Shaving Is an Epiphenomenon of Type I and II Anti-CD20-Mediated Phagocytosis, whereas Antigenic Modulation Limits Type I Monoclonal Antibody Efficacy.

Rituximab is an anti-CD20 mAb used in the treatment of B cell malignancies. Loss of surface CD20 Ag from the surface of target cells is thought to be one mechanism governing resistance to rituximab, but how this occurs is not completely understood. Two explanations for this have been proposed: antigenic modulation whereby mAb:CD20 complexes are internalized in a B cell intrinsic process and shaving, in which mAb:CD20 complexes undergo trogocytic removal by effector cells, such as macrophages. However, there is conflicting evidence as to which predominates in clinical scenarios and hence the best strategies to overcome resistance. In this study, we investigated the relative importance of modulation and shaving in the downregulation of surface mAb:CD20. We used both murine and human systems and treated ex vivo macrophages with varying concentrations of non-FcγR-interacting beads to achieve differential macrophage saturation states, hence controllably suppressing further phagocytosis of target cells. We then monitored the level and localization of mAb:CD20 using a quenching assay. Suppression of phagocytosis with bead treatment decreased shaving and increased modulation, suggesting that the two compete for surface rituximab:CD20. Under all conditions tested, modulation predominated in rituximab loss, whereas shaving represented an epiphenomenon to phagocytosis. We also demonstrate that the nonmodulating, glycoengineered, type II mAb obinutuzumab caused a modest but significant increase in shaving compared with type II BHH2 human IgG1 wild-type mAb. Therefore, shaving may represent an important mechanism of resistance when modulation is curtailed, and glycoengineering mAb to increase affinity for FcγR may enhance resistance because of shaving.

Understanding the immunogenicity and antigenicity of nanomaterials: Past, present and future.

Nanoparticle immunogenicity and antigenicity have been under investigation for many years. During the past decade, significant progress has been made in understanding what makes a nanoparticle immunogenic, how immune cells respond to nanoparticles, what consequences of nanoparticle-specific antibody formation exist and how they challenge the application of nanoparticles for drug delivery. Moreover, it has been recognized that accidental contamination of therapeutic protein formulations with nanosized particulate materials may contribute to the immunogenicity of this type of biotechnology products. While the immunological properties of engineered nanomaterials and their application as vaccine carriers and adjuvants have been given substantial consideration in the current literature, little attention has been paid to nanoparticle immuno- and antigenicity. To fill in this gap, we herein provide an overview of this subject to highlight the current state of the field, review past and present research, and discuss future research directions.

[Chemical modification of chymopapain with monomethoxypolyethylene glycol and its effect on enzymic activity and antigenicity].

OBJECTIVE: To study the effect of chemical modification of chymopapain with monomethoxypolyethylene glycol on enzymic activity and antigenicity. METHODS: Under the substrate protecting and non-substrate protecting, the chymopapain (Cp) was modified with two types of mPEG derivatives mPEG1 and mPEG2. The average ratio of modified-NH2 was tested by trinitrobenzenesulfonic acid (TNBS) method, the enzymic activity was tested with macromolecular casein and ATEE as substrates, the antigenicity of modified enzyme was determined by ELISA method. RESULTS: (1) Both mPEG1 and mPEG2 can reduce and eliminate antigenicity of Cp, the mPEG2 was better than mPEG1. (2) The enzymic activities of modified Cp were reduced, the enzymic activities of mPEG1-modified Cp were higher than that of mPEG2-modified Cp (especially macromolecular protein as its substrate). (3) The enzymic activities in present of ATEE were obviously higher than that in absent of substrate. CONCLUSION: When mPEG1 as the modifier and in present of ATEE, the antigenicity of Cp was completely eliminated, and the enzymic activities were still higher.

Carpobrotus edulis methanol extract inhibits the MDR efflux pumps, enhances killing of phagocytosed S. aureus and promotes immune modulation.

Although alkaloids from the family Aizoaceae have anticancer activity, species of this family have received little attention. Because these alkaloids also exhibit properties normally associated with compounds that have activity at the level of the plasma membrane, a methanol extract of Carpobrotus edulis, a common plant found along the Portuguese coast, was studied for properties normally associated with plasma membrane active compounds. The results of this study show that the extract is non-toxic at concentrations that inhibit a verapamil sensitive efflux pump of L5178 mouse T cell lymphoma cell line thereby rendering these multi-drug resistant cells susceptible to anticancer drugs. These non-toxic concentrations also prime THP-1 human monocyte-derived macrophages to kill ingested Staphylococcus aureus and to promote the release of lymphokines associated with cellular immune functions. The extract also induces the proliferation of THP-1 cells within 1 day of exposure to quantities normally associated with phytohaemagglutinin. The potential role of the compound(s) isolated from this plant in cancer biology is intriguing and is currently under investigation. It is supposed that the resistance modifier and immunomodulatory effect of this plant extract can be exploited in the experimental chemotherapy of cancer and bacterial or viral infections.

Differential effect of HIV-1 protease inhibitors on P-glycoprotein function in multidrug-resistant variants of the human CD4+ T lymphoblastoid CEM cell line.

BACKGROUND: P-glycoprotein causing multidrug resistance (MDR) and limiting the efficacy of antineoplastic drugs and protease inhibitors (PIs) is expressed in human CD4+ T lymphocytes, one of the main targets of HIV, in a range of pharmacological barriers and at varying degrees in non-Hodgkin's lymphoma and Kaposi's sarcoma. METHODS: The differential effect of PIs on P-glycoprotein function was studied by measuring drug efflux inhibition, MDR-reversing ability and MAb UIC2 epitope modulation in MDR variants of the human T lymphoblastoid CEM cell line. RESULTS: The treatment of MDR cells with PIs induces different UIC2 epitope modulations indicating a differential recognition and binding of these antiviral drugs by MDR1 P-glycoprotein. In fact, ritonavir, saquinavir and indinavir act differently to the P-glycoprotein blocker in CEM-VBL10 cells. The MDR level of these cells was markedly affected by ritonavir and saquinavir in the order, while the PI indinavir does not seem to compete with the P-glycoprotein drug transport function. In CEM-VBL100 cells, expressing a very high number of P-glycoprotein molecules, only ritonavir acts as an efficient drug efflux inhibitor and MDR-reversing agent. CONCLUSION: The HIV-1 PIs ritonavir and saquinavir even at different levels act as genuine P-glycoprotein substrates by inhibiting dye substrate efflux, modulating UIC2 epitope and reversing drug resistance. Conversely, at least in the in vitro system used in the present study, the PI indinavir does not significantly alter P-glycoprotein drug transport activities and function.

Modulation in vitro of keratinocyte integrins by interferon-alpha and interferon-gamma.

BACKGROUND: Interferon-alpha and -gamma are glycoproteins with antiviral and immunoregulatory properties. In vitro studies have shown a role for these cytokines in the regulation of epidermal keratinocyte growth and differentiation. In the same way, integrins are adhesion molecules which regulate keratinocyte proliferation and differentiation. AIM: To determine whether the regulatory activity of interferons on keratinocyte proliferation and differentiation is related to a modulation of keratinocyte integrins. METHODS: Two different methods were used: monolayers and reconstituted skin, incubated either with 1,200 U/mL interferon-alpha or 500 U/mL interferon-gamma or control medium for 48 h. The integrin expression was assessed by flow cytometry and immunohistochemistry. RESULTS: In monolayers, only the alpha3 subunit was significantly inhibited by interferon-gamma. In reconstituted skin, where keratinocytes are differentiated, both interferons had an inductive effect on beta1 expression and interferon-alpha had an inhibitory effect on alpha6 expression. CONCLUSION: Interferon-alpha and -gamma induce a modulatory effect on alpha3, alpha6 and beta1 which appears to be related to the state of differentiation. Moreover, the decreased expression of alpha6 and alpha3 could be one of the mechanisms involved in the formation of bullous lesions during long-term interferon therapy.

Differentiation between the complement modulating effects of an arabinogalactan-protein from Echinacea purpurea and heparin.

Due to the important physiological role of the complement system, complement modulation, either inhibition or stimulation, is an interesting target for drug development. Several plant polysaccharides are known to exhibit complement modulating activities. Sometimes these effects are described as complement inhibition, although the basic mechanism is a stimulation of the complement activation. This misinterpretation is due to the observed reduced haemolysis in the widely used haemolytic complement assay, which does not allow to differentiate between complement activators and inhibitors, when it is performed in the classical manner. The aim of the presented study was to demonstrate that by simple modifications of the classical procedure this assay becomes an efficient tool to distinguish between real complement inhibitors and complement activating compounds without performing expensive, molecular mechanistic investigations. As practical examples heparin with proven complement inhibiting activity and AGP, a new arabinogalacatan-protein type II isolated from pressed juice of the aerial parts of Echinacea purpurea, as a potential complement activating compound were included in the study. By means of varying the preincubation time of the test compound with complement, AGP was clearly identified as a stimulator of both the classical and alternative pathway of complement activation. These findings correspond to the results of molecular mechanistic investigations. Selective removal of the arabinose side chains of AGP resulted in considerably reduced activity. Therefore, the three-dimensional structure of the polysaccharide, i. e., a backbone branched by side chains, is supposed to be important for the interactions with the complement system. The complement activating effects of AGP may contribute to the well-established immunostimulating effects of the pressed juice from Echinacea purpurea. Abbreviations. AGP:arabinogalactan-protein AGP-hydr.:hydrolysed arabinogalactan-protein AP-CA:haemolytic complement assay for the alternative pathway CP-CA:haemolytic complement assay for the classical pathway EGTA-VB:veronal buffered saline containing EGTA and Mg 2+HPS:human pooled serum RT:room temperature LPS:lipopolysaccharide RaE:rabbit erythrocytes RT:room temperature ShE(A):(sensitised) sheep erythrocytes VB:veronal buffered saline containing Ca 2+ and Mg 2+

Establishment of a human thymic myoid cell line. Phenotypic and functional characteristics.

The subset of myoid cells is a normal component of the thymic stroma. To characterize these cells, we immortalized stromal cells from human thymus by using a plasmid vector encoding the SV40 T oncogene. Among the eight cell lines obtained, one had myoid characteristics including desmin and troponin antigens. This new line was designated MITC (myoid immortalized thymic cells). These cells expressed both the fetal and adult forms of muscle acetylcholine receptor (AChR) at the mRNA level, as well as the myogenic transcription factor MyoD1. alpha-Subunit AChR protein expression was detected by flow cytometry and the AChR was functional in patch-clamp studies. In addition, AChR expression was down-modulated by myasthenia gravis sera or by monoclonal antibody anti-AChR on MITC line similarly to TE671 rhabdomyosarcoma cells, making the MITC line an interesting tool for AChR antigenic modulation experiments. Finally, the MITC line expressed LFA-3, produced several cytokines able to act on T cells, and protected total thymocytes from spontaneous apoptosis in vitro. These results are compatible with a role of thymic myoid cells in some steps of thymocyte development. Therefore MITC line appears to be a useful tool to investigate the physiological role of thymic myoid cells.

Alteration of a model antigen by Au(III) leads to T cell sensitization to cryptic peptides.

Certain metal ions are known to be potent sensitizers, but the self proteins modified by metal ions and the self peptides recognized by 'metal-specific' T cells are unknown. In humans and mice treatment with gold anti-rheumatic drugs, containing Au(I), may lead to allergic and autoimmune side effects. Human and murine T cells do not react to Au(I), however, but to the reactive metabolite Au(III). Here we show that alteration by Au(III) of a model antigen, bovine ribonuclease (RNase)A, results in T cell sensitization to cryptic peptides of this protein. Upon immunization of mice with Au(III)-pretreated RNase [RNase/Au(III)], CD4+ T cell hybridomas specific for RNase/Au(III) were obtained in addition to those recognizing the immunodominant peptide RNase 74-88; the latter also were obtained after immunization with native RNase. RNase/Au(III)-specific T cell hybridomas reacted against RNase/Au(III) and RNase denatured by S-sulfonation of cysteine residues, but not against native RNase, or RNase pretreated with Au(I), A1(III), Cu(II), Fe(II), Fe(III), Ni(II), Mn(II), or Zn(II). Using a panel of overlapping, synthetic RNase peptides which were devoid of gold or gold-induced modifications, epitope mapping revealed that RNase/Au(III)-specific T cell hybridomas recognized the cryptic peptides 7-21 and 94-108, respectively. Comparison of the proliferative response of bulk CD4+ T cells, prepared from splenocytes after immunization with either RNase/Au(III) or native RNase, revealed that Au(III) pretreatment of RNase led to a markedly enhanced response to the two cryptic peptides while it did not influence the response to the immunodominant peptide. The cryptic peptides were also presented after preincubation of bone marrow-derived macrophages with RNase and Au(I), but not with RNase alone, suggesting that oxidation of Au(I) to Au(III) and subsequent protein alteration by Au(III) can happen in mononuclear phagocytes. We conclude that Au(III) alteration of proteins alters antigen processing and, thus leads to presentation of cryptic peptides. This mechanism may shed light on the development of allergic and autoimmune side effects of Au(I) anti-rheumatic drugs. In addition, it might provide a general mechanism of how metal ions act as T cell sensitizers.

Involvement of multiple protein kinases in CD3-mediated activation of human T lymphocytes.

The role of different protein kinases in the process of T cell activation has been studied using several inhibitors. The model we adopted was the activation of PBMC by monoclonal antibody OKT3. The results obtained confirm that PKC and PTK are involved. Thus, the inhibitors H-7, staurosporine, and genistein exerted a dose-dependent inhibition of CD2 up-regulation, CD25 expression, IL-2 production, and cellular proliferation. On the other hand, our data indicate that PKA is not involved since the inhibitor HA1004 was ineffective. W-7, an inhibitor of Ca(2+)-CaM protein kinases, inhibited OKT3-induced modulation of cell-surface markers and PBMC proliferation, whereas a slight increase in IL-2 release was detected at the highest dose used (20 microM). Using the MLCK inhibitor ML-9, we extended our studies to the myosin light chain kinase, which influences the organization of the cytoskeleton. ML-9-inhibited PBMC activation in terms of modulation of cell-surface markers and proliferation but stimulated IL-2 production. Similar results were obtained using the cytoskeleton disruptors demecolcine and cytochalasin B. Taken together the data described herein indicate that T cell activation is a complex event in which, aside from classical signal transduction-associated kinases PKC and PTK, at least two other kinases, Ca(2+)-CaM kinases and MLCK, seem to be involved, the latter probably through correct assembly of the cytoskeleton.

Different mechanisms regulate the monoclonal antibody-induced modulation of CD2, CD3, and CD5 in human lymphocytes.

The CD2, CD3, and CD5 antigens are down-modulated from the cell surface of peripheral blood mononuclear cells after a 24-hr incubation with specific monoclonal antibodies (mAb). Here we show that active (phorbol myristate acetate, phorbol dibutyrate acetate, and mezerein) but not inactive (4 beta-phorbol) tumor-promoting agents inhibit the mAb-induced modulation of CD2 and CD5, but not CD3, without concomitant changes in the surface distribution of these antigens (such as capping). This inhibitory effect is not protein synthesis dependent and is reversed by protein kinase C inhibitors (staurosporine and H-7). The use of cytoskeleton-disrupting agents shows the existence of different cytoskeletal interactions driving the mAb-induced modulation of CD2 and CD5 with respect to CD3. Treatment with cytochalasin D (an agent that inhibits microfilament polymerization) but not colchicine (an agent that inhibits microtubule polymerization) reproduced the effect of TPA on the mAb-induced modulation of CD2, CD3, and CD5. Our results indicate that the mAb-induced modulation of CD2 and CD5 is dependent on microfilament (namely actin) polymerization and PKC activation, while the modulation of CD3 is not.

Modulation of phosphatidylserine epitope expression by BeWo cells during forskolin treatment.

The adenylcyclase activator forskolin induces the human choriocarcinoma line, BeWo, to undergo differentiation and fusion within 48 to 72 h. Using three monoclonal antibodies that differentiate between the anionic phospholipids cardiolipin (CL) and phosphatidylserine (PS) and immunoperoxidase techniques we investigated the expression of PS by BeWo during 48 h of forskolin treatment. We observed that BeWo cells not exposed to forskolin express an epitope of pS that reacts strongly with monoclonal antibody BA3B5C4 (CL+/PS+), whereas following treatment with forskolin there is a decrease in reactivity with BA3B5C4 and a concurrent increased activity with a second PS-reactive monoclonal antibody, 3SB9b (CL-/PS+). A third monoclonal antibody, D11A4 (CL+/PS-), that reacted with all anionic phospholipids except PS did not bind to BeWo cells, whether forskolin treated or not. These observations support previous interpretations using human placenta that during cytotrophoblast differentiation two antigenic forms of PS are expressed. Based on the described relationship of PS with cellular fusion events in other systems and the association of naturally occurring antibodies against PS with pregnancy loss and intrauterine growth retardation in humans, we propose that altered expression of PS during normal placental development and in BeWo after exposure to forskolin may be critical in the cytotrophoblast differentiation process.

"Neutral allografts"--lack of allogeneic stimulation by cultured human cells expressing MHC class I and class II antigens.

Cultured human dermal fibroblasts, smooth muscle cells, epidermal cells and endothelial cells were tested for immunogenicity in an in vitro allostimulation assay. Gamma interferon was used to induce MHC class II expression, since these cells constitutively express class I but not class II antigens. In contrast to human dermal fibroblasts, smooth muscle cells and epidermal cells, endothelial cells, were able to stimulate a significant proliferative response in normal allogeneic lymphocytes. Since ICAM-1 was also expressed on these cells, this inability to initiate allostimulation was probably not due to the absence of adhesion molecules. Addition of exogenous cytokines such as IL-1, IL-2, and TNF did not restore T cell proliferation in the test system. Therefore the inability of dermal fibroblasts, smooth muscle cells, and epidermal cells to initiate significant allostimulation was also not due to lack of cytokine production. It appears that certain cells lack as-yet-undefined costimulatory factors required for their effective recognition as foreign. These results support the notion that cultured human fibroblasts, smooth muscle cells, and epidermal cells could serve as building blocks of engineered "neutral allografts" for use across MHC barriers.

Modulation by interferon alpha and gamma of the expression of a melanoma-associated antigen detected by autologous antibody.

Interferons (IFN) are known to alter the expression of histocompatibility and tumour-associated antigens. We have reported the isolation and purification of a 66-kD melanoma-associated antigen (MAA) that is recognized by the host. Competitive binding with MAA reduced autologous antibody binding to five melanoma cell lines, suggesting that a similar antigen is detected by other patients with melanoma. Nine melanoma cell lines were incubated for 3 days with 0.01-100 units/ml of interferon alpha (IFN-alpha) or interferon gamma (IFN-gamma) and the maximum titre of autologous antibody reactivity was determined by protein A haemadsorption. Incubation with IFN-alpha or IFN-gamma resulted in a decrease in maximum titre of autologous antibody reactivity directed against all melanoma cell lines. A 3-day incubation of three melanoma cell lines with IFN-gamma augmented the expression of HLA-DR, as has been reported by others. Incubation with spent media from autologous melanoma cells exposed to IFN-alpha inhibited autologous antibody binding less than control media from melanoma cells to which no IFN was added, indicative of decreased production or internalization of MAA. Conversely, incubation with spent media obtained after exposure to IFN-gamma inhibited autologous antibody binding to a greater degree than control spent media, consistent with increased shedding of antigen. These results suggest that IFN-alpha and IFN-gamma down-regulate the expression of MAA detected by autologous antibody by different mechanisms of action.