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We were interested to study the relationship between DNA lesions, DNA repair, mutation fixation, and tumour development. Therefore, mice harbouring lacZ reporter genes and being either wild-type or defective in the DNA excision repair gene XPA, were treated with the genotoxic carcinogen benzo[a]pyrene at an oral dose of 13 mg/kg b.w. (3 times/week). At different time points, i.e. 1, 5, 9 or 13 weeks after start of the oral administration, levels of BPDE-N2-dG adducts (the major formed DNA adduct by benzo[a]pyrene in mice), and lacZ mutation frequencies were measured both in target (spleen) and non-target (lung and liver) tissues. Both in wild-type and XPA-deficient mice, benzo[a]pyrene treatment resulted in increased BPDE-N2-dG adduct levels in all three tissues analysed. In XPA-deficient mice, BPDE-N2-dG adduct levels still increased up to 13 weeks of oral benzo[a]pyrene treatment, whereas in DNA repair proficient mice steady-state levels were reached after 5 weeks of treatment. After 13 weeks, the BPDE-N2-dG adduct levels observed in XPA-/- mice, were 2- to 3-fold higher than the steady state levels observed in XPA+/+ mice in the same tissues. Mutation frequencies in the lacZ reporter gene were the same in wild-type and XPA-deficient mice that were treated with the solvent only. Oral benzo[a]pyrene treatment resulted in an increase in mutation frequency in the lacZ marker gene in all three tissues, but this increase was most profound in the spleen. After 13 weeks of treatment, a 7-fold increase in lacZ mutation frequency was detected in the spleen of wild-type mice as compared to mutation frequencies in control mice. At the same time point, a 15-fold increase in lacZ mutation frequency was observed in the spleen of XPA-deficient mice. The data presented here show, that a defect in NER mainly results in enhanced mutation frequencies in lymphocytic cells after oral treatment with the genotoxic compound benzo[a]pyrene. Interestingly, as we established in a previously performed carcinogenicity assay, the same oral treatment with benzo[a]pyrene induced lymphomas residing in the spleen of XPA-deficient mice. [ABSTRACT FROM PUBLISHER]

We studied the distribution of FcγRIII (CD16) in human lymphoid and non-lymphoid tissue by immunohistochemistry and two-colour immunofluorescence. In all tissues except stomach, skin, muscle and heart, infiltrating cells were stained. In tonsil and thymus, the CDI6 antibody CLBgran1 labelled some cells with high endothelial morphology in T-cell areas. Two-colour fluorescence combination of CD16 and antibody to Von Willebrand factor confirmed the endothelial nature of the CLBgran1-positive cells. FcγRIII expression was also demonstrated by antibody CLBgran11, which detects a product of the Fc&#x03B3RIII-B gene of only the NAI haplotype. A series of 95 endomyocardial biopsies (EM B) taken after heart transplantation was evaluated for FcγRIII expression. In 18 cases FcγRIII was unambiguously detected on endothelial cells by CD 16 antibody CLBgran1. There was a significant correlation between capillary CD16 expression and transplant rejection. We conclude that FcγR expression on endothelium represents endothelial activation found in immunopathological and inflammatory conditions. [ABSTRACT FROM AUTHOR]

Interleukin 6 (IL-6) is a cytokine with multiple biological activities on various tissues and cells. We have investigated the effect of recombinant human IL-6 (rnIL-6) on growth and differentiation of U937, Recombinant human IL-6 induced a dose-dependent growth inhibition, apparent around day 4. of up to 50%, by day 8 of culture. Concomitant with this, changes in cytochemical activities were observed, indicative of differentiation, A panel of U937 antigens was analysed after culture with rhIL-6, Expression of the majority of these membrane antigens was unaffected, with the exception of two classes. First, rhIL-6 had a profound effect on members of the leucouytic cell adhesion molecules (Leu CAM) family. Expression of the a-chain of CR3 (complement receptor 3; CD11b) was enhanced in a dose-dependent fashion, with maximal expression around day 7, Parallel to this a simultaneous increase in β-chain (CD18) expression was found. Furthermore, enhanced expression of CR3 was accompanied by increased rosetting with sheep erythrocytes sensitized with C3bi, A much lower, but significant, enhancement was observed for the a-chain of the p 150.95 antigen (CD11c). Expression of leucocyte function-associated antigen-1, (LFA-1). (CD11a/CD18) remained unchanged. Remarkably, however, expression of a ligand of LFA-1. intercellular adhesion molecule-1 (ICAM-1; CD54), was enhanced with similar kinetics as CR3 and p150.95. A specific anti-rnIL-6 antiserum completely inhibited the IL-6 effect. These observations provide further support for an important role of IL-6 in the regulation of myeloid cell development in man. [ABSTRACT FROM AUTHOR]

In this paper data are presented indicating that immunotherapy with monoclonal anti-idiotypic antibodies (MoAb anti-id) can provoke different responses in the B-cell tumour concerned. With respect to the course of disease during and after immunotherapy, the in vitro findings may very well explain the in vivo observations in the two patients (D.E.F., B.O.R.) with B-cell chronic lymphocytic leukaemia (B-CLL) who were treated with MoAb anti-id. After initial tumour reduction, there was a recurrence of tumour cells with altered functional and phenotypic properties. In both cases the recurring tumour cells still expressed the same idiotype. In one patient (D.F.F.) the phenotypic changes (a surface Ig change from IgM, IgG. igA. and IgD to weakly positive IgM and IgD) and functional changes (a 10-fold increase in [3H]thymidine uptake and a decreased idiotype secretion in vitro), together with the in vivo findings with respect to the course of disease-at relapse an impressive tumour regrowth rate with constant serum idiotype level-suggest that immunoselection might have taken place favouring the survival and relapse of a less mature, more aggressive tumour cell population with a lower idiotype expression. In the second patient (B.O.R.), the phenotypic changes (an isotype change from IgM and IgD to IgM with the loss of IgD, and a gradual decrease in expression of CD19 and CD24) and functional changes (a 10-fold increase of idiotype secretion in vitro), together with the in vivo finding that the serum idiotype level had increased 25-fold compared with the preimmunotherapy serum level with comparable tumour load, strongly suggest an immunotherapy-induced differentiation of the malignant B cell. We also describe an increased expression of CD74. detected by MoAb BoM22, on the recurring tumour cells of patient B.O R., whereas the expression of HLA-DP, -DQ and -DR did not change. The significance of this finding is unclear. [ABSTRACT FROM AUTHOR]

Graft-versus-host disease (OVHD) after allogeneic bone marrow transplantation (BMT) is initialed by immunocompetent T cells present in the graft. Selective elimination of distinct T-cell subsets or a sufficient, but not complete T-cell depletion, might abolish severe GVHD without graft rejection and loss of the anti-tumour potential. In this study we analysed the efficacy of different monoclonal antibodies (MoAb) WT32 (CD3). OKT4 (CD4), T101 (CD5), WT1 (CD7), and WT82 (CD8) with respect to their cytotoxicity to T cells either as immunotoxin (IT) or in combination with complement. The cytotoxic potential was assessed by protein synthesis inhibition and clonogenic assays. The ricin A conjugated MoAb exerted only a minor effect on blood or bone marrow T cells, although they were highly inhibitory to T-cell lines. However, in the presence of 20 mM ammonium chloride. IT directed against CD3, CD5, and CD7 were highly cytotoxic. IT directed against CD4 and CDS were less effective, due to a low internalization. The complement-mediated cytotoxicity was efficient for all antigens used. The natural killer (NK) activity, as measured by cytotoxicity to K562, was hardly depressed by anti-CD3, anti-CD4, anti CD5, and anti-CD8, but was eliminated by anti-CD7. All procedures used had only a minimal effect on haematopoietic progenitors us measured by CFU-GM and BFU-E assays. We concluded that, although the T-cell population can be eliminated with the combination of anti-CD3, anti-CD5, and anti-CD7 antibodies plus complement, IT with 20 mM NH4Cl appear to kill higher amounts of T cells. Selective elimination of CD4- and CD5-positive cells is effectively obtained by MoAb with complement. [ABSTRACT FROM AUTHOR]

Anti-Ig antibodies can have two opposing effects on B-cell proliferation, resulting either in stimulation or inhibition. We have examined the proliferative response of 30 B-cell tumours to anti-Ig in the presence and absence of B-cell growth factor. Three reaction patterns were observed. In 12 cases a dose-dependent synergism between anti-Ig and B-cell growth factor was present in the induction of proliferation, in ten cases anti-Ig did not induce any response, and in eight eases anti-Ig suppressed the B-cell growth factor (BCGF)-induced proliferation. Similar responses to anti-Ig were found in the absence of BCGF. When these B-cell tumours were typed for expression of Ig isotypes, HLA class II antigens, several B-cell markers, activation markers, complement receptors, Fc receptors, cell size, and cell cycle phase, no correlation could be found with the proliferative response of these tumour B cells to anti-Ig. T cells or T-cell factors were not involved, because T-cell depletion did not change the tumour B-cell proliferative response to incubation with anti-Ig. The observed inhibition of proliferation did not correlate with the expression of Fc receptors, indicating the involvement of suppressor mechanisms other than the cross-linking of Fc receptors with surface immunoglobulins. Tumour B cells, for which monoclonal anti-idotypic antibodies (MoAb anti-id) were available, responded to MoAb anti-id in the same way as they did to anti-Ig. In view of the treatment of B-cell malignancies with MoAb anti-id, the question of whether these responses in vitro correlate with in vivo clinical outcome of anti-id therapy is of interest. So far our data show that the proliferative response of B-cell tumours to anti-Ig or MoAb anti-id is heterogeneous and cannot be linked to phenotype, is T cell-independent, and is most likely an intrinsic property of the malignant cell. [ABSTRACT FROM AUTHOR]

In the context of ex vivo T cell elimination from bone marrow, the anti-T cell cytotoxic potential of immunotoxins (IT) prepared by conjugation of the monoclonal antibodies (MoAb) WT32 (CD3). T101 (CD5), and WT1 (CD7) to ricin A chain was evaluated. The cytotoxicity of IT was based on protein synthesis inhibition in human T cell lines: CH1, CEM, HPB-ALL, and Jurkat, and appeared closely related to the antigen density and internalization rate of the IT. Normal unstimulated T cells appeared to be rather insensitive to IT not due to a low antigen density or decreased internalization. The cytotoxicity of IT to T cells could be enhanced considerably by NH4Cl. Treatment of T cells with a cocktail of IT (10-8 M) and 20 mM NH4Cl resulted in a 5000-fold cytoreduction as measured by clonogenic assays of limiting T cell dilutions, whereas the haematopoietic progenitor cells remained unaltered. Stimulation of T cells with phytohaemagglutinin (PHA) prior to incubation with IT considerably increased the sensitivity to IT treatment. Thus, normal I cells are less sensitive to anti-T cell IT than T cell lines and activated T cells. This suggests that a low protein synthesis is responsible for the resistance to IT. However, a high specific cytotoxicity of 11 to normal 1 cells can be achieved in the presence of 20 mM ammonium chloride. [ABSTRACT FROM AUTHOR]

We have previously reported a polymorphism in the mitogenic effect of murine (m) IgGI anti-CD3 monoclonal antibodies. This polymorphism was genetically determined and could be attributed to polymorphism of the Fc receptor (FcR) for mIgGl present on human monocytes. We have now extended these studies by quantitating FcR expression on monocytes and cell lines by it recently developed EA rosette assay, using the erythrocyte-associated pseudoperoxidase activity. The data show that the polymorphism of the monocyte FcR for mIg2a-based on a quantitative rather than an absolute difference. Furthermore, this FcR is specific for mIgGI and does not bind mIgG2a nor mIgG2b nor surprisingly, human IgG. The expression of this FcR on cell lines correlates with their accessory function in IgG I anti-CD3-induced T cell proliferation mIgG2a can inhibit the rosetting of monocytes with erythrocytes sensitized with human IgG. The FcR detected by this rosette technique can interact with all four human IgG subclasses but not with mIgG1 or mIgG2b. The expression of this type of FcR in human cell lines correlates well with their ability to support mIgG2a anti-CD3-induced mitogenesis. These direct measurements of FcR expression support the concept that human monocytes have two independent FcR with affinity for mouse IgG: one receptor specific for mIgG2a (which also binds human IgG), and a second specific for mIgG1. [ABSTRACT FROM AUTHOR]

The Raji-cell test is one of the most widely used methods for the detection and quantitation of immune complexes. Immune complexes and not 7 S IgG bind via C3 to complement receptors on the cell membrane of the Raji cell, During sucrose gradient fractionation of human and murine systemic lupus erythematosus sera, with a high Raji cell-binding activity, we could not demonstrate immune complexes in these sera. Subsequent analysis showed that the major part of the Raji cell binding was used by 7 S IgG with an anti-DNA specificity. Blocking experiments with complement-bearing aggregated IgG revealed that complenicni and Fc receptors were not involved in the binding of these anti-DNA antibodies to Raji cells. We conclude that the Raji cell test is not suitable for the detection and quantitation of immune complexes in sera containing anti-DNA antibodies. [ABSTRACT FROM AUTHOR]

Anti-CD3 MoAbs arc widely used in T cell activation studies, and are effective in immunosuppressive therapy. We used a panel of mouse (m) anti-CD3 switch variant MoAbs of five different isotypes to study IL-6 release from accessory cells. Incubation of human (h) mononuclear cells with anti-CD3 MoAbs resulted in increased lL-6 levels with MoAbs of mIgGI and mIgG2a isotypes. with no effect of mIgG2b or mIgA. This suggested involvement of IgG Fc receptors (FcγR) in triggering IL-6 production. To evaluate the role of different FcγR molecules individually we used a panel of hFcγR-transfected mouse fibroblasts. and Jurkat T cells as a model. IL-6 secretion by CD32 transfectants expressing the hFcγRHa high-responder (HR) allelic form was triggered by mIgGl anti-CD3 MoAb, with no effect of four other isotypes. None of theanti-CD3 MoAbs induced IL-6 secretion by CD32 transfectants expressing either a variant of this receptor, containing only a single intracellular amino acid (CT-). the hFcγRIIa low-responder (LR) allelic form, or hFcγRIIb1. hFcγRI (CD64) transfectants exhibited IL-6 production after incubation with mIgG2a anti-CD3 MoAb, and to a lesser extent with mlgG2b. and mIgGI MoAb. Indirect involvement of T cells in triggering IL-6 secretion could be excluded by experiments in which transfectants were cultured with immobilized anti-CD3 MoAb. These data indicate that cross-linking of either hFcγRI, or hFcγRIIaHR by appropriate anti-CD3 MoAbs triggers IL-6 production of accessory cells, and not T cells. This may also take place in vivo during immunosuppressive therapy with anti-CD3 MoAbs. and related antibody-mediated immune responses. [ABSTRACT FROM AUTHOR]

Ten sera of patients with systemic lupus erythematosus (SLE) were tested in an enzyme linked immunosorbent assay for their ability to react with glycosaminoglycans, constituents of proteoglycans, in relation to their anti-DNA reactivity. The SLE sera reacted with hyaluronic acid and chondroitin sulphate and this reactivity correlated with the anti-DNA activity of these sera. By contrast, sera obtained from patients with other autoimmune diseases or normal sera lacked any of these reactivities. Anti-DNA antibodies purified by affinity chromatography with either oligo dT cellulose or Cibracon blue F3Ga Sepharose reacted with DNA as well as with hyaluronic acid. The cross-reactivity of anti-DNA antibodies could be confirmed by the reaction of a mouse monoclonal anti-DNA antibody with DNA, hyaluronic acid, and chondroitin sulphate. This pattern of cross-reactivities of anti-DNA antibodies suggests that several compounds can function as antigenic targets for these antibodies provided that their structures contain repeating negatively charged groups. [ABSTRACT FROM AUTHOR]

WT 1, an IgG2a subclass monoclonal antibody, recognizes a human T lineage specific antigen (mol. wt 40,000). This antigen is strongly expressed on thymic T blasts, and on peripheral T cells activated by phytohaemagglutinin, whereas cortical thymocytes and peripheral T cells are moderately positive for WT 1, In contrast, B lymphocytes, myeloid and erythroid cells, including the progenitor cells of these lineages, and terminal deoxynucleotidyl transferase positive cells in the bone marrow, arc all WT 1 negative. Binding of WT 1 to T cells is blocked by a previously described antibody (3A1) suggesting that both antibodies bind to the same antigen present on human T cells. WT 1, however, is also reactive with T lymphocytes from rhesus monkeys whereas 3A1 is not. Therefore, the biological effects of WT 1 could be studied in a monkey model. In a skin allograft model. WT 1 was immunosuppressive and induced a marked prolongation of graft survival. [ABSTRACT FROM AUTHOR]

Using pump-probe spectroscopy, we study the propagation of laser-excited longitudinal, high-amplitude, coherent picosecond acoustic phonon wave packets in thin sapphire slabs at room temperature. We observe significant stretching of the wave packet up to several tens of picoseconds, accompanied by strong steepening of the wave fronts. The results demonstrate the supersonic and subsonic nature of propagation in viscous nonlinear media and more specifically the presence of acoustic frequencies in the wave packet as high as 500 GHz. All experimental data are in excellent agreement with simulations based on Burgers’ equation. [ABSTRACT FROM AUTHOR]

A correction to the article "Optical generation and detection of shock waves in sapphire at room temperature," published on September 29, 2006 issue is presented.