Bovine monoclonal alloantibodies to blood group antigens prepared by murine x bovine or (murine x bovine) x bovine interspecies fusions.

In order to obtain monoclonal alloantibodies against bovine blood group antigens, lymph node cells from calves immunized with bovine red blood cells (RBC) were fused with either murine NSO/1 myeloma cells or a HAT sensitive murine x bovine heterohybridoma cell line. Both fusion partners resulted in heterohybridoma cell lines, producing monoclonal alloantibodies against bovine red blood cell antigens. Several clones produced antibodies against identical antigens and some of these clones have been further analysed. The antibodies produced by these selected cell lines have been compared with conventional polyclonal antisera used in bovine blood typing service. Thus extensive tests--including the ISAG Comparison Tests 1989/90 and 1991/92--have proved that monoclonal alloantibodies specific for the internationally recognized bovine red cell antigens A2, I1, O1, Q, A', B', Q', C1, R1, X1, S and Z have been produced. The Q, A', B', and C1 antibodies react weakly with certain phenogroups, whereas the A2, I1, O1, Q', R1, X1, S and Z antibodies have proved to be excellent blood typing reagents and have now substituted the polyclonal antisera in routine bovine blood typing in our laboratory.     

The role of CD47 in neutrophil transmigration. Increased rate of migration correlates with increased cell surface expression of CD47

CD47, a cell surface glycoprotein, plays an important role in modulating neutrophil (PMN) migration across endothelial and epithelial monolayers. Here we show that anti-CD47 monoclonal antibodies (mAbs) delay PMN migration across collagen-coated filters or T84 epithelial monolayers toward the chemoattractant formylmethionylleucylphenylalanine (fMLP). Despite delayed transmigration by anti-CD47 mAbs, the numbers of PMN migrating across in either condition were the same as in the presence of control non-inhibitory mAbs. Cell surface labeling and immunoprecipitation demonstrated upregulation of CD47 to the PMN cell surface with kinetics similar to those of the transmigration response. Subcellular fractionation studies revealed redistribution of CD47 from intracellular compartments that co-sediment with secondary granules to plasma membrane-containing fractions after fMLP stimulation. Experiments performed to investigate potential signaling pathways revealed that inhibition of tyrosine phosphorylation with genistein reversed the anti-CD47-mediated PMN migration delay, whereas inhibition of phosphatidylinositol 3-kinase only partially reversed anti-CD47 effects that correlated with a rapid increase in PMN cell surface CD47. Analysis of the contribution of epithelial-expressed CD47 to PMN transmigration revealed that PMN migration across CD47-deficient epithelial monolayers (CaCO2) was significantly increased after stable transfection with CD47. These results suggest that cell surface CD47 and downstream tyrosine phosphorylation signaling events regulate, in part, the rate of PMN migration during the inflammatory response.     

Hormonal and immunological changes in blood and mammary secretion in the sow at parturition

The purpose of the present study was to record possible variations of estradiol-17 beta (E2) and cortisol concentrations, and parameters related to granulocyte phagocytosis in mammary secretions from healthy sows at parturition. The study was comprised 8 primiparous sows (Landrace x Yorkshire). Blood and mammary secretion samples were collected twice daily from 3 d before (only blood) until 3 d after farrowing. Estradiol-17 beta and cortisol concentrations were determined in plasma and in cell-depleted skimmed mammary secretions. Phagocytic capacity of polymorphonuclear cells (PMN) was assessed in whole blood and in cell suspensions derived from mammary secretions. Opsonic activity was assessed in serum and in cell-depleted skimmed mammary secretions. The 2 assays were based on chemiluminescence. Estradiol-17 beta concentration in plasma decreased (P < 0.001) directly after parturition. In skimmed secretions, the highest E2 concentration was recorded in the first sample after parturition and decreased (P < 0.01) thereafter. The highest cortisol concentration in plasma was recorded in the evening before parturition (P < 0.01). In skimmed secretions, there was no significant variation in cortisol concentration. The concentrations of both steroid hormones were lower in mammary secretions than in plasma. The phagocytic capacity of PMN in blood and mammary secretion, expressed as peak chemiluminescence per PMN, showed no significant change. This was also true for the opsonic activity in serum. In skimmed secretions the opsonic activity increased (P < 0.01) after parturition. These data emphasize the differences between plasma and mammary secretion concentrations of steroid hormones as well as between systemic and mammary gland immune competence. Regarding the phagocytosis process in mammary secretions, the part directly related to the PMN function seemed not to be altered at parturition compared with later on in lactation, whereas the part related to opsonic activity seemed to be impaired at parturition. The latter may play a role in the development of coliform mastitis at this time.     

Alloantibodies prevent the induction of transplantation tolerance by enhancing alloreactive T cell priming

Circulating alloantibodies in transplant recipients are often associated with increased Ab-mediated as well as cellular rejection. We tested the hypothesis that alloantibodies facilitate cellular rejection by functioning as opsonins to enhance T cell activation using a BALB/c to C57BL/6 heart or skin transplant model. Long-term heart and skin survival induced with anti-CD154 alone or in combination with donor-specific transfusion (DST), respectively, was abrogated by the presence of anti-K(d) mAbs, and alloreactive T cell activation as well as acute rejection was observed. The prevention of graft acceptance in the skin model was dependent on anti-K(d) binding to and converting DST from tolerigenic to immunogenic. Adoptive transfer of CFSE-labeled TCR-transgenic T cells into B6 recipients treated with anti-CD154/DST revealed the ability of anti-K(d) to enhance the proliferation of anti-K(d)-specific T cells via the indirect pathway as well as of non-K(d)-reactive, recipient MHC-restricted CD4(+) and CD8(+) T cells. Thus, alloantibodies with restricted specificity are able to facilitate the indirect presentation as well as the cross-presentation of a larger repertoire of "linked" donor-derived Ags. These observations highlight the ability of alloantibodies to function not only in classical humoral rejection but also as opsonins that facilitate the CD40-CD154-independent activation of alloreactive T cells.     

Cytochemical investigation of neutral proteases in polymorphonuclear (PMN) neutrophils in acute inflammatory diseases

Summary Neutral proteases can be released from PMN neutrophils in blood smears from healthy subjects by incubation with NaCl-borate buffer. The activity of the PMN proteases can be revealed by the degradation of erythrocytes and plasma within ring-shaped areas centered around each neutrophil (halo effect). During the acute stage of various inflammatory diseases (pneumonia, meningitis, cholecystitis, etc.) the activity of neutral PMN proteases is substantially reduced, as reflected by reduced halo formation. After recovery, halo formation returns to normal. Temporary lowering of neutral PMN proteases is thus one of a series of functional defects of PMN neutrophils which are detectable in the course of acute infectious diseases. These include reduced phagocytosis, altered chemotaxis and reduced bactericidal function. The cytochemical test for neutrophilic granulocyte function used in the present investigation is especially practical by comparison with the other techniques: it saves time and is simple to perform.Dedicated to Prof. W. Graumann on the occasion of his 65th birthday     

Study of equivalents of rhesus antigens in non-human primates]

Human alloantibodies specific of some Rh antigens cross-react with non human primates red blood cells. These crossreactions demonstrated that only African apes express equivalents of Rho (D) and hr' (c). The antigenic resemblance between these two human antigens and their primate homologues is confirmed by the reactivities of human anti-D and anti-c monoclonal antibodies. The use of a human Rh cDNA probe allowed to confirm by Southern blot hybridization that nonhuman primates possess Rh-like genes. The number of Rh-like genes per haploid genome was deduced from the results obtained with exon-specific probes.     

Rapid diagnosis of cytomegalovirus in immunocompromised patients using monoclonal antibodies that detect early viral antigens

A technique was applied to detect early fluorescent antigens (DEFA) of cytomegalovirus (CMV) using the E13 monoclonal antibodies in 52 immuno-compromised patients hospitalized in the Nephrology Institute of Havana. Of the 75 urine or blood (buffy coat) samples taken, 15 were found positive to CMV. Using classical diploid human fibroblast isolation technique, 12 CMV strains were isolation of previously detected positive samples by DEFA. In addition, CMV was isolated from one sample reported to be negative by DEFA. A coincidence of 80% was found between both techniques. With the ELISA test, all the sample studied have IgG antibodies to CMV.     

Endotoxaemia, production of tumour necrosis factor alpha and polymorphonuclear neutrophil activation following strenuous exercise in humans

To examine whether endotoxaemia accompanying long-term, strenuous physical exercise is involved in exercise-induced increase in plasma tumour necrosis factor alpha (TNF-alpha) concentration and polymorphonuclear neutrophil (PMN) activation, 14 male recreational athletes [mean age 28 (SEM 1) years] were studied. Exercise consisted of a 1.5-km river swim, a 40-km bicycle race, and a 10-km road race. Mean time to complete the race was 149.8 (SEM 4.8) min. The plasma concentrations of granulocyte myeloperoxidase (MPO) and TNF-alpha were significantly higher than baseline values immediately and 1 h after exercise (P<0.001). Both variables returned to pre-race levels the day after exercise. Marked, transient decreases in plasma concentrations of anti-lipopolysaccharide (LPS) immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies directed against a panel of selected smooth gram-negative LPS were observed after the race, reaching in most cases minimal values in the blood sample drawn immediately following the completion of the triathlon. There was no significant correlation between the magnitude of PMN activation, as assessed by the increase in plasma concentrations of MPO, and the humoral markers of endotoxaemia and TNF-alpha. An inverse, highly significant relationship between the increase in plasma TNF-alpha concentrations and the changes in circulating anti-LPS IgM antibodies concentrations was observed (r = -0.7; P<0.01). These findings suggest that exercise-induced endotoxaemia was involved in the release of TNF-alpha, that the magnitude of the TNF-alpha response to exercise was down-regulated by anti-LPS antibodies of the IgM class, and that the production of TNF-alpha and endotoxaemia did not seem to play a role in the activation of circulating PMN in the exercising subjects.     

Possible in vivo tolerance of human polymorphonuclear neutrophil to low-grade exercise-induced endotoxaemia.

To address the question of whether translocation of bacterial lipopolysaccharide (LPS) into the blood could be involved in the process of exercise-induced polymorphonuclear neutrophil (PMN) activation, 12 healthy male subjects who took part in a sprint triathlon (1.5 km river swim, 40 km bicycle race, 10 km road race) were studied. While there was no detectable amount of endotoxin in the blood samples drawn at rest, exercise was followed by the appearance of circulating endotoxin molecules at the end of competition in four subjects, and after one and 24 h recovery in three and seven athletes, respectively. The concentrations of plasma granulocyte myeloperoxidase ([MPO]), were significantly higher immediately after exercise and one hour later-than baseline values (P<0.001). This variable returned to pre-race levels the day after exercise, despite the presence of detectable amounts of LPS, at that time, in seven athletes. The absence of significant correlation (r=0.26; P=0.383) and temporal association between [MPO] and plasma endotoxin levels led us to conclude that endotoxaemia was not involved in the process of exercise-induced PMN degranulation observed in our subjects.     

Sero-prevalence of RBC alloantibodies among Han and minority patients in Xinjiang

The aim of the study was to examine the sero-prevalence and frequency of red blood cell (RBC) alloantibodies (RAAbs) and to investigate the risk factors for producing RAAbs among the Han and Uyghurs in Xinjiang. The RBC antibody screening test and identification were conducted for 45,163 Han and 70,633 Uyghurs admitted to the First Affiliated Hospital of Xinjiang Medical University from October 2010 to December 2014. RBC alloantibodies against the Rh antigens were the most frequent, including anti-E(21.7% in the Han, 21.6% in Uyghurs) and anti-D(18.5% in the Han, 18.2% in Uyghurs). Notably, the sero-prevalence of anti-K and anti-Fy^a was also high in Xinjiang. Transfusion and pregnancy were risk factors among both the Han and Uyghurs; furthermore, Uyghurs had a higher sero-prevalence of RBC antibodies compared to that of Han because of a higher incidence of these risk factors. We concluded that RBC alloantibodies against the Rh factor showed the highest frequency, and antibodies against Asian-related high-frequency antigens, including Fy^a and low-frequency antigens, such as K were notably high in Xinjiang.     

Involvement of CD44 and the cytoskeletal linker protein ankyrin in human neutrophil bacterial phagocytosis

The leukocyte CD44 and CD45 cell surface receptors are associated via the linker proteins ankyrin and fodrin with the cytoskeleton, which itself is important in immune cell functions such as adherence, chemotaxis, and phagocytosis. The effects of rat antihuman CD44 and CD45 monoclonal antibodies on phagocytosis of fluoresceinated heat-killed Staphylococcus aureus 502A by normal human neutrophils (PMNs) during 2 hr incubation in RPMI-1640 was studied via flow cytometry and confocal microscopy. Flow cytometry was performed using an excitation wavelength of 488 nm, fluorescence being measured at 515–560 nm on 50,000 PMNs per sample. Confocal microscopy was performed on samples after further incubation with rhodamine-conjugated antiankyrin. Anti-CD44 resulted in an increase of 27–31% compared to control (P = 0.004) in the proportion of PMNs fluorescing, an increase of 17–24% (P = 0.001) in mean intracellular fluorescence per PMN, and an increase in total PMN fluorescence of 50–58% compared to control (P < 0.001). In contrast, anti-CD45 had little effect on phagocytosis. Colchicine (a microtubule-disrupting agent) enhanced, whereas cytochalasin-D (a microfilament inhibitor) inhibited bacterial phagocytosis; cytochalasin-D completely abrogated the effect of anti-CD44 on this PMN function. Hyaluronic acid augmented phagocytosis by an increment similar to that observed with anti-CD44. Two-color flow cytometry and confocal microscopy demonstrated that ankyrin always colocalized with ingested fluorescein isothiocyanate (FITC)-labeled bacteria. These data strongly suggest that CD44 is involved in bacterial phagocytosis, provide further evidence of CD44 receptor linkage to cytoskeletal elements in human leukocytes, and suggest that ankyrin has a significant role in the transport of phagosomes. © 1996 Wiley-Liss, Inc.     

TCEP-HCl can resolve the daratumumab interference

Daratumumab (DARA) is a human CD38 IgG1 monoclonal antibody that has significant therapeutic effects on multiple myeloma (MM). However, DARA may interfere with the indirect antiglobulin test (IAT) used to test for MM. At present, there are defects in the popular strategies for IAT anti-human globulin reactions caused by anti-CD38 monoclonal antibody drugs. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) is a new and effective solution to eliminate DARA's interference with the IAT. To verify its effect, the following experiments were done. After TCEP-HCl was administered to red blood cells (RBCs), the expression level of CD38 antigen on RBCs was determined by flow cytometry. Serological experiments were performed to verify the effects of TCEP-HCl on different blood groups of RBCs. The results showed that DARA could cause false-positive reaction in IAT, and the CD38 antigen on the membranes of RBCs decreased after TCEP-HCl treatment. Serological results showed that TCEP-HCl did not damage other blood group antigens on RBCs, except for Kell antigens. Indirect antiglobulin tests appeared normal after treatment with the TCEP-HCl reagent. The conclusion is that TCEP-HCl can resolve daratumumab interference in the IAT.     

Delayed leukocytosis after hard strength and endurance exercise: Aspects of regulatory mechanisms

Background  

During infections, polymorphonuclear neutrophilic granulocytes (PMN) are mobilized from their bone marrow stores, travel with blood to the affected tissue, and kill invading microbes there. The signal(s) from the inflammatory site to the marrow are unknown, even though a number of humoral factors that can mobilize PMN, are well known. We have employed a standardized, non-infectious human model to elucidate relevant PMN mobilizers. Well-trained athletes performed a 60-min strenuous strength workout of leg muscles. Blood samples were drawn before, during and just after exercise, and then repeatedly during the following day. Cortisol, GH, ACTH, complement factors, high-sensitive CRP (muCRP), IL-6, G-CSF, IL-8 (CXCL8) and MIP-1β (CCL4) were measured in blood samples. PMN chemotaxins in test plasma was assessed with a micropore membrane technique.     

Lysis of fresh solid tumor targets in the presence of Con A is mediated primarily by Leu 7+ peripheral blood T lymphocytes: blocking by the anti-CD3 monoclonal antibody and comparison with recombinant interleukin 2-induced lysis by natural killer cells

We investigated the lysis of fresh human solid tumor cells by peripheral blood T lymphocytes in the presence of lectins and anti-CD3 monoclonal antibodies (mAb). Addition of certain lectins (Con A, PHA, or WGA) directly into the 4-hr 51Cr-release assay caused significant lysis of (P less than 0.001) noncultured solid tumor targets by enriched populations of granular lymphocytes (GL). Significant levels (P at least less than 0.001) of Con A- or PHA-dependent solid tumor lysis by GL-enriched lymphocytes were observed in 32 of 39 donors (82%) and 14 of 20 donors (70%), respectively. In contrast, the addition of other lectins (PNA, PWM, or LPS) or anti-CD3 mAb did not cause cytotoxicity. The levels of Con A-dependent lysis were comparable to those of interleukin 2 (IL-2)-induced lysis by Leu 11b+ natural killer (NK) cells. The presence of lectins at the effector phase, but not of recombinant IL-2 (rIL-2), was required for the lysis of solid tumor targets. Both Con A-dependent and rIL-2-induced lysis were totally inhibited by treatment of the effector cells with the lysosomotropic agent L-leucine methyl ester (LeuOMe). Effector cells responsible for Con A-dependent lysis of solid tumors expressed T3 (CD3), T8 (CD8), and Leu 7 antigens, but lacked T4 (CD4) and Leu 11 (CD16) antigens as determined by both negative and positive cell selection studies. Con A-dependent lysis was inhibited at the effector phase by anti-CD3 (OKT3 or anti-Leu 4) or anti-CD2 (OKT11) mAb. On the basis of their phenotype (Leu 7+ CD3+ CD8+ CD16-), we hypothesize that these effector cells may contain a population of cytotoxic T cells (CTL) generated in vivo against autologous modified cells that can lyse fresh solid tumor target cells under conditions where the recognition requirements for the CTL are bypassed by lectin approximation.     

Kinetics of neutrophil-releasing activity of filtration leukapheresis and postleukapheresis plasma

A rat model was used to study the effect of filtration leukapheresis on the kinetics of granulocyte mobilization in normal rat donors and in recipients of plasma obtained from these donors. Our earlier observations showed that infusion of postpheresis plasma (PPP) into normal homologous recipients is consistently capable of inducing a marked granulocytosis. The effects of such a transfusion is dose related and duration dependent. This study shows that the granulocytosis occurring subsequent to administration of PPP was directly related to the duration of pheresis in the donor. Its maximum effect occurred 2–3 hr after transfusion. Animals with a low granulocyte count were less capable of mobilizing granulocytes during filtration leukapheresis than animals with a higher prepheresis count, possibly due to low endogenous stores of granulopoietin. Additionally, rats < 300 g were capable of mobilizing fewer granulocytes per volume of circulating blood than were larger (older?) animals. The maximum ability of PPP to increase circulating granulocyte numbers was found when postpheresis plasma was injected along with concurrent removal of circulating granulocytes by filtration leukapheresis.     

Immunomagnetic bead separation of mononuclear cells from contaminating granulocytes in cryopreserved blood samples

Density gradient centrifugation usually allows efficient separation of mononuclear cells from granulocytes using fresh human blood samples. However, we have found that with cryopreserved blood samples, density gradient centrifugation fails to separate granulocytes from mononuclear cells and have explored using immunomagnetic anti-CD15 microbeads as an alternate method to separate these cell populations. Using cryopreserved blood samples from 10 healthy donors we have shown that granulocytes express a significantly higher level of CD15 antigen than monocytes and lymphocytes, which allows for their efficient separation from mononuclear cells using anti-CD15 microbeads. This procedure is critical for purification of individual cell populations from cryopreserved leukocyte samples and could also potentially be applied to avoid granulocyte contamination of mononuclear cells isolated from stored blood and from patients with sepsis or thermal injury.     

Minimal peripheral blood cells carrying clonal markers of B cell disorders: evidence for monoclonality of circulating lymphocytes in patients with multiple myeloma

Peripheral blood lymphocytes (PBL) of 20 patients with multiple myeloma (MM) were assayed for clonality by Southern blot and cell surface marker analysis. Eight samples showed monoclonal origin of circulating lymphocytes by demonstrating rearrangements of the heavy chain immunoglobulin gene (IgH). In selected experiments, comparison of IgH rearrangements of bone marrow plasma cells and peripheral blood-derived mononuclear cells, highly enriched for B lymphocytes, proved to be identical. However, monoclonal circulating cells could not be detected in samples with rearranged IgH genes by surface marker phenotyping using one-color immunofluorescence analysis and a panel of monoclonal and polyclonal antibodies to various B lineage-associated antigens. These results indicate that in a substantial proportion of MM, monoclonal growth involves circulating B lymphocytes and underscores the clinical usefulness of Southern analysis of IgH gene rearrangements for monitoring this disease.     

Recovery of bulky DNA adducts by the regular and a modified 32P-postlabelling assay; influence of the DNA-isolation method

Bulky DNA adducts are widely used as biomarkers of human exposure to complex mixtures of environmental genotoxicants including polycyclic aromatic hydrocarbons. The 32P-postlabelling method is highly sensitive for the detection of bulky DNA adducts, but its relatively low throughput poses limits to its use in large-scale molecular epidemiological studies. The objectives of this study were to compare the impact of DNA-sample preparation with a commercial DNA-isolation kit or with the classical phenol-extraction procedure on the measurement of bulky DNA adducts by 32P-postlabelling, and to increase the throughput of the 32P-postlabelling method--whilst maintaining radio-safety--by reducing the radioisotope requirement per sample. The test DNA samples were prepared from MCF-7 cells treated with benzo[a]pyrene and from human peripheral blood lymphocytes, buffy coat, and peripheral lung tissue. The modified 32P-postlabelling procedure involved an evaporation-to-dryness step after the enzymatic digestions of the DNA, and radio-labelling with a reduced amount of [γ-32P]ATP substrate in a reduced reaction volume compared with the regular method. Higher levels of DNA adducts were measured in the MCF-7 cells and in the lung-tissue samples after isolation with the kit than after solvent extraction. A seven-fold higher level of adducts was detected in the buffy-coat DNA samples isolated with the kit than with the phenol extraction procedure (p<0.001). Reduction of the amount of [γ-32P]ATP from 50 μCi to 25 μCi (>6000 Ci/mmol specific radioactivity) per sample in the modified 32P-postlabelling procedure was generally applicable without loss of adduct recovery for all test samples prepared with both DNA isolation methods. The difference between the bulky DNA-adduct levels resulting from the two DNA-isolation procedures requires further systematic investigation. The modified 32P-postlabelling procedure allows a 50% reduction of radioisotope requirement per sample, which facilitates increased throughput of the assay whilst maintaining radio-safety.     

Visual detection of granulocyte surface antigens using the avidin-biotin complex

A visual test for detection of granulocyte surface markers using the avidin-biotin complex (ABC) has been developed. That this assay is highly specific, reproducible, and sensitive was determined by studying the expression of HLA antigens on granulocytes with monoclonal antibodies. Further, using granulocyte specific alloantisera, the results of the ABC test compared well to data from leukoagglutination assays and indirect immunofluorescence tests. The assay is particularly advantageous in that granulocytes can be stored, only small amounts of cells and sera are needed, and heterogeneous cell populations can easily be studied.     

Developing and validating a modified enzyme linked immunosorbent assay method for detecting HEV IgG antibody from dried blood spot (DBS) samples in endemic settings

Serological analysis is an integral part of laboratory practice nowadays. The present study was aimed to develop and validate a modified Enzyme linked Immunosorbent Assay (ELISA) for determination of IgG antibody against Hepatitis E Virus (HEV) using dried blood spots (DBS) and corresponding plasma samples. A total of 65 samples (45 HEV patients, 20 healthy controls) were analyzed. DBS and plasma samples demonstrated equivalent optical densities for detecting anti-HEV IgG. A highly significant correlation was observed between plasma and DBS sample absorbances (R² = 0.98; p < 0.001) at dilution 1:200, indicating true agreement between the two procedures. The assay exhibited decent linearity and showed no effect of physiological hematocrit on assay performance. Data suggested recommendable promise in using DBS as a suitable alternative to plasma samples to determine HEV IgG antibody evidenced by significant correlation with plasma results. Therefore, identical method for processing DBS specimens including it's proper storage is recommended for implementation of a modified ELISA in different settings.