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Background/Aims: Monocyte activation and subsequent cytokine generation is presumed to be involved in haemodialysis (HD)-related morbidity. The present study was designed to investigate HD-induced changes in monocytes, with respect to their phenotypic profile and cytokine release, both in peripheral blood (PB) and dialyser eluates (DE). In addition, the effect of the type of dialyser on monocyte activation was assessed. Methods: Dialyser elution was performed in 8 patients after 3 h of HD, using cuprammonium (CU) and polysulfon (PS) dialysers in a randomised cross-over design. PB samples and DE were analysed for both the expression of a variety of monocyte cell surface markers (CD62L, CD11b, CD25, HLA-DR, CD64 and CD14) by flow cytometry and IL-1β levels. Monocytes were identified by dual labelling with antibodies against CD14. Results: In PB, the expression of CD11b increased during HD with both devices, but was more pronounced with CU (CU versus PS: p < 0.05). CD62L decreased during HD, but only significantly for PS (p < 0.02). HLA-DR was downregulated during HD with CU (p = 0.056). The expression of CD64 was higher during HD with CU (p = 0.02). Finally, CD14 increased during HD with both dialysers (p < 0.03). DE yielded a mean cell count of 51 × 10[sup 6] cells. The proportion of monocytes in DE was 3% for CU and 4% for PS. In eluted monocytes, a significant upregulation of CD11b, CD25, and HLA-DR was observed. CD62L was downregulated when compared to PB at t[sub 180] (p < 0.001). In DE, no correlation was found between the type of dialyser and the phenotypic changes. In 10 of 16 DE supernatants, 6 CU and 4 PS, IL-1β release could be demonstrated, CU yielding significantly more of this cytokine than PS (p = 0.03). Conclusions: According to both their phenotypic profile and cytokine release, monocytes sticking to the dialyser membrane after HD are considerably more activated than circulating monocytes. Activation of eluted monocytes appeared independent of the type of dialyser, suggesting an effect of mechanical stress rather than bioincompatibility. In contrast, phenotypic activation of peripheral blood monocytes and cytokine release in the DE supernatant were mainly dialyser-dependent.Copyright © 2002 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: Degranulation of polymorphonuclear leukocytes (PMN) during hemodialysis (HD) is usually assessed by measuring degranulation products. However, this process might also be estimated by the assessment of cell surface markers. In this study, the relationship between the expression of PMN degranulation markers (CD63 and CD66b) and the release of degranulation products [myeloperoxidase (MPO) and lactoferrin (LF)] was investigated during clinical HD in order to evaluate cell surface markers as a useful index of PMN degranulation. Methods: The expression of CD63 and CD66b on PMN and the release of MPO and LF were investigated in 10 chronic HD patients, during both heparin (HD[sub hep] ) and trisodium citrate anticoagulation (HD[sub cit] ), in a randomized order. Samples were drawn from both the efferent and afferent lines of the dialyzer at 0, 7.5, and 180 min. Results: During HD[sub hep] at first passage, a major increase in MPO (from 158 ± 32 to 448 ± 177 µg/l, p = 0.001) and LF (from 134 ± 52 to 260 ± 120 µg/l, p = 0.01) was found across the dialyzer, whereas marked changes were not observed during HD[sub cit] . The expression of CD63 and CD66b increased across the dialyzer during both anticoagulation modalities, but was only significant in the case of HD[sub hep] (CD63: mean fluorescence intensity from 247 ± 61 to 331 ± 118, p < 0.01; CD66b: mean fluorescence intensity from 340 ± 76 to 434 ± 103, p = 0.01). During HD[sub hep] a correlation was noted between the degranulation products and markers of both azurophilic and specific granules (MPO and CD63: r = 0.35; p < 0.01; LF and CD66b: r = 0.39, p < 0.01). Significant differences in the expression of CD63 and CD66b between HD[sub hep] and HD[sub cit] were not observed. When analyzing the combined data for both HD[sub hep] and HD[sub cit] , no correlation was observed between degranulation products and markers. Conclusion: Our data suggest that the measurements of cell surface markers may not be a reliable indicator of the degree of HD-induced PMN degranulation.Copyright © 2000 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

CBA/N mice harbour an X-linked B cell defect which is transmitted by CBA/N female mice to their hybrid male progeny. These mice mount normal responses to thymus-dependent (TD) and some thymus-independent (TI-1) antigens, while the response to TI-2 antigens is absent. Hapten-specific plaque-forming cell (PFC) responses to TD antigens can be blockaded by concomitant exposure of these mice to TI-2 antigens bearing the same hapten. This paper investigates in defective mice the blockade of their response to TNP3-LPS (trinitrophenylated lipopolysaccharide, a TI-1 antigen), imposed by DNP59-Ficoll (dinitrophenylated Ficoll, a TI-2 antigen). The effectiveness of the blocking agent, DNP59-Ficoll, differed in various inbred mouse strains: CBA/N × C3H/HeN F1 male > CBA/N female > CBA/N · C3H/HeN F1 female. The role of T cells in the observed hapten-specific blockade phenomenon was investigated using athymic CBA/N nude mice and a B cell tolerogen. Our findings indicate that T cell participation is not essential for the blockade of CBA/N PFC responses and they suggest that direct blockade of TI- and TD-responsive B cell populations is likely to occur. [ABSTRACT FROM AUTHOR]

CBA/N mice have an X-linked S-cell defect which prevents them from responding to non- mitogenic thymus-independent (T1-2) antigens such as haptenated Ficoll. The CBA/N mice do, however, respond to another group of thymus-independent (TI-2) antigens among which are haptenated liposomes. The F1 male progeny of CBA/N female mice express the same characteristics. Hapten-specific plaque-forming cell (PFC) responses to haptenated proteins (thymus-dependent, TD, antigens) by CSA/N mice and CBA/N × C3H/HeN F1 male mice can be blocked by concomitant exposure to TI-2 antigens bearing the same hapten. Simultaneous exposure to haptenated liposomes (TI- I antigen) and TD antigens, however, results in a synergistic response to the hapten if the antigens share common epitopes. The tripeptide-enlarged hapten dinitrophenyl-β-alanylgly- cylglycine (J), conjugated to phosphatidyl-ethanol- amine (PE) and incorporated into liposomal model membranes, was used throughout the experiments. In F1 male mice the moderate responses to TD antigens could be restored to values equivalent to those seen in F1 female mice. This adjuvant effect of haptenated liposomes is hapten-specific, time-dependent, and restricted to certain structural forms of the liposomes. [ABSTRACT FROM AUTHOR]

This paper describes the effect of altering liposomal membrane composition on humoral and cellular immunogenicity of haptenated liposomes in mice. Antibody formation was determined by enumeration of direct, plaque-forming cells in the spleen and delayed-type hypersensitivity (DH) was measured with a footpad swelling test. Humoral immunogenicity of haptenated liposomes was strongly influenced by membrane phospholipid, cholesterol and charged amphiphile composition. Haptenated liposomes prepared from phospholipids with a low (diolcoyl- and dilauroyl-phosphatidylcho- line) or high (distearoyl phosphatidylcholine) phase- transition temperature were less immunogenic than those prepared from phospholipids with an intermediate phase-transition temperature (dipalmitoyl phosphatidylcholine and sphingomyelin). In general, increasing the amount of liposomal membrane cholesterol induced a higher humoral response. These results are discussed in relation to liposomal membrane fluidity. Induction of an optimal DH with haptenated liposomes did not occur in the absence of the adjuvant dimethyl dioctadecyl ammonium bromide (DDA). When DDA was used, alterations in membrane composition did not influence cellular immunogenicity. From these results it was concluded that ‘intermediate’ liposomal membrane fluidity is the most important requirement for induction of optimal antibody formation with haptenated liposomes and that a certain physicochemical configuration of the antigen, provided by the adjuvant DDA, is a prerequisite for induction of DH. [ABSTRACT FROM AUTHOR]

This paper describes the induction of IgM immune memory to haptenated liposomes in mice. The tripeptide-enlarged haptens 3-(p-azo-benzene-arsonate)-N-acetyi-L-tyrosylglycyiglycine (A) and N-(2,4-dinitrophenyl)-β-alanylglycylglycine (J) were coupled to phosphatidylethanola mine (PE) and the conjugates A-PE and J-PE were incorporated into separate liposomal membranes (monofunctional A-PE-liposomes or J-PE-Liposomes) or into the same liposomal membranes [bifunctional (A,J)PE-liposomes]. The magnitude of the humoral response was measured by the appearance of direct and indirect plaque-forming cells in the spleens of immunized mice. Intracutaneous priming of mice with A-PE-liposomes mixed with the adjuvant dimethyl dioctadecyl ammonium bromide (DDA) and secondary immunization with bifunctional (A.J)-PE-liposomes resulted in enhanced hapten A- and J-specific IgM responses. However, no switch from IgM to IgG antibodies was observed in these mice. The J-specific IgM response was enhanced only when hapten I was incorporated into a bifunctional liposome which also contained A-epitopes. in mice primed with A-PE-liposomes in the absence of DDA, a greatly diminished memory to both hapten A and J was observed. This finding indicates a crucial role for the adjuvant in the induction of memory. J-PE-liposomes and DDA were not able to induce memory to monofunctional J-PE-liposomes or bifunctional (A,J)-PE-liposomes. The possibility that hapten A-specific B lymphocytes were responsible for the induction of memory was excluded by hapten-specific blockade of these cells with a B-cell tolerogen. These data, in addition to the observation that no IgM memory could be induced in congenitally athymic nude mice, suggest that the observed memory can be ascribed to priming of hapten A-specific T lymphocytes. [ABSTRACT FROM AUTHOR]

This paper describes the specificity of delayed-type hypersensitivity (DH) and antibody formation in the mouse to the tripeptide-enlarged hapten, 3-(p-azobenzenearsonate)-N-acetyl-L-tyrosylglycylglycine (A). Hapten A was coupled to phosphatidylethanolamine (PE) and incorporated into liposomal membranes (A PE-liposomes). DH was measured as footpad swelling and antibody formation by the enumeration of direct plaque-forming cells in the spleen. A-PE-liposomes mixed with the cationic, surface-active lipid, dimethyl dioctadecyl ammonium bromide (DDA) induce, on intracutaneous injection in mice, hapten-specific DH without a contribution by the carrier. With other haptenated liposomes it was not possible to induce DH to those haptens, including the closely related hapten 3-(p-azobenzenesulphonate)-N-acetyl-L-tyrosylglycylglycine (S). A-PE- and S-PE-liposomes evoke, after intravenous injection in mice, a humoral response. The antibody formation to A-PE-liposomes was thymus-independent. In this response a considerable cross reaction between haptens A and S was observed. [ABSTRACT FROM AUTHOR]

This paper describes the induction of delayed-type hypersensitivity (DH) in the mouse and guinea-pig to haptenated liposomes. The tripeptide- enlarged hapten 3-(p-azobenzenearsonate)-N-acetyl- L-tyrosylglycylglycine (A) was coupled to phosphatidylethanolamine (PE) and incorporated into liposomal membranes (A PE-liposomes). In mice DH was measured as footpad swelling and in guinea pigs by skin testing. To induce hapten A-specific DH in mice with A-PE- liposomes the application of the cationic, surface- active lipid, dimethyl dioctadecyl ammonium bromide (DDA) was necessary. The use of Freund's complete adjuvant (FCA) did not result in the induction of DH to hapten A. In guinea-pigs, however, FCA and DDA had equally good adjuvant properties in the induction of DH. The time course of the DH and the optimal time interval between immunization and elicitation were determined for the mouse system. Also, the effect of dose and epitope density was studied in that system. Cyclophosphamide treatment, before immunizing mice with A-PE-liposomes and DDA, resulted in greatly impaired DH, probably caused by the short lifetime of the integrity of liposomes after intracutaneous administration to mice. The results make it very likely that presentation of hapten A in a liposomal or micellar structure is required to induce a cellular immune response to this hapten in mice. [ABSTRACT FROM AUTHOR]

This paper describes a rather simple coupling method for tripeptide enlarged haptens to phosphatidylethanolamine (PE) and the incorporation of these conjugates into liposomal model membranes (haptenated liposomes). These haptenated liposomes evoke a hapten-specific humoral immune response in mice. The magnitude of the response as measured by the appearance of direct plaque forming cells in the spleen is dependent on the route of immunization and the dose and epitope density of the hapten-PE derivatives. It was not possible to evoke an IgG response after either primary or secondary immunization with haptenated liposomes (as measured by the production of indirect plaques or mercaptoethanol-resistant antibody). These data, in addition to the observations that mice depleted of, or deficient in thymus-derived (T) lymphocytes respond to haptenated liposomes, indicate that these haptenated liposomes are T-cell independent antigens. [ABSTRACT FROM AUTHOR]

The primary neutralizing antibody response to encephalomyocarditis (EMC) virus and Semliki Forest virus (SFV) is enhanced by addition of either negatively or positively charged liposomes. The purified u.v. light inactivated viruses were merely mixed with liposomes and injected intraperitoneally (i.p.) into mice. In contrast, neutral liposomes were unable to enhance the primary response to these viruses. Furthermore primary immunization with inactivated SFV mixed with either neutral, positively or negatively charged liposomes was associated with an enhancement of the secondary hurnoral response after i.p. booster injection of mice with inactivated virus alone. But neutral liposomes seemed to be less effective in this respect than either positively or negatively charged liposomes. [ABSTRACT FROM AUTHOR]

Mass spectra of the three isomeric vinylimidazoles have been compared and the structures of the fragment ions [C3H4N2]+· and [C5H5N2]+ have been investigated by collisionally activated dissociation mass spectrometry. The greater part of the non-decomposing ions m/ z 68 from 2-vinylimidazole and from 2-imidazolecarboxylic acid methyl ester, and a minor part of this ion formed from the free acid, all have the same structure: the imidazole ring system, with hydrogens at both nitrogen atoms but none at C(2). An analogous structure, with an ethyl group at C(2), is proposed for the m/ z 93 ion from 2-vinylimidazole. [ABSTRACT FROM AUTHOR]

ions generated from a number of different presursors have been studied by high kinetie energy ion-molecule reations. It has been shown that at least four distinct stable species oeeur, of which acetonitrile and methyl isoeyanide retain their original structure. With imidazole or pyrazole as precursors, a mixture of open thain radical cations, not identical to the above species and probably interconvertible via the 1 H-azirine radocal cation, is formed. From butrynitrile, pyrrole, crotonitrile, allyl interconvertible via the and cyanocyopropane a fourth species, probably the vinylidenimine ion, is formed. [ABSTRACT FROM AUTHOR]

The relative losses of unlabelled vs. labelled HCN from the [M]+˙ and [M - 1]+ ions of a number of specifically labelled 1-methylimidazoles (I) and 1-methylpyrazoles (II) have been determined. Hydrogen randomisation in the molecular ions prior to fragmentation is insignificant. Expulsion of HCN follows two distinct pathways: elimination involving positions 2 and 3 (predominant in I) and elimination involving the methyl group and the nitrogen atom at position 1 (predominant in II). The molecular ions eject H˙ from the methyl groups to a high degree of specificity. In both cases some contribution by position 5 is observed. The resultant [M - 1]+ ions exhibit extensive, but incomplete hydrogen randomisation. Loss of HCN from these ions is consistent with intermediacy of ring-expanded ions, but notably in II a proportion of the HCN is generated from the group. A mechanism for this observation is presented. [ABSTRACT FROM AUTHOR]

The behaviour of a number of deuterated imidazoles upon electron-impact has been studied. Loss of H⋅ from the molecular ion occurs specifically at the 4(5)-position. Loss of HCN is less specific, the order being 2 > 4(5) ≫ 1. [ABSTRACT FROM AUTHOR]

The mass spectra of deuterated pyrazoles show that loss of H. and of HCN from the molecular ion occurs with a very high specificity from the 3(5)-position. For the two processes isotope effects and preference factors have been determined. Metastable ion decompositions involving the loss of HCN from the [M - H] -fragment indicate that the identity of the hydrogen atoms in this fragment is lost to a large extent. [ABSTRACT FROM AUTHOR]

The mass spectra of ten pyrazole compounds have been determined. Fragmentation schemes have been derived by means of the metastable defocusing method. The predominant process is cleavage of the nitrogen-nitrogen bond resulting in expulsion of HCN. The process second in prominence is the loss of a nitrogen molecule after initial removal of a hydrogen radical or a substituent, giving the species [C3H2R]+, probably a cyclopropenyl ion. In general, the fragmentation pattern is strongly influenced by the substituent. [ABSTRACT FROM AUTHOR]

The analysis of hemodialysis (HD)-related bioincompatibility is focused mainly on phenomena observed in peripheral blood. However, since biocompatibility originates inside the dialyzer, white blood cells (WBC) adhering to the dialyzer are probably most subject to the influence of both dialyzer membrane and dialysate. In order to collect membrane-adherent cells, a reliable and reproducible elution technique was developed. After 3 h of HD, blood was returned to the patient with 0.9% NaCl. Then, dialyzers were eluted by recirculation of phosphate-buffered saline (PBS) or PBS/3 mM EDTA for 20 min, with or without prior flushing with 200 ml PBS. Finally, remaining adherent cells were collected by an afterwash with 10% trypsin. These solutions, as well as blood samples, were analyzed for WBC count, viability and differentiation. Random eluate samples were analyzed by flow cytometry, and the influence of elution on PMN activation was tested in a separate control experiment. WBC numbers decreased by flushing before elution, whereas cell numbers were maximal after elution with PBS/3 mM EDTA (30 x 10(6)). Trypsin afterwash resulted in a further yield of 12 x 10(6) cells. The eluates contained 81% PMN (blood 68%, p < 0.01), with a degranulated appearance, and only 12% lymphocytes (blood 21%, p < 0.05); cell viability in the eluates was > 95%. The eluted cells could be analyzed by flow cytometry, and the procedure itself induced only minimal PMN activation. In conclusion, a maximal number of adherent cells, consisting mainly of PMN, was obtained by direct elution with PBS/3 mM EDTA. The method itself did not induce marked PMN activation, and the cells obtained were suitable for further investigations, including flow cytometry.

This paper describes a comparison between the microscopic brilliant cresyl blue and the flow cytometric thiazol orange (FACScan) and auramine O (Sysmex) methods for enumeration of reticulocytes. The mean intra-assay coefficients of variation for the microscopic, FACScan and Sysmex methods were established to be 33.9 and 5% respectively. A rather poor correlation was observed between the microscopic count and both flow cytometric methods (FACScan r = 0.61; Sysmex r = 0.57). However, the correlation between the flow cytometric methods was satisfactory (r = 0.79). Reference ranges for the reticulocyte count, corresponding with the 2.5th and 97.5th percentile, were determined for the microscopic (8-30/1000), FACScan (11-27/1000) and Sysmex (8-18/1000) methods. Sysmex R-3000 methodology definitely revealed the lowest and narrowest reference range. In conclusion, because of higher reproducibility, flow cytometric analysis of reticulocytes is an attractive alternative procedure for the microscopic enumeration method.

IgG subclass measurements are generally performed with the radial immunodiffusion (RID) technique. With this method, results are obtained after an incubation period of 48-72 hours. We developed nephelometric assays on the Behring Nephelometer Analyzer (BNA) that allow a quantification of IgG subclass concentrations in a large number of samples quickly (less than 15 minutes for a complete IgG subclass profile) and reproducibly (intra-assay variation 2.5-5.5%, interassay variation 3.4-6.0% and inter-lab variation 5.4-10.3%). The nephelometric method was compared with the RID technique by analyzing the IgG subclass levels in sixty selected samples. For all IgG subclasses identical results and high correlation coefficients (r > 0.93) were found. In addition, the detection limits of the nephelometric method for all four IgG subclasses were identical or lower than those of the RID technique. Furthermore, the interlab variations of the nephelometric IgG subclass assays are lower than those of the RID method. However, the major advantages of the nephelometric assay are the speed, the minimal workload (automated IgG subclass determinations) and the possibility for automated bidirectional data transmission. Recently we have established new reference ranges for the human IgG subclasses in sera of adults and children. In order to validate these reference values we have measured the IgG subclasses in sera from 112 healthy children with the nephelometric method. In 1992, more than 2000 patient sera were tested by the nephelometric assay. A predominance of IgG2 abnormality was observed. In 9.8% of these sera the IgG2 concentration was decreased. Elevated IgG2 concentrations were found in 1.9% of the sera. Furthermore, the sum of the quantitated four IgG subclasses was similar to that of total IgG (less than 20% difference).

Allergen-specific IgE concentrations for 6 inhalant allergens were compared in 54 subjects, using the Pharmacia CAP System and the DPC AlaSTAT System. Similar results were obtained in 91.6% of the tests. In 8% of the tests a positive score was obtained for the Pharmacia CAP System whereas a negative result was found by use of the DPC AlaSTAT System. A negative Pharmacia CAP System result combined with a positive DPC AlaSTAT System result occurred only in 0.4% of the tests. Sensitivity and specificity was established for the Pharmacia CAP System and the DPC AlaSTAT System by comparison with results of the skin prick test. Results obtained with the Pharmacia CAP System were found to be of slightly higher sensitivity than those obtained by DPC AlaSTAT System and of similar or slightly lower specificity.

T- and B-lymphocyte populations in peripheral lymphoid tissues occur in distinct compartments (e.g., the periarteriolar lymphocyte sheath of the splenic white pulp is a T-cell area). The authors report on two patients with severe combined immunodeficiency (SCID) and one patient with immunodeficiency after anti-T-cell treatment for rejection of a heart transplant, in which the area surrounding the central arteriole in spleen white pulp was well-populated despite T-cell deficiency (documented by, for example, severe depletion of lymph node paracortex). Immunologic phenotyping showed the B-lymphoid lineage of lymphocytes at this location. The framework in the periarteriolar area consisted of follicular dendritic cells, which are typical framework components of B-cell areas. We conclude that assessment of only conventional histopathology of the spleen in these patients leads to erroneous conclusions about the type of immunodeficiency and that immunologic phenotyping is required to document the exact nature of the deficiency.

This paper describes the immunogenicity of haptenated liposomes in a number of inbred mouse strains. The tripeptide-enlarged hapten, dinitrophenyl-beta-alanylglycylglycine (J) was conjugated to phosphatidyl-ethanolamine (PE) and incorporated into liposomal model membranes. The magnitude of the response to this thymus-independent (TI) antigen was measured by the appearance of direct plaque-forming cells in the spleen. The response to J-PE-liposomes in the different mouse strains varied sufficiently to suggest the existence of high, intermediate and low responders. On the basis of this preliminary survey no correlation was found between the immunogenicity of J-PE-liposomes and H-2 haplotype. The positive responses obtained in CBA/N mice appear to classify J-PE-liposomes as a TI-1 antigen. Further characterization of this response was carried out in CBA/N X C3H/HeN F1 male and female mice. The magnitude of the response was dependent on dose and epitope density of the hapten-PE derivative. The response could be blocked in F1 male mice by prior immunization with the TI-2 antigen J53-Ficoll. This suggests that J-PE-liposomes have no intrinsic mitogenic properties. Non-haptenated liposomes could not be used as an adjuvant to restore the response in F1 male mice to haptenated Ficoll (TI-2) preparations.