Uptake and biodistribution of technetium methylene-[P]diphosphonate during endosteal healing around titanium, stainless steel and hydroxyapatite implants in rat tibial bone

Early evaluation of intraosseous implant success and failure is critical, but, until now, there have been no reliable systems of measurement. The present study assessed whether the use of ^99mtechnetium methylene-[³²P]diphosphonate (^99mTcMD³²P), a marker for both bone formation and mineralization, can indicate if an implant is bone-bonding or non-bonding. Moreover, this study examined how bone-bonding (titanium and hydroxyapatite) and non-bonding (stainless steel) implants affected the normal healing of bone after marrow ablation, as measured by uptake of ^99mTc and ³²P. Titanium, hydroxyapatite and stainless steel implants were placed in the right tibiae of Sabra strain rats following ablation of the marrow, and ^99mTcMD³²P was injected 18h before harvest. At 3, 6, 14, 21 and 42 d (and in some experiments, on days 28 and 35) post-injury, the treated and contralateral tibiae were removed and cleaned of soft tissue. The uptake of ^99mTc and ³²P was measured in the whole bone, as well as in its organic and inorganic phases. Effects of the implants were assessed by comparing the treated to the untreated tibia in each rat. The distribution of ^99mTc and ³²P varied with each implant. After the insertion of titanium, increased ^99mTc uptake was seen in whole bone and in the inorganic and organic phases at days 6–14. ³²P uptake in whole bone and in the inorganic phase increased only at day 6, and ³²P uptake was decreased in the organic phase at that time. In tibiae implanted with hydroxyapatite, ^99mTc and ³²P uptake was seen in the whole bone at days 6 and 14. While ^99mTc uptake was increased in both the organic and inorganic phases, ³²P uptake into the organic phase was decreased at both day 6 and day 14. In tibiae implanted with stainless steel, effects were observed only on day 6. The increased ^99mTc uptake in whole bone reflected increases in both the organic and mineral phases. Increased ³²P uptake was observed in whole bone as well, due to an increase in the ³²P uptake in the mineral phase only; incorporation of ³²P in the organic phase was comparable to that found in the contralateral limb. The results of this study indicate that implants alter bone healing, as indicated by the uptake of ^99mTc and ³²P in the different bone compartments. Moreover, decreased ³²P uptake by the organic phase in the presence of bone-bonding implants suggests that cleavage of ^99mTcMD³²P into its technetium and methylene diphosphonate moieties was inhibited, perhaps as a function of the onset of calcification in the newly synthesized osteoid. The effect of the implants on bone healing was observed on days 6–14, when active bone formation and mineralization were occurring, supporting the hypothesis that these materials modulate events associated with initial calcification. Uptake of ^99mTc varies as a function of time, and uptake of ³²P varies with time and distribution in the mineral or organic phase of bone, suggesting that these parameters may be useful as indicators of bone-bonding.     

Heparin binding and release properties of DEAE cellulose membranes

Two cellulose-based membranes, containing 5 and 20% N, N-diethyl-aminoethyl cellulose (DEAE) respectively, were investigated for ionic attachment of heparin and heparin release. Heparinization was achieved by recirculation of a heparin-containing glycine buffer over each membrane. Heparin was determined by chemical analysis or by ^99mTc labelling. The heparin uptake was shown to be linearly dependent on the heparin content in the contacting fluid. Heparin release was studied by contacting heparinized membranes with saline, glycine buffer, phosphate buffer and plasma. Incubation with plasma brought about the release of 50% of the attached heparin. Crosslinking of the heparinized membrane with glutaraldehyde reduced the heparin release by one half. The release reaction is more critical in the case of increased heparin uptake and a more efficient immobilization of heparin appears necessary.     

Interaction between `sensitized lymphocytes' and antigen in vitro: IV. Studies on the mechanism of release of skin reactive and macrophage migration-inhibitory factors

Peritoneal exudate lymphocytes (PEL) of guinea-pigs injected with Freund's complete adjuvant (FCA) were capable of liberating skin reactive factor (SRF) and macrophage migration-inhibitory factor (MIF) when cultured for 24 hours in the presence of sheep red cells (SRC) preincubated with lymph node cells (LNC) of guinea-pigs immunized with SRC in FCA. Similar release of soluble mediators was found in the following situations: (a) incubation of non-immune PEL with SRC sensitized with the exclusion peak of Sephadex G-200 fractionated immune LNC supernatant; (b) incubation of non-immune LNC with complexes of SRC and anti-SRC IgG, prepared from serum of animals immunized with SRC in CFA, and (c) incubation of non-immune LNC with a mixture of SRC urea extract and anti-SRC IgG. SRC incubated for 24 hours with immune LNC and freed of lymphocytes by selective filtration through a Millipore filter, were able to provoke an inflammatory reaction in the skin of normal guinea-pigs, which was delayed in character. These findings are interpreted as suggestive evidence for the non-identity of the mediator-producing and antigen-reactive cells and as support for the hypothesis that a complex composed of antigen and an antibody-like material, secreted by stimulated antigen-reactive lymphocytes, is responsible for the release of soluble mediators in cell-mediated immune reactions.     

Mechanisms of Cell-mediated Cytotoxicity in Mice Rejecting Xenogeneic Human Lymphoblastoid Cells

The in vivo immunization of mice with human lymphoblastoid cell line LHN13 generates direct cell-mediated cytotoxicity by spleen cells. The lytic activity appears as early as day 3 after the intraperitoneal inoculation of 7.5 × 10⁵ cells and persists at least until day 11. The killer cells do not adhere to plastic and are not retained on nylon wool columns or on Degalan heads coaled with mouse Ig plus rabbit-anti-mouse Ig The effector cells are partly inhibited by treatment with anti-Thy-1.2 serum plus complement, but this inhibition appears to be non-specific since antiserum alone or normal mouse serum plus complement have the same effects. Heat-aggregated IgG strongly inhibit cytotoxicity, indicating that the effector cells are Fc-positive and that such receptors are implicated in lysis. Altogether, these features strongly argue for an ADCC phenomenon. The involvement of antibodies is demonstrated by the fact that eluates (56°C, 30 min) from immune cells alone induce lysis in the presence of normal spleen cells as effectors The lytic activity of these eluates can be removed by specific adsorption on protein A coupled to Sepharose heads and on the human lymphoid target cells. Positive complementation between immune and non-immune spleen cells suggests that the arming process may occur in vitro during the assay, when antibodies are released by plasmacytes     

Complement and cell-mediated lysis of haptenated erythrocytes sensitized with oligomers of rabbit igg antibody

Trinitrophenylated (TNP)‡ chicken erythrocytes were coated with various amounts of radiolabeled, affinity cross-linked oligomers of rabbit anti-TNP IgG. These cells were used as targets for lysis by complement or by several types of effector cells, normal splenocytes, cells of a macrophage line (P388D₁) or cells of two lymphoma lines (PL-1 and ABLS-5).With complement as mediator, lysis increased with the number and size of target-bound molecules. In contrast, antibody-dependent, cell-mediated cytolysis (ADCC) depended only on the number of sub-units bound, and was independent of oligomer size. Moreover, complement-mediated lysis required a threshold level of bound antibody before lysis occurred, whereas at low levels of sensitization ADCC increased linearly with increasing antibody. These results suggest that IgG clusters on the target cell are required for complement-mediated lysis but not for cell-mediated lysis. Furthermore, the data suggest that the crosslinking of neighboring Fc receptors on the surface of effector cells is not required for initiating ADCC.Non-immune IgM and small oligomers of non-immune IgG were used to block lysis mediated by each type of effector. ADCC was readily inhibited by small oligomers but not by IgM, while complement was inhibited by only larger IgG oligomers and by IgM. These results suggest that ADCC is not blocked by IgM, but is much more susceptible to inhibition by small IgG immune complexes than complement-mediated lysis.     

Modulation of lethal and persistent rat parvovirus infection by antibody

Summary Two day-old athymic (rnu/rnu) and euthymic (rnu/+) rat pups nursing immune or non-immune dams were inoculated oronasally with the Yale strain of rat virus (RV-Y). All athymic and euthymic pups (57/57) from immune dams remained clinically normal, whereas 51 of 66 athymic and euthymic pups from non-immune dams died within 30 days. Infectious RV was detected by explant culture in 12 of 15 surviving pups of both genotypes from non-immune dams 30 days after inoculation, but in none of the 57 surviving pups from immune dams. RV-Y DNA was detected by Southern blotting in kidneys of surviving athymic pups from non-immune dams but was not detected in pups from immune dams. Euthymic pups from immune dams appeared not to produce endogenous antibody to RV after virus challenge, whereas euthymic pups from non-immune dams produced high-titered RV immune serum. Pups of both genotypes given immune serum prior to or with RV were fully protected from disease and persistent infection, whereas pups given immune serum 24 hours after RV were partially protected. These studies show that RV antibody offers significant protection against lethal and persistent RV infection.     

An automated micromethod for detection of lytic antimycobacterial antibodies by immune lysis of liposomes sensitized to tuberculin

Calcein was concentrated inside multilamellar liposomes (MLV) by encapsulation through an osmotic gradient in order to increase its quenching. Immune lysis of these MLV sensitized to PPD by direct encapsulation or by exposure to preformed vesicles was studied in the presence of rabbit anti-BCG serum and of a non-immune rabbit serum as a source of complement, successively added in the wells of a microplate to a final volume of 0.1 ml. A 40 to 50% release of encapsulated calcein was observed after 5 min using a Titertek Fluoroskan II. Preformed liposomes exposed to PPD were more sensitive to anti-BCG serum than liposomes formed in PPD. Trials with human sera revealed a significantly higher level of specific lytic immunoglobulins in tuberculous patients than in non-tuberculous subjects. Key words: Liposome immune lysis assay; lytic antimycobacterial antibodies; tuberculosis, human; test automation.     

OSTEOCLAST-LIKE GIANT CELL TUMOR OF THE LIVER

A 66-year-old male with osteoclast-like giant cell tumor of the liver that arose in the non-cirrhotlc liver is presented. The liver tests were almost normal, and plasma levels of alpha-fetoprotein and carcinoembryonic antigen were within normal limits. The findings of liver scan by ^99mTc phytate, celiac angiography, and CT scans are described for the first time for this rare neoplasm, showing a large, unresectable liver tumor. Histologically, the tumor mainly consisted of osteoclast-like giant cells and mononuclear cells, which were focally arranged in a vaguely trabecular pattern and sarcomatous pattern. By an electromicroscopic study, however, no definitive evidence was obtained whether it arose from epithelial cells or nonepithellal cells. Various clinicopathological features were described and compared with previously reported cases including two cases arising in the liver.     

Characteristics of the effector cells mediating cytotoxicity against antibody-coated target cells. I. Phagocytic and non-phagocytic effector cell activity against erythrocyte and tumour target cells in a 51Cr release cytotoxicity assay and [125I]IUdR growth inhibition assay.

Both phagocytic and non-phagocytic effector cells were able to kill rabbit antibody-coated chicken erythrocytes (CRBC) while only non-phagocytic effector cells were active against alloantibody-coated SL2 lymphoma. In addition to the variation in susceptibility of erythrocyte and tumour target cells to various effector cell populations, it was found that different tumour cells can vary markedly in their ability to be killed by non-immune spleen cells in the presence of antibody. It is postulated that both the type of antibody and certain characteristics of the cell membrane are important in determining whether target cells are susceptible to antibody-dependent cell-mediated cytotoxicity detected by the 51Cr release assay. It was also demonstrated that alloantibody-coated P-815-Y mastocytoma, which showed very little evidence of cytotoxicity in the 51Cr release assay, was markedly inhibited in its ability to incorporate [125I]IUdR after incubation with antiserum and non-immune spleen cells. This growth inhibition in the absence of cytotoxicity, or cytostasis, is discussed in relation to the potential mechanisms of target cell damage, and in the light of recent observations (Plata, Gomard, LeClerc and Levy, 1974; Newlands and Roitt, 1975) that cytotoxicity and growth inhibition assays detect different effector cell populations in tumour-bearing animals.     

IL-31与自身免疫性疾病

自身免疫性疾病(autoimmune disease,AID)是指机体对自身抗原发生免疫反应而导致自身组织损害所引起的疾病,其发病机制是T、B细胞过度活化。白细胞介素(interleukin,IL)-31是最近发现的一种四螺旋束细胞因子,主要由CD4 ＋T辅助细胞产生,并且在免疫细胞和非免疫细胞受体广泛表达。...     

Aujeszky's disease vaccination and infection of pigs with maternal immunity: Effects on cell- and antibody-mediated immunity

Summary SCC, ADV-SCC, ADV-ADCC and ADV-LYST as well as ND₅₀-titres of neutralizing serum antibodies were examined in 36 passively immune pigs, 25 of which were vaccinated at 3 weeks of age and partly revaccinated 3 weeks later. Twenty-five vaccinated animals and 8 non-immune control pigs were challenged with infectious ADV.Independent of the state of maternal immunity the cytotoxic response of the white blood cells from all the animals was low at WPP 3 but rose with increasing age. ADV-LYST occurred only in some of the animals. A single vaccination evoked no significant effect on our immune parameters, but revaccination led to higher ADV-LYST and ADV-ADCC. In pigs vaccinated at WPP3 the neutralizing serum titres decreased gradually, similar to unvaccinated animals, indicating that the antibodies were of maternal origin. However, after vaccination at WPP6, no further decline of ND₅₀-titres could be detected, pointing to a limited antibody production. Animals vaccinated at WPP3 and revaccinated 3 weeks later showed a significant increase of serum neutralizing titres.Whereas the controls showed typical symptoms of Aujeszky's disease, the immune animals, especially the unvaccinated passively immune pigs, showed only elevated temperatures and most of them excreted small amounts of ADV.The development of cellular immunity after infection was rather similar within the maternally immune group independent whether the animals had been vaccinated or not, but ADV-ADCC and ADV-LYST showed a more rapid progress within the vaccinated group than in the non-vaccinated group and the non-immune control group. Infection resulted in significantly higher ND₅₀ titres in vaccinated and revaccinated animals than in unvaccinated animals, indicating a secondary response in those pigs. Thus, ADV sensitization of lymphocytes had been evoked by vaccination despite the presence of maternal antibody.The interpretation of the results was complicated by great individual and litter-dependent variations of the immune parameters.Abbreviations ADV Aujeszky's disease virus - ADV-ADCC antibody-dependent cell-mediated cytotoxicity against ADV infected cells - ADV-LYST lymphocyte stimulation by ADV - ADV-SCC spontaneous cell-mediated cytotoxicity against ADV infected cells - CTI cytotoxic index(ices) - DPI day post infection - LYST lymphocyte stimulation - SCC spontaneous cell-mediated cytotoxicity - SI stimulation index(ices) - WPP week post partum - WPV week post vaccination - WPRV week post revaccination With 5 Figures     

Human Amoebiasis: The Interaction of Lymphocyte Surface-Bound Immune Complexes and Pmns Impairs T Cell Proliferative Responses to E. Histolytica Mitogen

In some of the sera from patients with amoebiasis circulating immune complexes are present which are thought to interact with lymphoid cells, enabling them to elicit a burst of oxygen consumption in PMNs. The intensity of chemiluminescence is related to the presence of C3⁺ and Fc IgG⁺ cells in the lymphoid cell suspensions employed. The generation and release of highly reactive oxygen derivatives from PMNs impair T lymphocyte proliferative responses to the E. histolytica mitogen. The Authors suggest that one of the mechanisms by which circulating immune complexes present in the sera of patients with amoebiasis may interfere with T cell-mediated immune responses, is through their binding to the surface of the C3⁺, Fc IgG⁺ cells with subsequent stimulation of the PMN oxidative metabolism.     

A novel flow cytometric assay for quantitation and multiparametric characterization of cell-mediated cytotoxicity

Cell-mediated cytotoxicity is a crucial mechanism involved in several fundamental immunological processes such as protection against intracellular pathogens or termination of an immune response. This phenomenon is classically evaluated by the 51Cr release assay, which requires a radioactive isotope and does not permit the characterization of cells involved in the cytotoxic reaction. We describe a new flow cytometry method, developed in the context of CD95-mediated cell death, which allows the precise quantitation of cell-mediated cytotoxicity and the detection of intracellular events involved in the cytotoxic process. This assay uses a combination of two dyes, i.e. 5- (and 6-) carboxyfluorescein diacetate succinimydyl ester (CFSE) to label effector cells and 7-amino actinomycin D (7-AAD) to stain apoptotic target cells. We show that this assay is more sensitive than the 51Cr release assay and makes it possible to quantitate the percentage of cell lysis and, concomitantly, to immunophenotype target cells. It also facilitates the analysis of some events of the apoptotic pathway such as caspase activation or the expression of mitochondrial molecules. This new assay should contribute to a better understanding of the mechanisms involved in cell-mediated cytotoxicity in normal and pathological situations.     

IL-3 in the development and function of basophils

The β common chain (βc) cytokine family includes granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) and IL-5, all of which use βc as key signaling receptor subunit. GM-CSF, IL-3 and IL-5 have specific roles as hematopoietic growth factors. IL-3 binds with high affinity to the IL-3 receptor α (IL-3Rα/CD123) and then associates with the βc subunit. IL-3 is mainly synthesized by different subsets of T cells, but is also produced by several other immune [basophils, dendritic cells (DCs), mast cells, etc.] and non-immune cells (microglia and astrocytes). The IL-3Rα is also expressed by immune (basophils, eosinophils, mast cells, DCs, monocytes, and megacaryocytes) and non-immune cells (endothelial cells and neuronal cells). IL-3 is the most important growth and activating factor for human and mouse basophils, primary effector cells of allergic disorders. IL-3-activated basophils and mast cells are also involved in different chronic inflammatory disorders, infections, and several types of cancer. IL-3 induces the release of cytokines (i.e., IL-4, IL-13, CXCL8) from human basophils and preincubation of basophils with IL-3 potentiates the release of proinflammatory mediators and cytokines from IgE- and C5a-activated basophils. IL-3 synergistically potentiates IL-33-induced mediator release from human basophils. IL-3 plays a pathogenic role in several hematologic cancers and may contribute to autoimmune and cardiac disorders. Several IL-3Rα/CD123 targeting molecules have shown some efficacy in the treatment of hematologic malignancies.     

Antibody-dependent cell-mediated cytotoxicity (ADCC) toward human O+ red cells coated with anti-D antibody: comparison between lymphocyte and monocyte ADCC activity

We investigated the antibody dependent cell-mediated cytotoxicity (ADCC) of lymphocytes and monocytes toward human O+ red cells coated with anti-D antibody using a 51Cr release assay. Lysis of sensitized red cells by lymphocytes occurred rapidly, but monocyte-mediated lysis occurred slowly. This difference might be due to postphagocytic 51Cr release by monocytes. ADCC of lymphocytes increased in proportion to the effector cell number, but large amounts of antibodies were required. In contrast, ADCC of monocytes was independent of the effector/target ratio and very small amounts of antibodies could produce red cell lysis. Large amounts of fluid phase IgG were required to inhibit the lymphocyte ADCC, whereas the monocyte ADCC was markedly inhibited by small amounts of IgG. Monocyte-mediated lysis was completely inhibited by the addition of 10% human AB serum, but lymphocyte-mediated lysis was only slightly inhibited. Purified IgG1 and IgG3 were much more inhibitory to the lysis by both effectors than IgG2 and IgG4 (IgG2 greater than IgG4). Erythrophagocytosis also was inhibited by IgG1 and IgG3. These studies demonstrate that lymphocytes as well as monocytes can cause the lysis of antibody sensitized red cells, and IgG1 and IgG3 subclasses are more important than IgG2 and IgG4 in causing lysis of anti-D coated red cells.     

DRAQ5-based, no-lyse, no-wash bone marrow aspirate evaluation by flow cytometry

Flow cytometry (FC) is a powerful tool for objective phenotyping of hematolymphoid neoplasia. Analysis of bone marrow aspirates and peripheral blood specimens by FC typically requires an erythrocyte lysis or gradient separation method to remove erythrocytes prior to analysis, which may result in the loss of certain populations, in particular nucleated erythroid cells. We developed a method to analyze bone marrow aspirates (BMAs) by FC without erythrocyte lysis or washing to minimize cell loss by exploiting the nuclear DRAQ5 fluorescence as a gating parameter (DRAQ5 protocol). We analyzed a total of 31 BMAs from patients with a variety of diagnoses utilizing the DRAQ5 protocol in combination with CD71 and CD45 antibodies to determine the marrow differentials. These were compared with differential counts obtained by morphologic study and erythrocyte lysis FC. The DRAQ5 protocol preserved the nucleated erythrocytes, allowing calculations of the myeloid to erythroid ratio and of blasts/abnormal cells that better reflect the morphologic nucleated cell differential than erythrocyte lysis FC.     

Natural cytotoxicity of human blood monocytes: production of monocyte cytotoxic factors (MCF) during interaction with tumor cells

Human blood monocytes obtained by EDTA-reversible adherence to autologous serum-coated plastic dishes expressed natural cytotoxicity against NK-sensitive K562 cells in a 4-h 51Cr release assay. These monocytes released soluble cytotoxic factors, termed monocyte cytotoxic factors (MCF), when cultured with target cells. In contrast, blood monocytes obtained by adherence to fetal calf serum-coated plastic surfaces failed to kill K562 cells and to produce MCF. Although some lysis could be detected at 18 h, optimal lysis of K562 cells by MCF was observed after 48 h incubation in a microcytotoxicity assay using trypan blue dye exclusion. The addition of actinomycin D to the cytotoxicity assay enhanced the sensitivity and then NCF activity was detectable in a 18-h Cr release assay. Neither supernatants produced by culture of monocytes alone nor lysates of monocytes were cytotoxic. In addition, cytochalasin A inhibited both direct cell-mediated lysis and generation of MCF. Optimal production of MCF occurred after 6-24 h of interaction with K562 cells, although significant activity was already present by 3 h. Treatment of monocytes with OKM1 monoclonal antibody plus complement abrogated both cell-mediated lysis and MCF generation, whereas Leu-11b plus complement were ineffective. These results indicate that human blood monocytes can release MCF during interaction with tumor cells and that this may be involved in the lytic mechanism of monocyte-mediated natural cytotoxicity.     

Specific antibody-dependent cellular cytotoxicity in human malaria.

A micromethod for the study of specific antibody-dependent cellular cytotoxicity (ADCC) in human malaria is described, using cultured, asexual Plasmodium falciparum parasites as viable target cells. Lymphocytes from children with acute malaria, uninfected immune adult Gambians and adult Gambians infected with P. falciparum were capable of killing P. falciparum in vitro in the presence of malaria antibody. A parasite growth-promoting factor, produced by lymphocytes in non-immune serum and at a lymphocyte--parasite ratio of 10:1, in immune serum, was found to produce three-fold increases in growth of P. falciparum. The mechanisms by which ADCC may occur are also discussed.     

Potentiation of NK cell-mediated cytotoxicity in human lung adenocarcinoma: role of NKG2D-dependent pathway

Natural cytotoxicity receptors and NKG2D correspond to major activating receptors involved in triggering of tumor cell lysis by human NK cells. In this report, we investigated the expression of NKG2D ligands (NKG2DLs), MHC class I-related chain (MIC) A, MICB and UL16-binding proteins 1, 2 and 3, on a panel of human non-small-cell lung carcinoma cell lines, and we analyzed their role in tumor cell susceptibility to NK cell lysis. Although adenocarcinoma (ADC) cells expressed heterogeneous levels of NKG2DLs, they were often resistant to NK cell-mediated killing. Resistance of a selected cell line, ADC-Coco, to allogeneic polyclonal NK cells and autologous NK cell clones correlated with shedding of NKG2DLs resulting from a matrix metalloproteinase (MMP) production. Treatment of ADC-Coco cells with a MMP inhibitor (MMPI) combined with IL-15 stimulation of autologous NK cell clones lead to a potentiation of NK cell-mediated cytotoxicity. This lysis is mainly NKG2D mediated, since it is abrogated by anti-NKG2D-neutralizing mAb. These results suggest that MMPIs, in combination with IL-15, may be useful for overcoming tumor cell escape from the innate immune response.     

Alteration of human blood cells and changes in plasma mediators produced by radiographic contrast media.

In vitro incubation of human blood cells with iodinated radiographic contrast media (RCM) produced marked effects which were dose-dependent: erythrocytes showed crenation which was reversible; neutrophil leukocytes released the lysosomal enzyme beta-glucuronidase; basophil leukocytes released histamine; and platelets released serotonin as well as beta-glucuronidase. The release reaction could not be attributed to cell lysis, as demonstrated by the release of the cytoplasmic enzyme lactic dehydrogenase (LDH). In normal human serum, RCM produced activation of the complement system with lysis of cells. This RCM-induced complement activation seemed to occur via the alternate pathway. Stabilizers and cations present in the clinically used RCM solutions did not produce any complement changes.