially in those with sepsis. Comorbidities (other than hematologic diseases) do not significantly influence these abnormalities. These alterations could contribute to the microcirculatory alterations

Red blood cells as modulators of T cell growth and survival.

PubMed

Arosa, Fernando A; Pereira, Carlos F; Fonseca, Ana M

2004-01-01

T cell homeostasis is largely controlled by a balance between cell death and survival and anomalies in either process account for a number of diseases linked to excessive or faulty T cell growth. Yet, the influence that cells outside the immunological system have on these processes has only recently received attention. Accumulated evidence indicate that homeostasis of the CD4+ and CD8+ T cell pools is highly dynamic and regulated by signals delivered by cells and molecules present in the different internal microenvironments. The major function of red blood cells (RBC) is generally considered to be oxygen and carbon dioxide transport. In recent years, however, RBC have been implicated in the regulation of basic physiological processes, from vascular contraction and platelet aggregation to T cell growth and survival. Regulation of T cell survival by RBC may influence the response of selected subsets of T cells to internal or external stimuli and may help explaining the immunomodulatory activities of red blood cells. By interfering in the balance between death and survival RBC become potential tools that can be manipulated to improve or reverse pathological situations characterized by anomalies in the control of T cell growth.

Red blood cell-deformability measurement: review of techniques.

PubMed

Musielak, M

2009-01-01

Cell-deformability characterization involves general measurement of highly complex relationships between cell biology and physical forces to which the cell is subjected. The review takes account of the modern technical solutions simulating the action of the force applied to the red blood cell in macro- and microcirculation. Diffraction ektacytometers and rheoscopes measure the mean deformability value for the total red blood cell population investigated and the deformation distribution index of individual cells, respectively. Deformation assays of a whole single cell are possible by means of optical tweezers. The single cell-measuring setups for micropipette aspiration and atomic force microscopy allow conducting a selective investigation of deformation parameters (e.g., cytoplasm viscosity, viscoelastic membrane properties). The distinction between instrument sensitivity to various RBC-rheological features as well as the influence of temperature on measurement are discussed. The reports quoted confront fascinating possibilities of the techniques with their medical applications since the RBC-deformability has the key position in the etiology of a wide range of conditions.

Fully human monoclonal antibodies from antibody secreting cells after vaccination with Pneumovax®23 are serotype specific and facilitate opsonophagocytosis.

PubMed

Smith, Kenneth; Muther, Jennifer J; Duke, Angie L; McKee, Emily; Zheng, Nai-Ying; Wilson, Patrick C; James, Judith A

2013-05-01

B lymphocyte memory generates antibody-secreting cells (ASCs) that represent a source of protective antibodies that may be exploited for therapeutics. Here we vaccinated four donors with Pneumovax®23 and produced human monoclonal antibodies (hmAbs) from ASCs. We have cloned 137 hmAbs and the specificities of these antibodies encompass 19 of the 23 serotypes in the vaccine, as well as cell wall polysaccharide (CWPS). Although the majority of the antibodies are serotype specific, 12% cross-react with two serotypes. The Pneumovax®23 ASC antibody sequences are highly mutated and clonal, indicating an anamnestic response, even though this was a primary vaccination. Hmabs from 64% of the clonal families facilitate opsonophagocytosis. Although 9% of the total antibodies bind to CWPS impurity in the vaccine, none of these clonal families showed opsonophagocytic activity. Overall, these studies have allowed us to address unanswered questions in the field of human immune responses to polysaccharide vaccines, including the cross-reactivity of individual antibodies between serotypes and the percentage of antibodies that are protective after vaccination with Pneumovax®23. Copyright © 2012 Elsevier GmbH. All rights reserved.

NMDA Receptor Activity in Circulating Red Blood Cells: Methods of Detection.

PubMed

Makhro, Asya; Kaestner, Lars; Bogdanova, Anna

2017-01-01

Abundance and activity of N-methyl-D-aspartate (NMDA) in circulating red blood cells contributes to the maintenance of intracellular Ca 2+ in these cells and, by doing that, controls red cell volume, membrane stability, and O 2 carrying capacity. Detection of the NMDA receptor activity in red blood cells is challenging as the number of its copies is low and shows substantial cell-to-cell heterogeneity. Receptor abundance is reliably assessed using the radiolabeled antagonist ([ 3 H]MK-801) binding technique. Uptake of Ca 2+ following the NMDA receptor activation is detected in cells loaded with Ca 2+ -sensitive fluorescent dye Fluo-4 AM. Both microfluorescence live-cell imaging and flow cytometry may be used for fluorescence intensity detection. Automated patch clamp is currently used for recording of electric currents triggered by the stimulation of the NMDA receptor. These currents are mediated by the Ca 2+ -sensitive K + (Gardos) channels that open upon Ca 2+ uptake via the active NMDA receptor. Furthermore, K + flux through the Gardos channels induced by the NMDA receptor stimulation in red blood cells may be detected using unidirectional K + ( 86 Rb + ) influx.

Prolonged storage of packed red blood cells for blood transfusion.

PubMed

Martí-Carvajal, Arturo J; Simancas-Racines, Daniel; Peña-González, Barbra S

2015-07-14

A blood transfusion is an acute intervention, used to address life- and health-threatening conditions on a short-term basis. Packed red blood cells are most often used for blood transfusion. Sometimes blood is transfused after prolonged storage but there is continuing debate as to whether transfusion of 'older' blood is as beneficial as transfusion of 'fresher' blood. To assess the clinical benefits and harms of prolonged storage of packed red blood cells, in comparison with fresh, on recipients of blood transfusion. We ran the search on 1st May 2014. We searched the Cochrane Injuries Group Specialized Register, Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library), MEDLINE (OvidSP), Embase (OvidSP), CINAHL (EBSCO Host) and two other databases. We also searched clinical trials registers and screened reference lists of the retrieved publications and reviews. We updated this search in June 2015 but these results have not yet been incorporated. Randomised clinical trials including participants assessed as requiring red blood cell transfusion were eligible for inclusion. Prolonged storage was defined as red blood cells stored for ≥ 21 days in a blood bank. We did not apply limits regarding the duration of follow-up, or country where the study took place. We excluded trials where patients received a combination of short- and long-stored blood products, and also trials without a clear definition of prolonged storage. We independently performed study selection, risk of bias assessment and data extraction by at least two review authors. The major outcomes were death from any cause, transfusion-related acute lung injury, and adverse events. We estimated relative risk for dichotomous outcomes. We measured statistical heterogeneity using I(2). We used a random-effects model to synthesise the findings. We identified three randomised clinical trials, involving a total of 120 participants, comparing packed red blood cells with ≥ 21 days storage

A Synthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies.

PubMed

Ruprecht, Colin; Bartetzko, Max P; Senf, Deborah; Dallabernadina, Pietro; Boos, Irene; Andersen, Mathias C F; Kotake, Toshihisa; Knox, J Paul; Hahn, Michael G; Clausen, Mads H; Pfrengle, Fabian

2017-11-01

In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories worldwide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types, and developmental stages. Despite their importance and broad use, the precise binding epitope has been determined for only a few of these antibodies. Here, we use a plant glycan microarray equipped with 88 synthetic oligosaccharides to comprehensively map the epitopes of plant cell wall glycan-directed antibodies. Our results reveal the binding epitopes for 78 arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan-directed antibodies. We demonstrate that, with knowledge of the exact epitopes recognized by individual antibodies, specific glycosyl hydrolases can be implemented into immunological cell wall analyses, providing a framework to obtain structural information on plant cell wall glycans with unprecedented molecular precision. © 2017 American Society of Plant Biologists. All Rights Reserved.

Cell membrane antigen-antibody complex dissociation by the widely used glycine-HCL method: an unreliable procedure for studying antibody internalization.

PubMed

Tsaltas, G; Ford, C H

1993-02-01

Methods following the process of binding and internalization of antibodies to cell surface antigens have often employed low pH isoosmolar buffers in order to dissociate surface antigen-antibody complexes. One of the most widely used buffers is a 0.05 M glycine-HCL buffer pH 2.8. Since the efficacy of action of this buffer was critical to a series of internalization experiments employing monoclonal antibodies (Mabs) to carcinoembryonic antigen (CEA) expressing cancer cell lines in this laboratory, we tested its performance in a number of different assays. Our results indicate that this buffer only partially dissociates antigen-antibody bonds and therefore can introduce major inaccuracies in internalization experiments.

Conditioning out-of-date bank-stored red blood cells using a cell-saver auto-transfusion device: effects on numbers of red cells and quality of suspension fluid.

PubMed

Read, M S; Coles, P; Pomeroy, M; Anderson, E; Aziz, M I

2014-11-01

We investigated the utility of a cell-saver device for processing out-of-date red blood cells, by washing twenty bags of red blood cells that had been stored for between 36 and 55 days. The volume of recovered cells, and the characteristics of the suspension fluid, were measured before and after treatment. The ratio of free haemoglobin to total haemoglobin was up to 0.02 before processing, and up to 0.011 afterwards, changing by between -0.013 and +0.003. This ratio met the current standard for free haemoglobin (less than 0.008 in more than 75% of samples), both before and after processing. Ninety-three percent of red blood cells survived the process. Potassium ion concentration fell from above 15 mmol.l(-1) in all cases, to a mean of 6.4 mmol.l(-1) (p < 0.001). The pH rose to a mean value of 6.44 (p = 0.001). Lactate ion concentration fell to a mean value of 14 mmol.l(-1) (p < 0.001). Sodium ion concentration rose from a mean value of 93 mmol.l(-1) to a mean value of 140 mmol.l(-1) (p < 0.001). A useful proportion of out-of-date red blood cells remained intact after conditioning using a cell-saver, and the process lowered concentrations of potentially toxic solutes in the fluid in which they were suspended. © 2014 The Association of Anaesthetists of Great Britain and Ireland.

HIV-1 Antibody Neutralization Breadth Is Associated with Enhanced HIV-Specific CD4+ T Cell Responses

PubMed Central

Soghoian, Damien Z.; Lindqvist, Madelene; Ghebremichael, Musie; Donaghey, Faith; Carrington, Mary; Seaman, Michael S.; Kaufmann, Daniel E.; Walker, Bruce D.

2015-01-01

ABSTRACT Antigen-specific CD4+ T helper cell responses have long been recognized to be a critical component of effective vaccine immunity. CD4+ T cells are necessary to generate and maintain humoral immune responses by providing help to antigen-specific B cells for the production of antibodies. In HIV infection, CD4+ T cells are thought to be necessary for the induction of Env-specific broadly neutralizing antibodies. However, few studies have investigated the role of HIV-specific CD4+ T cells in association with HIV neutralizing antibody activity in vaccination or natural infection settings. Here, we conducted a comprehensive analysis of HIV-specific CD4+ T cell responses in a cohort of 34 untreated HIV-infected controllers matched for viral load, with and without neutralizing antibody breadth to a panel of viral strains. Our results show that the breadth and magnitude of Gag-specific CD4+ T cell responses were significantly higher in individuals with neutralizing antibodies than in those without neutralizing antibodies. The breadth of Gag-specific CD4+ T cell responses was positively correlated with the breadth of neutralizing antibody activity. Furthermore, the breadth and magnitude of gp41-specific, but not gp120-specific, CD4+ T cell responses were significantly elevated in individuals with neutralizing antibodies. Together, these data suggest that robust Gag-specific CD4+ T cells and, to a lesser extent, gp41-specific CD4+ T cells may provide important intermolecular help to Env-specific B cells that promote the generation or maintenance of Env-specific neutralizing antibodies. IMPORTANCE One of the earliest discoveries related to CD4+ T cell function was their provision of help to B cells in the development of antibody responses. Yet little is known about the role of CD4+ T helper responses in the setting of HIV infection, and no studies to date have evaluated the impact of HIV-specific CD4+ T cells on the generation of antibodies that can neutralize

Integration of red cell genotyping into the blood supply chain: a population-based study.

PubMed

Flegel, Willy A; Gottschall, Jerome L; Denomme, Gregory A

2015-07-01

When problems with compatibility arise, transfusion services often use time-consuming serological tests to identify antigen-negative red cell units for safe transfusion. New methods have made red cell genotyping possible for all clinically relevant blood group antigens. We did mass-scale genotyping of donor blood and provided hospitals with access to a large red cell database to meet the demand for antigen-negative red cell units beyond ABO and Rh blood typing. We established a red cell genotype database at the BloodCenter of Wisconsin on July 17, 2010. All self-declared African American, Asian, Hispanic, and Native American blood donors were eligible irrespective of their ABO and Rh type or history of donation. Additionally, blood donors who were groups O, A, and B, irrespective of their Rh phenotype, were eligible for inclusion only if they had a history of at least three donations in the previous 3 years, with one donation in the previous 12 months at the BloodCenter of Wisconsin. We did red cell genotyping with a nanofluidic microarray system, using 32 single nucleotide polymorphisms to predict 42 blood group antigens. An additional 14 antigens were identified via serological phenotype. We monitored the ability of the red cell genotype database to meet demand for compatible blood during 3 years. In addition to the central database at the BloodCenter of Wisconsin, we gave seven hospitals online access to a web-based antigen query portal on May 1, 2013, to help them to locate antigen-negative red cell units in their own inventories. We analysed genotype data for 43,066 blood donors. Requests were filled for 5661 (99.8%) of 5672 patient encounters in which antigen-negative red cell units were needed. Red cell genotyping met the demand for antigen-negative blood in 5339 (94.1%) of 5672 patient encounters, and the remaining 333 (5.9%) requests were filled by use of serological data. Using the 42 antigens represented in our red cell genotype database, we were able to

Fluorescent immunolabeling of cancer cells by quantum dots and antibody scFv fragment.

PubMed

Zdobnova, Tatiana A; Dorofeev, Sergey G; Tananaev, Piter N; Vasiliev, Roman B; Balandin, Taras G; Edelweiss, Eveline F; Stremovskiy, Oleg A; Balalaeva, Irina V; Turchin, Ilya V; Lebedenko, Ekaterina N; Zlomanov, Vladimir P; Deyev, Sergey M

2009-01-01

Semiconductor quantum dots (QDs) coupled with cancer-specific targeting ligands are new promising agents for fluorescent visualization of cancer cells. Human epidermal growth factor receptor 2/neu (HER2/neu), overexpressed on the surface of many cancer cells, is an important target for cancer diagnostics. Antibody scFv fragments as a targeting agent for direct delivery of fluorophores offer significant advantages over full-size antibodies due to their small size, lower cross-reactivity, and immunogenicity. We have used quantum dots linked to anti-HER2/neu 4D5 scFv antibody to label HER2/neu-overexpressing live cells. Labeling of target cells was shown to have high brightness, photostability, and specificity. The results indicate that construction based on quantum dots and scFv antibody can be successfully used for cancer cell visualization.

Mechanisms Linking Red Blood Cell Disorders and Cardiovascular Diseases

PubMed Central

2015-01-01

The present paper aims to review the main pathophysiological links between red blood cell disorders and cardiovascular diseases, provides a brief description of the latest studies in this area, and considers implications for clinical practice and therapy. Anemia is associated with a special risk in proatherosclerotic conditions and heart disease and became a new therapeutic target. Guidelines must be updated for the management of patients with red blood cell disorders and cardiovascular diseases, and targets for hemoglobin level should be established. Risk scores in several cardiovascular diseases should include red blood cell count and RDW. Complete blood count and hemorheological parameters represent useful, inexpensive, widely available tools for the management and prognosis of patients with coronary heart disease, heart failure, hypertension, arrhythmias, and stroke. Hypoxia and iron accumulation cause the most important cardiovascular effects of sickle cell disease and thalassemia. Patients with congenital chronic hemolytic anemia undergoing splenectomy should be monitored, considering thromboembolic and cardiovascular risk. PMID:25710019

[Identification of human monoclonal HIV-1-neutralizing antibodies from phage antibody library by cell-based screening].

PubMed

Zhang, Na; Man, Lai; Sun, Jian-ping; Meng, Jia-zi; He, Yu-xian

2013-09-01

To identify human monoclonal HIV-l-neutralizing antibodies from an HIV-1 CRF07BC specific phage display antibody library by cell-based screening. 293T cells were transfected by pCH064. 2-Env plas mid and then used to biopan the phage antibody library. The positive phage clones were screened by cell based ELISA and sequenced for the variable region of heavy (VH) and light (VL) chains. The expressed Fabs were purified by Ni(+2) -NTA column and analyzed by SDS-PAGE. The cell- and gp120 protein-based ELISA as well as flow cytometry were used to measure Fab's binding activity. The neutralizing activity of Fabs was assessed by HIV-1 pseudoviruses. After 4-round biopanning, the binding phages to transfected cells were enriched about 650-folds. A total of 28 positive clones were screened out by cell ELISA and sequence analysis identified 5 different Fabs possessing unique VH and VL (2801, 2837, 2863, 2870 and 2920). Interestingly, these Fabs reacted with the Env-transfected 293T cells but not soluble gp120 proteins, suggesting that they might target conformation-dependent epitopes presenting on viral Env complex. We found that three Fabs (2801, 2863, 2870) exhibited potent neutralizing activity against CRF07_BC isolate CH120. 6 with IC50 of 2.24, 0.89 and 3.09 microg/mL respectively, and that 2801 and 2863 cross-neutral ized the subtype B isolate SF162 at IC50 of 0.69 and 3.52 microg/mL respectively. In conclusion, the HIV-1 Env-transfected 293T cells can be used to efficiently enrich and screen the phage antibody library and isolate human monoclonal HIV-1-neutralizing Fabs that target the Env complex-dependent conformational epitopes. Therefore, our studies provide a powerful platform for exploring the mechanism of HIV-1 neu tralizing response and for designing AIDS vaccines.

Partial Red Blood Cell Exchange in Children and Young Patients with Sickle Cell Disease: Manual Versus Automated Procedure.

PubMed

Escobar, Carlos; Moniz, Marta; Nunes, Pedro; Abadesso, Clara; Ferreira, Teresa; Barra, António; Lichtner, Anabela; Loureiro, Helena; Dias, Alexandra; Almeida, Helena

2017-10-31

The benefits of manual versus automated red blood cell exchange have rarely been documented and studies in young sickle cell disease patients are scarce. We aim to describe and compare our experience in these two procedures. Young patients (≤ 21 years old) who underwent manual- or automated-red blood cell exchange for prevention or treatment of sickle cell disease complications were included. Clinical, technical and hematological data were prospectively recorded and analyzed. Ninety-four red blood cell exchange sessions were performed over a period of 68 months, including 57 manual and 37 automated, 63 for chronic complications prevention, 30 for acute complications and one in the pre-operative setting. Mean decrease in sickle hemoglobin levels was higher in automated-red blood cell exchange (p < 0.001) and permitted a higher sickle hemoglobin level decrease per volume removed (p < 0.001), while hemoglobin and hematocrit remained stable. Ferritin levels on chronic patients decreased 54%. Most frequent concern was catheter outflow obstruction on manual-red blood cell exchange and access alarm on automated-red blood cell exchange. No major complication or alloimunization was recorded. Automated-red blood cell exchange decreased sickle hemoglobin levels more efficiently than manual procedure in the setting of acute and chronic complications of sickle cell disease, with minor technical concerns mainly due to vascular access. The threshold of sickle hemoglobin should be individualized for clinical and hematological goals. In our cohort of young patients, the need for an acceptable venous access was a limiting factor, but iron-overload was avoided. Automated red blood cell exchange is safe and well tolerated. It permits a higher sickle hemoglobin removal efficacy, better volume status control and iron-overload avoidance.

Reflectance confocal microscopy of red blood cells: simulation and experiment (Conference Presentation)

NASA Astrophysics Data System (ADS)

Zeidan, Adel; Yeheskely-Hayon, Daniella; Minai, Limor; Yelin, Dvir

2016-03-01

The properties of red blood cells are a remarkable indicator of the body's physiological condition; their density could indicate anemia or polycythemia, their absorption spectrum correlates with blood oxygenation, and their morphology is highly sensitive to various pathologic states including iron deficiency, ovalocytosis, and sickle cell disease. Therefore, measuring the morphology of red blood cells is important for clinical diagnosis, providing valuable indications on a patient's health. In this work, we simulated the appearance of normal red blood cells under a reflectance confocal microscope and discovered unique relations between the cells' morphological parameters and the resulting characteristic interference patterns. The simulation results showed good agreement with in vitro reflectance confocal images of red blood cells, acquired using spectrally encoded flow cytometry (SEFC) that imaged the cells during linear flow and without artificial staining. By matching the simulated patterns to the SEFC images of the cells, the cells' three-dimensional shapes were evaluated and their volumes were calculated. Potential applications include measurement of the mean corpuscular volume, cell morphological abnormalities, cell stiffness under mechanical stimuli, and the detection of various hematological diseases.

Inability to detect transferrin receptors on P. falciparum parasitized red cells.

PubMed

Pollack, S; Schnelle, V

1988-01-01

The mechanism by which P. falciparum takes up iron from transferrin has been explored. Binding of 125I labelled transferrin to parasitized red cells at 37 degrees C is two-fold greater than to control cells; at 0 degrees C there is no significant difference. The binding is non-specific as judged from the following: it is not saturable; it is not limited to transferrin as lactoferrin (which has iron binding domains) and bovine serum albumin (which does not) also bind in excess to parasitized red cells. A transferrin receptor complex could not be demonstrated when parasitized red cells, to which 125I transferrin was bound, were solubilized in Triton X100. Previous observation showed that uptake of transferrin iron by parasitized red cells is not accompanied by equimolar uptake of transferrin protein. We therefore suggest that nonspecifically bound transferrin is endocytosed, that the protein is degraded and the iron selectively retained.

The negative regulation of red cell mass by neocytolysis: physiologic and pathophysiologic manifestations.

PubMed

Rice, Lawrence; Alfrey, Clarence P

2005-01-01

We have uncovered a physiologic process which negatively regulates the red cell mass by selectively hemolyzing young circulating red blood cells. This allows fine control of the number of circulating red blood cells under steady-state conditions and relatively rapid adaptation to new environments. Neocytolysis is initiated by a fall in erythropoietin levels, so this hormone remains the major regulator of red cell mass both with anemia and with red cell excess. Physiologic situations in which there is increased neocytolysis include the emergence of newborns from the hypoxic uterine environment and the descent of polycythemic high-altitude dwellers to sea level. The process first became apparent while investigating the mechanism of the anemia that invariably occurs after spaceflight. Astronauts experience acute central plethora on entering microgravity resulting in erythropoietin suppression and neocytolysis, but the reduced blood volume and red cell mass become suddenly maladaptive on re-entry to earth's gravity. The pathologic erythropoietin deficiency of renal disease precipitates neocytolysis, which explains the prolongation of red cell survival consistently resulting from erythropoietin therapy and points to optimally efficient erythropoietin dosing schedules. Implications should extend to a number of other physiologic and pathologic situations including polycythemias, hemolytic anemias, 'blood-doping' by elite athletes, and oxygen therapy. It is likely that erythropoietin influences endothelial cells which in turn signal reticuloendothelial phagocytes to destroy or permit the survival of young red cells marked by surface molecules. Ongoing studies to identify the molecular targets and cytokine intermediaries should facilitate detection, dissection and eventual therapeutic manipulation of the process. Copyright (c) 2005 S. Karger AG, Basel.

Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax

PubMed Central

Zimmerman, Peter A.; Ferreira, Marcelo U.; Howes, Rosalind E.; Mercereau-Puijalon, Odile

2013-01-01

Resistance to Plasmodium vivax blood-stage infection has been widely recognised to result from absence of the Duffy (Fy) blood group from the surface of red blood cells (RBCs) in individuals of African descent. Interestingly, recent studies from different malaria-endemic regions have begun to reveal new perspectives on the association between Duffy gene polymorphism and P. vivax malaria. In Papua New Guinea and the Americas, heterozygous carriers of a Duffy-negative allele are less susceptible to P. vivax infection than Duffy-positive homozygotes. In Brazil, studies show that the Fya antigen, compared to Fyb, is associated with lower binding to the P. vivax Duffy-binding protein and reduced susceptibility to vivax malaria. Additionally, it is interesting that numerous studies have now shown that P. vivax can infect RBCs and cause clinical disease in Duffy-negative people. This suggests that the relationship between P. vivax and the Duffy antigen is more complex than customarily described. Evidence of P. vivax Duffy-independent red cell invasion indicates that the parasite must be evolving alternative red cell invasion pathways. In this chapter, we review the evidence for P. vivax Duffy-dependent and Duffy-independent red cell invasion. We also consider the influence of further host gene polymorphism associated with malaria endemicity on susceptibility to vivax malaria. The interaction between the parasite and the RBC has significant potential to influence the effectiveness of P. vivax-specific vaccines and drug treatments. Ultimately, the relationships between red cell polymorphisms and P. vivax blood-stage infection will influence our estimates on the population at risk and efforts to eliminate vivax malaria. PMID:23384621

Structural analysis of B-cell epitopes in antibody:protein complexes

PubMed Central

Kringelum, Jens Vindahl; Nielsen, Morten; Padkjær, Søren Berg; Lund, Ole

2012-01-01

The binding of antigens to antibodies is one of the key events in an immune response against foreign molecules and is a critical element of several biomedical applications including vaccines and immunotherapeutics. For development of such applications, the identification of antibody binding sites (B-cell epitopes) is essential. However experimental epitope mapping is highly cost-intensive and computer-aided methods do in general have moderate performance. One major reason for this moderate performance is an incomplete understanding of what characterizes an epitope. To fill this gap, we here developed a novel framework for comparing and superimposing B-cell epitopes and applied it on a dataset of 107 non-similar antigen:antibody structures extracted from the PDB database. With the presented framework, we were able to describe the general B-cell epitope as a flat, oblong, oval shaped volume consisting of predominantly hydrophobic amino acids in the center flanked by charged residues. The average epitope was found to be made up of ~15 residues with one linear stretch of 5 or more residues constituting more than half of the epitope size. Furthermore, the epitope area is predominantly constrained to a plane above the antibody tip, in which the epitope is orientated in a −30 to 60 degree angle relative to the light to heavy chain antibody direction. Contrary to previously findings, we did not find a significant deviation between the amino acid composition in epitopes and the composition of equally exposed parts of the antigen surface. Our results, in combination with previously findings, give a detailed picture of the B-cell epitope that may be used in development of improved B-cell prediction methods. PMID:22784991

The stress-free shape of the red blood cell membrane.

PubMed Central

Fischer, T M; Haest, C W; Stöhr-Liesen, M; Schmid-Schönbein, H; Skalak, R

1981-01-01

The two main proposals found in the literature for the stress-free shape of the red cell membrane are (a) the bioconcave shape and (b) the sphere of the same surface area. These possibilities are evaluated in this paper using theoretical modeling of equilibrium membrane shapes according to Zarda et al. (1977. J. Biomech. 10:211-221) and by comparison to experiments on red cells whose membrane shear modulus has been increased by treatment with diamide. Neither proposal is found to be compatible with all the experimental behaviour of native red cells. Neither proposal is found to be compatible with all the experimental behaviour of native red cells. To account for this discrepancy we propose that either the shear modulus of the native membrane is dependent on the membrane strain or that the bending stiffness is higher than estimated by Evans (1980. Biophys. J. 30:265-286). These studies suggest that the bioconcave disk is the more likely possibility for the stress-free shape. Images FIGURE 4 FIGURE 5 FIGURE 6 FIGURE 7 PMID:7248469

Interpretation of the Raji cell assay in sera containing anti-nuclear antibodies and immune complexes.

PubMed Central

Horsfall, A C; Venables, P J; Mumford, P A; Maini, R N

1981-01-01

The Raji cell assay is regarded as a test for the detection and quantitation of immune complexes. It is frequently positive in sera from patients with SLE. We have demonstrated a relationship between Raji cell binding and antibodies to DNA and soluble cellular antigens. In five sera containing high titres of antibodies of known single specificity, most of the Raji cell binding occurred in the 7S IgG fraction where the majority of anti-nuclear antibody was also found. When each of these sera was incubated with its specific antigen, Raji cell binding increased. Subsequent fractionation showed that this binding was in the high molecular weight fraction (greater than 200,000 daltons) and that Raji cell binding and antibody activity were abolished in the 7S fraction. These data confirm that Raji cell bind immune complexes but also indicate that 7S anti-nuclear antibodies may interact directly with Raji cells by an unknown mechanism. Therefore, in sera of patients with anti-nuclear antibodies, binding to Raji cells does not necessarily imply the presence of immune complexes alone. PMID:6975676

[Promising technologies of packed red blood cells production and storage].

PubMed

Maksimov, A G; Golota, A S; Krassiĭ, A B

2013-10-01

The current article is dedicated to promising technologies of packed red blood cells production and storage. The following new technical approaches are presented: (1) erythrocytes storage in strict anaerobic argon-hydrogen environment, (2) lyophilization of erythrocyte suspension by its atomization in nitrogen gas, (3) lyophilization of erythrocytes by directional freezing under the influence of radio frequency radiation, (4) automated pharming of antigen free packed red blood cells from progenitor cell directly at the battlefield.

Antibody-immobilized column for quick cell separation based on cell rolling.

PubMed

Mahara, Atsushi; Yamaoka, Tetsuji

2010-01-01

Cell separation using methodological standards that ensure high purity is a very important step in cell transplantation for regenerative medicine and for stem cell research. A separation protocol using magnetic beads has been widely used for cell separation to isolate negative and positive cells. However, not only the surface marker pattern, e.g., negative or positive, but also the density of a cell depends on its developmental stage and differentiation ability. Rapid and label-free separation procedures based on surface marker density are the focus of our interest. In this study, we have successfully developed an antiCD34 antibody-immobilized cell-rolling column, that can separate cells depending on the CD34 density of the cell surfaces. Various conditions for the cell-rolling column were optimized including graft copolymerization, and adjustment of the column tilt angle, and medium flow rate. Using CD34-positive and -negative cell lines, the cell separation potential of the column was established. We observed a difference in the rolling velocities between CD34-positive and CD34-negative cells on antibody-immobilized microfluidic device. Cell separation was achieved by tilting the surface 20 degrees and the increasing medium flow. Surface marker characteristics of the isolated cells in each fraction were analyzed using a cell-sorting system, and it was found that populations containing high density of CD34 were eluted in the delayed fractions. These results demonstrate that cells with a given surface marker density can be continuously separated using the cell rolling column.

Autologous Hematopoietic Stem Cell Transplantation to Prevent Antibody Mediated Rejection After Vascularized Composite Allotransplantation

DTIC Science & Technology

2017-10-01

Award Number: W81XWH-16-1-0664 TITLE: Autologous Hematopoietic Stem Cell Transplantation to Prevent Antibody-Mediated Rejection after...Annual 3. DATES COVERED 15 Sep 2016 – 14 Sep 2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Autologous Hematopoietic Stem Cell Transplantation to...sensitization, autologous hematopoietic stem cell transplantation, antibody mediated rejection, donor specific antibodies 16. SECURITY CLASSIFICATION OF

Effects of red-cell storage duration on patients undergoing cardiac surgery.

PubMed

Steiner, Marie E; Ness, Paul M; Assmann, Susan F; Triulzi, Darrell J; Sloan, Steven R; Delaney, Meghan; Granger, Suzanne; Bennett-Guerrero, Elliott; Blajchman, Morris A; Scavo, Vincent; Carson, Jeffrey L; Levy, Jerrold H; Whitman, Glenn; D'Andrea, Pamela; Pulkrabek, Shelley; Ortel, Thomas L; Bornikova, Larissa; Raife, Thomas; Puca, Kathleen E; Kaufman, Richard M; Nuttall, Gregory A; Young, Pampee P; Youssef, Samuel; Engelman, Richard; Greilich, Philip E; Miles, Ronald; Josephson, Cassandra D; Bracey, Arthur; Cooke, Rhonda; McCullough, Jeffrey; Hunsaker, Robert; Uhl, Lynne; McFarland, Janice G; Park, Yara; Cushing, Melissa M; Klodell, Charles T; Karanam, Ravindra; Roberts, Pamela R; Dyke, Cornelius; Hod, Eldad A; Stowell, Christopher P

2015-04-09

Some observational studies have reported that transfusion of red-cell units that have been stored for more than 2 to 3 weeks is associated with serious, even fatal, adverse events. Patients undergoing cardiac surgery may be especially vulnerable to the adverse effects of transfusion. We conducted a randomized trial at multiple sites from 2010 to 2014. Participants 12 years of age or older who were undergoing complex cardiac surgery and were likely to undergo transfusion of red cells were randomly assigned to receive leukocyte-reduced red cells stored for 10 days or less (shorter-term storage group) or for 21 days or more (longer-term storage group) for all intraoperative and postoperative transfusions. The primary outcome was the change in Multiple Organ Dysfunction Score (MODS; range, 0 to 24, with higher scores indicating more severe organ dysfunction) from the preoperative score to the highest composite score through day 7 or the time of death or discharge. The median storage time of red-cell units provided to the 1098 participants who received red-cell transfusion was 7 days in the shorter-term storage group and 28 days in the longer-term storage group. The mean change in MODS was an increase of 8.5 and 8.7 points, respectively (95% confidence interval for the difference, -0.6 to 0.3; P=0.44). The 7-day mortality was 2.8% in the shorter-term storage group and 2.0% in the longer-term storage group (P=0.43); 28-day mortality was 4.4% and 5.3%, respectively (P=0.57). Adverse events did not differ significantly between groups except that hyperbilirubinemia was more common in the longer-term storage group. The duration of red-cell storage was not associated with significant differences in the change in MODS. We did not find that the transfusion of red cells stored for 10 days or less was superior to the transfusion of red cells stored for 21 days or more among patients 12 years of age or older who were undergoing complex cardiac surgery. (Funded by the National

[Preparation of chicken red blood cells for calibration of flow cytometry].

PubMed

Yin, Jian; Zhao, Shutao; Wu, Xiaodong; Wang, Ce; Wu, Yunliang

2013-01-01

To prepare stable chicken red blood cells for the calibration of flow cytometry. The traditional isolation method of chicken red blood cells was modified by incorporating gelatin technique, Ca2+-free HBSS treatment and low-speed centrifugation. The effect of fluorescence staining of the cells was improved by the addition of TritonX-100 to enhance the membrane permeability and Rnase enzymes to disintegrate RNA tiles. The modified method was compared with the traditional method for viability of the freshly isolated cells and the DNA content coefficient of variation (CV) of the fixed cells. Chicken red blood cells obtained by the modified method showed a significantly higher viability than those obtained by the traditional method [(98.5∓3.5)% vs (93.5∓2.7)%, P<0.05]. After glutaraldehyde fixation, the isolated cells with the modified method were stable during the 90-day preservation with a significantly lower CV than the cells obtained by the traditional method [(6.0∓0.3)% to 6.2∓0.4% vs (8.6∓0.5)% to (13.1∓1.4)%, P<0.01]. The chicken red blood cells isolated using the modified method can be applicable for calibration of flow cytometry.

Treatment of mcf-7 breast cancer cells with a red grape wine polyphenol fraction results in disruption of calcium homeostasis and cell cycle arrest causing selective cytotoxicity.

PubMed

Hakimuddin, Fatima; Paliyath, Gopinadhan; Meckling, Kelly

2006-10-04

Food components influence the physiology by modulating gene expression and biochemical pathways within the human body. The disease-preventive roles of several fruit and vegetable components have been related to such properties. Polyphenolic components such as flavonoids are strong antioxidants and induce the expression of several xenobiotic-detoxifying enzymes. The mechanism of selective cytotoxicity induced by red grape wine polyphenols against MCF-7 breast cancer cells was investigated in relation to their interference with calcium homeostasis. MCF-7 cells showed an increase in cytosolic calcium levels within 10 min of treatment with the polyphenols. Immunohistochemical localization of calmodulin with secondary gold-labeled antibodies showed similar levels of gold labeling in both MCF-7 cells and the spontaneously immortalized, normal MCF-10A cell line. MCF-7 cells treated with the red wine polyphenol fraction (RWPF) showed swelling of endoplasmic reticulum, dissolution of the nucleus, and loss of plasma membrane integrity as well as reduced mitochondrial membrane potential. These cells were arrested at the G2/M interphase. By contrast, MCF-10A cells did not show such changes after RWPF treatment. The results suggest that polyphenol-induced calcium release may disrupt mitochondrial function and cause membrane damage, resulting in selective cytotoxicity toward MCF-7 cells. This property could further be developed toward breast cancer prevention strategies either independently or in conjunction with conventional prevention therapies where a positive drug-nutrient interaction can be demonstrated.

Immunity War: A Novel Therapy for Lymphoma Using T-cell Bispecific Antibodies.

PubMed

Prakash, Ajay; Diefenbach, Catherine S

2018-06-08

The activity of T cell mediated immunotherapies in B cell lymphoma has been limited to date. The novel bi-specific antibody CD20-TCB, has a 2:1 antibody design to maximize T cell engagement, and demonstrates activity in preclinical models. This may represent a novel therapeutic approach for patients with relapsed/refractory NHL. Copyright ©2018, American Association for Cancer Research.

Effects of acute hypoxic exposure on oxygen affinity of human red blood cells.

PubMed

Chowdhury, Aniket; Dasgupta, Raktim

2017-01-20

Adaptation of red blood cells subjected to acute hypoxia, crucial for managing high altitude syndrome and pulmonary diseases, has been investigated. For this, red blood cells were exposed to the acute hypoxic condition by purging nitrogen over increasing time periods from 15 to 60 min and thereafter equilibrated with atmospheric oxygen for 10 min. Raman spectra of these red blood cells were then recorded and analyzed to look for changes in the level of oxygenation compared to unexposed cells. A decreasing oxygen affinity for the cells was observed with increasing time of exposure to the hypoxic condition. This change in oxygen affinity for the red blood cells may result from metabolic adjustment of the cells under the hypoxic condition to promote increased concentration of intracellular 2, 3-diphosphoglycerate.

Red cell aspartate aminotransferase saturation with oral pyridoxine intake.

PubMed

Oshiro, Marilena; Nonoyama, Kimiyo; Oliveira, Raimundo Antônio Gomes; Barretto, Orlando Cesar de Oliveira

2005-03-02

The coenzyme of aspartate aminotransferase is pyridoxal phosphate, generated from fresh vegetables containing pyridoxine. Vitamin B6-responsive sideroblastic anemia, myelofibrosis and Peyronies syndrome respond to high pyridoxine doses. The objective was to investigate the oral pyridoxine oral dose that would lead to maximized pyridoxal phosphate saturation of red cell aspartate aminotransferase. Controlled trial, in Hematology Division of Instituto Adolfo Lutz. Red cell aspartate aminotransferase activity was assayed (before and after) in normal volunteers who were given oral pyridoxine for 15-18 days (30 mg, 100 mg and 200 mg daily). In vitro study of blood from seven normal volunteers was also performed, with before and after assaying of aspartate aminotransferase activity. The in vivo study showed increasing aspartate aminotransferase saturation with increasing pyridoxine doses. 83% saturation was reached with 30 mg daily, 88% with 100 mg, and 93% with 200 mg after 20 days of oral supplementation. The in vitro study did not reach 100% saturation. Neither in vivo nor in vitro study demonstrated thorough aspartate aminotransferase saturation with its coenzyme pyridoxal phosphate in red cells, from increasing pyridoxine supplementation. However, the 200-mg dose could be employed safely in vitamin B6-responsive sideroblastic anemia, myelofibrosis and Peyronies syndrome treatment. Although maximum saturation in circulating red cells is not achieved, erythroblasts and other nucleated and cytoplasmic organelles containing cells certainly will reach thorough saturation, which possibly explains the results obtained in these diseases.

Neutralizing antibodies against West Nile virus identified directly from human B cells by single-cell analysis and next generation sequencing

PubMed Central

Tsioris, Konstantinos; Gupta, Namita T.; Ogunniyi, Adebola O.; Zimnisky, Ross M.; Qian, Feng; Yao, Yi; Wang, Xiaomei; Stern, Joel N. H.; Chari, Raj; Briggs, Adrian W.; Clouser, Christopher R.; Vigneault, Francois; Church, George M.; Garcia, Melissa N.; Murray, Kristy O.; Montgomery, Ruth R.; Kleinstein, Steven H.; Love, J. Christopher

2015-01-01

West Nile virus infection (WNV) is an emerging mosquito-borne disease that can lead to severe neurological illness and currently has no available treatment or vaccine. Using microengraving, an integrated single-cell analysis method, we analyzed a cohort of subjects infected with WNV - recently infected and post-convalescent subjects - and efficiently identified four novel WNV neutralizing antibodies. We also assessed the humoral response to WNV on a single-cell and repertoire level by integrating next generation sequencing (NGS) into our analysis. The results from single-cell analysis indicate persistence of WNV-specific memory B cells and antibody-secreting cells in post-convalescent subjects. These cells exhibited class-switched antibody isotypes. Furthermore, the results suggest that the antibody response itself does not predict the clinical severity of the disease (asymptomatic or symptomatic). Using the nucleotide coding sequences for WNV-specific antibodies derived from single cells, we revealed the ontogeny of expanded WNV-specific clones in the repertoires of recently infected subjects through NGS and bioinformatic analysis. This analysis also indicated that the humoral response to WNV did not depend on an anamnestic response, due to an unlikely previous exposure to the virus. The innovative and integrative approach presented here to analyze the evolution of neutralizing antibodies from natural infection on a single-cell and repertoire level can also be applied to vaccine studies, and could potentially aid the development of therapeutic antibodies and our basic understanding of other infectious diseases. PMID:26481611

Neutralizing antibodies against West Nile virus identified directly from human B cells by single-cell analysis and next generation sequencing.

PubMed

Tsioris, Konstantinos; Gupta, Namita T; Ogunniyi, Adebola O; Zimnisky, Ross M; Qian, Feng; Yao, Yi; Wang, Xiaomei; Stern, Joel N H; Chari, Raj; Briggs, Adrian W; Clouser, Christopher R; Vigneault, Francois; Church, George M; Garcia, Melissa N; Murray, Kristy O; Montgomery, Ruth R; Kleinstein, Steven H; Love, J Christopher

2015-12-01

West Nile virus (WNV) infection is an emerging mosquito-borne disease that can lead to severe neurological illness and currently has no available treatment or vaccine. Using microengraving, an integrated single-cell analysis method, we analyzed a cohort of subjects infected with WNV - recently infected and post-convalescent subjects - and efficiently identified four novel WNV neutralizing antibodies. We also assessed the humoral response to WNV on a single-cell and repertoire level by integrating next generation sequencing (NGS) into our analysis. The results from single-cell analysis indicate persistence of WNV-specific memory B cells and antibody-secreting cells in post-convalescent subjects. These cells exhibited class-switched antibody isotypes. Furthermore, the results suggest that the antibody response itself does not predict the clinical severity of the disease (asymptomatic or symptomatic). Using the nucleotide coding sequences for WNV-specific antibodies derived from single cells, we revealed the ontogeny of expanded WNV-specific clones in the repertoires of recently infected subjects through NGS and bioinformatic analysis. This analysis also indicated that the humoral response to WNV did not depend on an anamnestic response, due to an unlikely previous exposure to the virus. The innovative and integrative approach presented here to analyze the evolution of neutralizing antibodies from natural infection on a single-cell and repertoire level can also be applied to vaccine studies, and could potentially aid the development of therapeutic antibodies and our basic understanding of other infectious diseases.

Polyreactive Antibodies Plus Complement Enhance the Phagocytosis of Cells Made Apoptotic by UV-Light or HIV

PubMed Central

Zhou, Zhao-hua; Wild, Teresa; Xiong, Ying; Sylvers, Peter; Zhang, Yahong; Zhang, Luxia; Wahl, Larry; Wahl, Sharon M.; Kozlowski, Steven; Notkins, Abner L.

2013-01-01

Polyreactive antibodies are a major component of the natural antibody repertoire and are capable of binding a variety of structurally unrelated antigens. Many of the properties attributed to natural antibodies, in fact, are turning out to be due to polyreactive antibodies. In humans, each day, billions of cells undergo apoptosis. In the present experiments, we show by ImageStream technology that although polyreactive antibodies do not bind to live T cells they bind to both the plasma membrane and cytoplasm of late apoptotic cells, fix complement, generate the anaphylatoxin C5a and increase by as much as 5 fold complement-mediated phagocytosis by macrophages. Of particular importance, T cells undergoing apoptosis following infection with HIV also bind polyreactive antibodies and are phagocytosed. We conclude that the polyreactive antibodies in the natural antibody repertoire contribute in a major way to the clearance of cells made apoptotic by a variety of natural and infectious processes. PMID:23881356

Antibody screening & identification in the general patient population at a tertiary care hospital in New Delhi, India.

PubMed

Makroo, Raj Nath; Bhatia, Aakanksha; Hegde, Vikas; Chowdhry, Mohit; Thakur, Uday Kumar; Rosamma, N L

2014-09-01

The development of alloantibodies can significantly complicate transfusion therapy and results in difficulties in cross-matching of blood. Most literature on alloimmunization is limited to multitransfused individuals, with very few studies on the general hospital patients. This study was aimed at assessing the frequency and type of unexpected red cell antibodies in the general patient population at a multispecialty tertiary care centre in New Delhi, India. The results of 49,077 antibody screening tests carried out on patients, from January 2009 to December 2012 were analyzed. The clinical and transfusion records were reviewed. The data were compiled and statistically analysed. A total of 49,077 (29,917; 60.96% males and 19,160; 39.04% females) patient samples were screened for the presence of unexpected antibodies. Antibody screening was positive in 403 patients (0.82%). In the serum samples of 164 patients only autoantibodies were identified, 27 revealed autoantibodies with one or more underlying alloantibodies, while 212 patients had only alloantibody/ies in their serum. The overall alloimmunization rate was 0.49 per cent. Antibodies against the Rh system were the most frequent (64.1%), the most common alloantibody identified being anti E (37.2%), followed by anti D (19.2%). Since clinically significant antibodies are frequently detected in our patient population, antibody screening and if required, identification is the need of the hour. Since antibodies against the common Rh and Kell blood group antigens are the most frequent, provision of Rh and Kell matched red cells may be of protective value.

Antibody-Independent Control of γ-Herpesvirus Latency via B Cell Induction of Anti-Viral T Cell Responses

PubMed Central

McClellan, Kelly B; Gangappa, Shivaprakash; Speck, Samuel H; Virgin, Herbert W.

2006-01-01

B cells can use antibody-dependent mechanisms to control latent viral infections. It is unknown whether this represents the sole function of B cells during chronic viral infection. We report here that hen egg lysozyme (HEL)-specific B cells can contribute to the control of murine γ-herpesvirus 68 (γHV68) latency without producing anti-viral antibody. HEL-specific B cells normalized defects in T cell numbers and proliferation observed in B cell−/− mice during the early phase of γHV68 latency. HEL-specific B cells also reversed defects in CD8 and CD4 T cell cytokine production observed in B cell−/− mice, generating CD8 and CD4 T cells necessary for control of latency. Furthermore, HEL-specific B cells were able to present virally encoded antigen to CD8 T cells. Therefore, B cells have antibody independent functions, including antigen presentation, that are important for control of γ-herpesvirus latency. Exploitation of this property of B cells may allow enhanced vaccine responses to chronic virus infection. PMID:16789842

Red blood cells in thrombosis.

PubMed

Byrnes, James R; Wolberg, Alisa S

2017-10-19

Red blood cells (RBCs) have historically been considered passive bystanders in thrombosis. However, clinical and epidemiological studies have associated quantitative and qualitative abnormalities in RBCs, including altered hematocrit, sickle cell disease, thalassemia, hemolytic anemias, and malaria, with both arterial and venous thrombosis. A growing body of mechanistic studies suggests that RBCs can promote thrombus formation and enhance thrombus stability. These findings suggest that RBCs may contribute to thrombosis pathophysiology and reveal potential strategies for therapeutically targeting RBCs to reduce thrombosis. © 2017 by The American Society of Hematology.

Cell growth inhibition and apoptotic effects of a specific anti-RTFscFv antibody on prostate cancer, but not glioblastoma, cells

PubMed Central

Nejatollahi, Foroogh; Bayat, Payam; Moazen, Bahareh

2017-01-01

Background: Single chain antibody (scFv) has shown interesting results in cancer immunotargeting approaches, due to its advantages over monoclonal antibodies. Regeneration and tolerance factor (RTF) is one of the most important regulators of extracellular and intracellular pH in eukaryotic cells. In this study, the inhibitory effects of a specific anti-RTF scFv were investigated and compared between three types of prostate cancer and two types of glioblastoma cells.  Methods: A phage antibody display library of scFv was used to select specific scFvs against RTF using panning process. The reactivity of a selected scFv was assessed by phage ELISA. The anti-proliferative and apoptotic effects of the antibody on prostate cancer (PC-3, Du-145 and LNCaP) and glioblastoma (U-87 MG and A-172) cell lines were investigated by MTT and Annexin V/PI assays.  Results: A specific scFv with frequency 35% was selected against RTF epitope. This significantly inhibited the proliferation of the prostate cells after 24 h. The percentages of cell viability (using 1000 scFv/cell) were 52, 61 and 73% for PC-3, Du-145 and LNCaP cells, respectively, compared to untreated cells. The antibody (1000 scFv/cell) induced apoptosis at 50, 40 and 25% in PC-3, Du-145 and LNCaP cells, respectively. No growth inhibition and apoptotic induction was detected for U-87 and A172 glioblastoma cells.  Conclusions: Anti-RTFscFv significantly reduced the proliferation of the prostate cancer cells. The inhibition of cell growth and apoptotic induction effects in PC-3 cells were greater than Du-145 and LNCaP cells. This might be due to higher expression of RTF antigen in PC-3 cells and/or better accessibility of RTF to scFv antibody. The resistance of glioblastoma cells to anti-RTF scFv offers the existence of mechanism(s) that abrogate the inhibitory effect(s) of the antibody to RTF. The results suggest that the selected anti-RTF scFv antibody could be an effective new alternative for prostate cancer

Generation of monoclonal antibodies specific for ORF68 of koi herpesvirus.

PubMed

Aoki, Takashi; Takano, Tomokazu; Unajak, Sasimnanas; Takagi, Madoka; Kim, Young Rim; Park, Seong Bin; Kondo, Hidehiro; Hirono, Ikuo; Saito-Taki, Tatsuo; Hikima, Jun-Ichi; Jung, Tae Sung

2011-05-01

Outbreaks of koi herpesvirus (KHV) infection in carp are still a serious problem worldwide. KHV is closely related to other two cyprinid herpesviruses, pox herpesvirus (CHV) and haematopoietic necrosis herpesvirus (CyHV-2) in goldfish. In this study, two major KHV antigenic proteins (ORF62 and ORF68) were identified by immunoscreening using a KHV-specific polyclonal antibody, and then monoclonal antibodies were generated for immunodiagnostic studies. After screening hybridoma cells, one mAb against ORF68 (mAb-7C6) was obtained but no mAbs against ORF62. mAb-7C6 specifically reacted with a lysate of KHV-infected koi fin cells (KF-1 cells) but not with lysates of CHV- or CyHV-2-infected KF-1 cells in an immuno-blotting analysis. Similar results were shown in the following tests: (1) a indirect fluorescent antibody test using infected KF-1 cells and (2) an immunohistochemical investigation by fast red stain (infected liver) or FITC detection (infected spleen). These results suggested that mAb-7C6 specifically reacts with KHV ORF68 protein. Copyright © 2010 Elsevier Ltd. All rights reserved.

CD133 antibody conjugation to decellularized human heart valves intended for circulating cell capture.

PubMed

Vossler, John D; Min Ju, Young; Williams, J Koudy; Goldstein, Steven; Hamlin, James; Lee, Sang Jin; Yoo, James J; Atala, Anthony

2015-09-03

The long term efficacy of tissue based heart valve grafts may be limited by progressive degeneration characterized by immune mediated inflammation and calcification. To avoid this degeneration, decellularized heart valves with functionalized surfaces capable of rapid in vivo endothelialization have been developed. The aim of this study is to examine the capacity of CD133 antibody-conjugated valve tissue to capture circulating endothelial progenitor cells (EPCs). Decellularized human pulmonary valve tissue was conjugated with CD133 antibody at varying concentrations and exposed to CD133 expressing NTERA-2 cl.D1 (NT2) cells in a microflow chamber. The amount of CD133 antibody conjugated on the valve tissue surface and the number of NT2 cells captured in the presence of shear stress was measured. Both the amount of CD133 antibody conjugated to the valve leaflet surface and the number of adherent NT2 cells increased as the concentration of CD133 antibody present in the surface immobilization procedure increased. The data presented in this study support the hypothesis that the rate of CD133(+) cell adhesion in the presence of shear stress to decellularized heart valve tissue functionalized by CD133 antibody conjugation increases as the quantity of CD133 antibody conjugated to the tissue surface increases.

From stem cell to red blood cells in vitro: "the 12 labors of Hercules".