Immature monocytes from G-CSF-mobilized peripheral blood stem cell collections carry surface-bound IL-10 and have the potential to modulate alloreactivity.

Production of the anti-inflammatory cytokine IL-10 by monocytes has been implicated as a probable negative regulator of graft-versus-host disease (GvHD) in patients undergoing allogeneic stem cell transplants (SCT). Monocytes from G-CSF-mobilized peripheral blood stem cell (gmPBSC) collections have been reported to produce more IL-10 than unmobilized monocytes in response to proinflammatory factors such as LPS. Why this should occur is unclear. In this study, monocyte phenotype and IL-10 localization and release were investigated in PB mononuclear cells (MNC) from 27 healthy donors mobilized for allogeneic SCT and from 13 patients with hematological malignancies mobilized for autologous SCT. All isolates contained elevated total percentages of monocytes in comparison with unmobilized PB, a high proportion of which displayed an immature phenotype. Stimulation of gmPB MNC with an inflammatory stimulus [fixed Staphylococcus aureus cells (SAC)] induced rapid up-regulation of CD14, indicating conversion to mature status. Localization studies indicated that IL-10 was predominantly present, bound on the surface of CD64(+)/CD14(low/neg) immature monocytes. Inflammatory stimuli (LPS, polyinosinic:polycytidylic acid, or SAC) induced release of variable quantities of IL-10 from the cell surface. MNC, separated into surface IL-10-positive or -negative fractions, differed in their ability to stimulate alloreactivity in MLR, and IL-10(+) MNC induced significantly lower levels of proliferation than IL-10(-) MNC. Thus, the subset of immature monocytes carrying surface-bound IL-10 in gmPB has the potential to modulate alloreactivity and GvHD after allogeneic SCT through cell-to-cell contact and released IL-10.

Evaluation of a panel of human monoclonal antibodies to D and exploration of the synergistic effects of blending IgG1 and IgG3 antibodies on their in vitro biologic function.

BACKGROUND: The D immunoprophylaxis program has successfully reduced the incidence of Rh hemolytic disease of the newborn (HDN), but it has also reduced the availability of plasma-derived polyclonal anti-D, which constitutes the current therapeutic product. Human monoclonal anti-D from hybridoma cell lines may be an acceptable alternative, and clinical efficacy of each anti-D is being evaluated in several centers. STUDY DESIGN AND METHODS: This study represents the largest assessment (outside of the International Workshops) of human D monoclonal antibodies for potential therapeutic use. The in vitro biologic activity and immunologic and serologic reactivity of a coded panel of 20 D antibodies (THERAD) was investigated. The bioassays used were lymphocyte (K-cell) antibody-dependent cell-mediated cytotoxicity (ADCC), monocyte ADCC, and monocyte chemiluminescence, which together reflect the processes involved in antibody-coated red cell destruction in vivo. From this panel, six antibodies (THERADs 14, 19, 22, 23, 27, and 28, comprising 3 IgG1 and 3 IgG3 D monoclonal antibodies) were further selected to investigate the effects of blending in the three bioassays. RESULTS: Several THERAD blends displayed greater activity than their component parts, in the range of 6 to 124 percent. There was no evidence to suggest functional blocking effects with this restricted panel of antibodies. CONCLUSION: The THERAD blends containing both IgG1 and IgG3 anti-D appeared to be the most functionally active, as did blends containing antibodies to two distinct D epitopes. This in vitro evidence has important implications for the future formulation of an effective monoclonal preparation for the prevention of Rh HDN.

A retrospective study of red cell maternal antibodies by chemiluminescence.

Plasma samples from 109 patients with maternal IgG alloantibodies were investigated using a chemiluminescence (CL) assay, a functional test, to predict which antibodies were clinically significant. The CL assay was able to distinguish between those patients who were unaffected or mildly affected requiring only phototherapy, and those patients with moderate or severe haemolytic disease of the fetus or newborn (HDN) requiring transfusion therapy. The CL result was compared with the anti-D quantification result, the number of IgG molecules bound per red cell and, in 80% of the cases, the monocyte monolayer assay. If mothers carrying Rh-negative fetuses were ignored, then the CL assay correctly predicted the outcome for 93.4% of all cases (including those other than D), while the AutoAnalyzer and monocyte assay predicted correctly 92.7% (of the anti-D patients) and 81.5%, respectively. Greater than 80% of patients with severe or moderate HDN had both IgG1 + IgG3 subclasses in the maternal plasma, while those infants who were unaffected or only mildly affected had a greater chance of having IgG1 only (44%) in the maternal plasma, IgG3 only (27%) or both subclasses (29%).

A comparison of the functional activity of monoclonal and polyclonal Rh antibodies.

The functional activity of monoclonal anti-Ds has been compared with that of non-D monoclonal antibodies. Approximately 50% of anti-Ds were more active than a positive control while more than 75% of non-D antibodies failed to reach 10% activity of the positive control. These figures do not correlate with the IgG bound per red cell which, however, does correlate closely with the antigen site density as would be expected.

A chemiluminescence assay for erythrophagocytosis.

A luminol-dependent chemiluminescence assay for the assessment of the phagocytosis of erythrocytes sensitized with anti-D IgG immunoglobulin by mononuclear leukocytes is described. The mononuclear leukocytes were obtained by apheresis enriched by centrifugation through a density gradient and stored in liquid nitrogen before use. The total reaction mixture, consisting of mononuclear leukocytes-luminol-erythrocytes (either anti-D IgG sensitized or unsensitized controls) was 500 microliters, light detection was by an LKB 1251 luminometer. Peak luminescence was seen between 35-45 minutes, the reaction being exhausted by 120 minutes. Determination of the reproducibility of the assay gave intra- and inter-assay coefficients of variation of 5% and 13% respectively. We found the chemiluminescent response to be affected by the number of erythrocytes used in the assay and by the composition of the medium in which the cells were resuspended, particularly the pH at the initiation of the assay. We also compared the chemiluminescence assay to a microscopic phagocytic assay and found the results virtually identical. However, the former chemiluminescence assay was much easier to perform, marginally more sensitive, less laborious and eliminated any possibility of subjective error.

Passive haemagglutination inhibition for quantitation of red cell associated IgG.

Passive haemagglutination inhibition (PHI) was adapted to quantitate red cell associated IgG. Twenty one patients with autoimmune haemolytic anaemia (AIHA) had a raised red cell associated IgG, mean (SD) = 5.783 (6.183) ng/10(6) red blood cell compared with that of 69 subjects with a red cell associated IgG of 0.433 (0.349) ng/10(6) red blood cell. Thirteen of 14 blood donors with a positive direct antiglobulin test (DAGT) had a normal red cell associated IgG. The only blood donor with positive DAGT and raised red cell associated IgG had AIHA. Studies of red cell associated IgG in other groups of patients were also undertaken. The technique is simple, does not require the use of sophisticated equipment, and is suitable as a routine test in hospital laboratories. The results of red cell associated IgG by PHI are reproducible and clinically relevant.

Waiting time in a multi-server cutoff-priority gueue, and its application to an urban ambulance service.

We consider a priority queue in steady state with N servers, two classes of customers, and a cutoff service discipline. Low priority arrivals are "cut off" (refused immediate service) and placed in a queue whenever N1 or more servers are busy, in order to keep N-N1 servers free for high priority arrivals. A Poisson arrival process for each class, and a common exponential service rate, are assumed. Two models are considered: one where high priority customers queue for service and one where they are lost if all servers are busy at an arrival epoch. Results are obtained for the probability of n servers busy, the expected low priority waiting time, and (in the case where high priority customers do not queue) the complete low priority waiting time distribution. The results are applied to determine the number of ambulances required in an urban fleet which serves both emergency calls and low priority patients transfers.