scholarly journals The anti-inflammatory, artificially programmed M3 macrophage phenotype restricts crystal-induced gouty inflammation in vitro

2020 ◽  
pp. 45-52
Author(s):  
С.В. Калиш ◽  
С.В. Лямина ◽  
Л.В. Кузнецова ◽  
О.П. Буданова ◽  
В.А. Логачев ◽  
...  
Keyword(s):  
Joint Damage ◽  
Fold Increase ◽  
Joint Inflammation ◽  
Calcium Pyrophosphate ◽  
Macrophage Phenotype ◽  
Monosodium Urate ◽  
Anti Inflammatory ◽  
Tgf Β1 ◽  
Msu Crystals

Подагра является частой причиной воспалительного артрита, разрушения суставов, образования камней в почках и почечной недостаточности. Причиной воспаления в суставе является взаимодействие кристаллов мочевой кислоты (monosodium urate - MSU) и пирофосфата кальция (calcium pyrophosphate - СаРР) с макрофагами. Мы предположили, что подавлять кристалл-индуцированное воспаление мог бы М3 фенотип макрофагов, который увеличивает продукцию антивоспалительных цитокинов при действии воспалительных индукторов (АВ-М3 фенотип). Цель работы состояла в проверке этой гипотезы. Методы. Подагрическое воспаление моделировали с помощью добавления кристаллов MSU и СаРР к культуре макрофагов. Для программирования АВ-М3 фенотипа использовали ингибитор инфламмасомы MCC950 и IL-4. Воспалительную реакцию оценивали по продукции IL-1b и TGFb1. Результаты. Реакция нативных (M0) макрофагов и на кристаллы MSU, и на кристаллы CaPP воспроизвела острое подагрическое воспаление в форме 3-кратного увеличения продукции IL-1b (p < 0,05). При добавлении кристаллов MSU к АВ-М3 макрофагам содержание IL-1b в культуральной среде было меньше в 25 раз (p < 0,05), а содержание TGFb больше почти в 2 раза (p < 0,05) по сравнению со средой М0 макрофагов с кристаллами MSU. Аналогичный результат был получен при добавлении кристаллов CaPP к АВ-М3 макрофагам. Заключение. Таким образом, АВ-М3 фенотип отвечает на провоспалительное действие кристаллов MSU и CaPP снижением продукции IL-1b и усилением продукции TGFb1. Результаты работы подтвердили гипотезу о том, что АВ-М3 фенотип может ограничить кристалл-индуцированное воспаление. Соответственно, разработка клинической версии технологии АВ-М3 макрофагов представляется весьма перспективной. Gout is a common cause of inflammatory arthritis, joint damage, kidney stones, and kidney failure. Joint inflammation is induced by interaction of uric acid (monosodium urate, MSU) crystals and calcium pyrophosphate (CaPP) crystals with macrophages. We hypothesized that the crystal-induced inflammation could be suppressed by the M3 switch phenotype, which increases production of anti-inflammatory cytokines under the action of inflammation inducers (AB-M3 phenotype). The aim of the present study was to test this hypothesis. Methods. Gouty inflammation was modeled by adding MSU and CaPP crystals to cultured macrophages. The inflammasome inhibitor, MCC950, and IL-4 were used for programming the AB-M3 phenotype. The inflammatory response was evaluated by production of IL-1β and TGFβ1. Results. The response of native (M0) macrophages to either MSU crystals or CaPP crystals reproduced acute gouty inflammation in the form of a 3-fold increase in IL-1 β production (p < 0.05). When MSU crystals were added to AB-M3 macrophages, the IL-1β content in the culture medium became 25 times lower (p < 0.05), and the TGF-β1 content became almost twice higher (p < 0.05) than the respective values in the M0 macrophage medium with MSU crystals. A similar result was obtained when CaPP crystals were added to AB-M3 macrophages. Conclusion. The AB-M3 phenotype responds to the pro-inflammatory action of MSU and CaPP crystals by decreasing the IL-1β production and increasing the TGF-β1 production. These results confirmed the hypothesis that the AB-M3 phenotype restricts the crystal-induced production of inflammatory IL-1β and increases the production of anti-inflammatory TGF-β1. Therefore, development of a clinical version of the AB-M3 macrophage technology is very promising.

cis-Aconitic Acid, a Constituent of Echinodorus grandiflorus Leaves, Inhibits Antigen-Induced Arthritis and Gout in Mice

Planta Medica ◽  
2021 ◽  
Author(s):  
Diego Pinto de Oliveira ◽  
Eliana de Faria Garcia ◽  
Mariana Assíria de Oliveira ◽  
Luiza C. M. Candido ◽  
Fernanda M. Coelho ◽  
...  
Keyword(s):  
Inflammatory Diseases ◽  
Oral Treatment ◽  
Joint Inflammation ◽  
Underlying Mechanism ◽  
Monosodium Urate ◽  
Joint Cavity ◽  
Leukocyte Accumulation ◽  
Aconitic Acid ◽  
Anti Inflammatory

Abstract cis-Aconitic acid is a constituent from the leaves of Echinodorus grandiflorus, a medicinal plant traditionally used in Brazil to treat inflammatory conditions, including arthritic diseases. The present study aimed to investigate the anti-arthritic effect of cis-aconitic acid in murine models of antigen-induced arthritis and monosodium urate-induced gout. The possible underlying mechanisms of action was evaluated in THP-1 macrophages. Oral treatment with cis-aconitic acid (10, 30, and 90 mg/kg) reduced leukocyte accumulation in the joint cavity and C-X-C motif chemokine ligand 1 and IL-1β levels in periarticular tissue. cis-Aconitic acid treatment reduced joint inflammation in tissue sections of antigen-induced arthritis mice and these effects were associated with decreased mechanical hypernociception. Administration of cis-aconitic acid (30 mg/kg p. o.) also reduced leukocyte accumulation in the joint cavity after the injection of monosodium urate crystals. cis-Aconitic acid reduced in vitro the release of TNF-α and phosphorylation of IκBα in lipopolysaccharide-stimulated THP-1 macrophages, suggesting that inhibition of nuclear factor kappa B activation was an underlying mechanism of cis-aconitic acid-induced anti-inflammatory effects. In conclusion, cis-aconitic acid has significant anti-inflammatory effects in antigen-induced arthritis and monosodium urate-induced arthritis in mice, suggesting its potential for the treatment of inflammatory diseases of the joint in humans. Additionally, our findings suggest that this compound may contribute to the anti-inflammatory effect previously reported for E. grandiflorus extracts.

scholarly journals THU0406 IDENTIFICATION OF INTRACELLULAR VACUOLES IN SYNOVIAL FLUID WITH CALCIUM PYROPHOSPHATE AND MONOSODIUM URATE CRYSTALS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 440.1-441
Author(s):  
M. L. Peral ◽  
I. Calabuig ◽  
A. Martín-Carratalá ◽  
M. Andrés ◽  
E. Pascual
Keyword(s):  
Clinical Practice ◽  
Synovial Fluid ◽  
Phase Contrast ◽  
Calcium Pyrophosphate ◽  
Monosodium Urate ◽  
Fluid Analysis ◽  
Cytoplasmic Vacuoles ◽  
Contrast Microscopy ◽  
Ordinary Light ◽  
Msu Crystals

Background:Synovial fluid analysis using polarized microscopy is the gold standard for the diagnosis of crystal-related arthritis. In our experience, we have noted that, when calcium pyrophosphate (CPP) crystals are observed, they sometimes appear within intracellular vacuoles. However, this phenomenon is not seen in those samples containing monosodium urate (MSU) crystals. This finding has been scantly reported in the literature, but may be useful in clinical practice to ensure accurate crystal identification.Objectives:Our study aims to assess whether the presence of vacuoles contributes to identifying the type of crystal, and also to gauge the frequency of their presentation.Methods:We conducted an observational study in a rheumatology unit between February and June of 2019. Synovial fluids containing CPP or MSU crystals, obtained in daily clinical practice, were consecutively included for analysis. Two observers simultaneously analyzed the presence of vacuoles by ordinary light and phase contrast microscopy in less than 24 hours after their extraction, using a microscope equipped with two viewing stations. The primary study variable was to determine whether CPP and MSU crystals are seen inside intracellular vacuoles, and to calculate the frequency of this finding for each type of crystal, estimating their 95% confidence interval (95% CI) and comparing rates using Fisher’s exact test.Results:Twenty-one samples were obtained. Data is given in the Table. MSU crystals were present in 7 (33.3%) and CPP crystals in 14 (66.6%). Interestingly, none of the MSU samples showed crystal-containing vacuoles (95% CI 0-35.4%). On the contrary, cytoplasmic vacuoles containing crystals were present in all of the CPP samples (95% CI 78.5-100%). The findings were confirmed by phase-contrast microscopy. Differences were statistically significant (p<0.001).Table.SAMPLES ACCORDING TO TYPE OF MICROCRYSTAL(n=21)SAMPLES WITH VACUOLS(UNDER ORDINARY LIGHT)SAMPLES WITH VACUOLS(UNDER PHASE CONTRAST)CPP (14; 66.6%)14 (100%)(95%CI 78.5-100%)14 (100%)(95%CI 78.5-100%)MSU (7; 33.3%)0 (0%)(95%CI 0-35.4%)0 (0%)(95%CI 0-35.4%)Conclusion:The presence of vacuoles may be a useful and easy way to differentiate MSU and CPP crystals when performing synovial fluid microscopy in clinical practice, since it appears to be a distinctive feature in CPP crystal fluids.References:[1]Kohn NN, Hughes RE, McCarty DJ Jr, Faires JS. The significance of calcium phosphate crystals in the synovial fluid of arthritic patients: the «pseudogout syndrome». II. Identification of crystals. Ann InternMed. 1962 May;56:738-45.[2]Pascual E, Sivera F, Andrés M. Synovial Fluid Analysis for Crystals. CurrOpRheumatol 2011;23:161-169.[3]McCarty DJ, Koopman WJ. Arthritis and allied conditions: A textbook of rheumatology, volumen 1. Lea &amp;Febiger. 1993.[4]Pascual E, Sivera F. Synovial fluid crystal Analysis. En Gout and other crystal arthropathies. Terkeltaub R ed. Elsevier; 2012: p.20-34.[5]Hwang HS, Yang CM, Park SJ, Kim HA. Monosodium Urate Crystal-Induced Chondrocyte Death via Autophagic Process. Int J Mol Sci. 2015 Dec 8;16(12):29265-77.Image 1. Microscopy with ordinary light. Cells with cytoplasmic vacuoles are observed, as well as abundant intra and extracellular CPP crystals.Image 2. Microscopy with phase contrast technique. Cells with intracellular vacuoles are observed inside which have microcrystals with parallelepiped morphology, compatible with CPP.Disclosure of Interests: :None declared

scholarly journals Influence of Cannabinoid on Interleukin-1β Treated Cartilage: An In Vitro Study

2020 ◽  
Vol 5 (4) ◽  
pp. 2473011420S0043
Author(s):  
Jiangyinzi Shang ◽  
Yuning Hu ◽  
Peter Alexander ◽  
MaCalus V. Hogan ◽  
Hang Lin ◽  
...  
Keyword(s):  
Real Time ◽  
Joint Damage ◽  
In Vitro Study ◽  
Interleukin 1Β ◽  
Human Chondrocytes ◽  
High Dose ◽  
Engineer Cartilage ◽  
Anti Inflammatory ◽  
Win 55

Category: Basic Sciences/Biologics Introduction/Purpose: Cannabinoids have been reported to possess the analgesic, immunomodulatory and anti-inflammatory properties. Recent studied further shown that cannabinoids attenuated joint damage in animal models of arthritis. However, the underlying mechanism has been completely understood. Interleukin-1β (IL-1β), a proinflammatory cytokine that can result in the degradation of cartilage, is associated with the pathogenesis of osteoarthritis. In this study, we hypothesize that cannabinoid can mitigate the detrimental effect of IL-1β on cartilage, thus reduce the progression of osteoarthritis. To test the hypothesis, we insulted human chondrocyte-derived cartilage with IL-1 β for 48 hours and then applied a synthetic cannabinoid agonist, Win- 55,212-2(Win-55), into the culture. The tissue phenotypes were assessed by real time polymerase chain reaction (PCR), histology and immunostaining. Methods: With the approval from CORID, human chondrocytes were isolated from healthy articular cartilage. P2 cells were used. MTS assay were employed to test the half-maximal (50%) inhibitory concentration (IC50). To generate cartilage in vitro, chondrocytes were pelleted and subjected to 14 days chondrogenic culture. The engineered cartilages were stimulated with 10 ng/ml IL-1β for 48 hours and then treated with different concentration of Win-55 (0.01, 0.1, or 1 µM) for another 48 hours. The tissue phenotype was assessed by glycosaminoglycan (GAG) assay, real-time PCR and histology. Results: We tested 10 doses, from 0.001µM up to 10 µM, and determined that the IC 50 of Win-55 on human chondrocytes for 2 days was ˜ 2 µM. Interestingly, this dose is significantly lower than the doses reported in similar studies. As shown in Figure 1, treatment with 2µM Win-55 causes the complete loss of GAG from engineer cartilage. In a relatively safe dose (<=1 µM), we did not observe obvious changes in all tested genes after the treatments of Win-55 (Figure 2). Conclusion: High dose of Win-55 may directly cause the degeneration of cartilage, while low dose of Win-55 doesn’t show beneficial influence on the phenotype of IL1-β-insulted cartilage. The reported anti-inflammatory effect of Win 55 on chondrocytes may due to the cytotoxicity or global inhibition of high dose Win 55 on cell activities. Therefore, if cannabinoid can be used to treat OA requires further investigation.

scholarly journals Probiotic Conditioned Media Induces an Anti-Inflammatory Macrophage Phenotype In Vivo and In Vitro

2011 ◽  
Vol 140 (5) ◽  
pp. S-19
Author(s):  
Michelle Taylor ◽  
Vandana Gambhir ◽  
Curtis Noordhof ◽  
Oliver Jones ◽  
Shu-Mei He ◽  
...  
Keyword(s):  
Conditioned Media ◽  
Macrophage Phenotype ◽  
Anti Inflammatory ◽  
Inflammatory Macrophage

Persistence of Crystals in Stored Synovial Fluid Samples

2020 ◽  
Vol 47 (9) ◽  
pp. 1416-1423
Author(s):  
Sonia Pastor ◽  
José-Antonio Bernal ◽  
Rocío Caño ◽  
Silvia Gómez-Sabater ◽  
Fernando Borras ◽  
...  
Keyword(s):  
Synovial Fluid ◽  
Light Microscopy ◽  
Room Temperature ◽  
Polarized Light ◽  
Calcium Pyrophosphate ◽  
Polarized Light Microscopy ◽  
Tetraacetic Acid ◽  
Monosodium Urate ◽  
Prospective Longitudinal ◽  
Msu Crystals

Objective.Lack of access to polarized light microscopy is often cited as an argument to justify the clinical diagnosis of crystal-related arthritis. We assessed the influence of time since sampling and preservation methods on crystal identification in synovial fluid (SF) samples under polarized light microscopy.Methods.This was a prospective, longitudinal, observational factorial study, analyzing 30 SF samples: 12 with monosodium urate (MSU) crystals and 18 with calcium pyrophosphate (CPP) crystals. Each SF sample was divided into 4 subsamples (120 subsamples in total). Two were stored in each type of preserving agent, heparin or ethylenediamine tetraacetic acid (EDTA), at room temperature or at 4°C. Samples were analyzed the following day (T1), at 3 days (T2), and at 7 days (T3) by simple polarized light microscopy, and the presence of crystals was recorded.Results.The identification of crystals in the MSU group was similar between groups, with crystals observed in 11/12 (91.7%) room temperature samples and in 12/12 (100%) refrigerated samples at T3. Identification of CPP crystals tended to decrease in all conditions, especially when preserved with EDTA at room temperature [12/18 (66.7%) at T3], while less reduction was seen in refrigerated heparin-containing tubes.Conclusion.Preserving samples with heparin in refrigerated conditions allows delayed microscopic examination for crystals. Avoiding crystal-proven diagnosis because of the immediate unavailability of microscopy no longer appears justified.

scholarly journals Fucoxanthin, A Carotenoid Derived from Phaeodactylum tricornutum Exerts Antiproliferative and Antioxidant Activities In Vitro

Antioxidants ◽  
2019 ◽  
Vol 8 (6) ◽  
pp. 183 ◽  
Author(s):  
Ulrike Neumann ◽  
Felix Derwenskus ◽  
Verena Flaiz Flister ◽  
Ulrike Schmid-Staiger ◽  
Thomas Hirth ◽  
...  
Keyword(s):  
Health Effects ◽  
Cell Lines ◽  
Metabolic Activity ◽  
Phaeodactylum Tricornutum ◽  
Caspase 3 ◽  
Mononuclear Cells ◽  
Antioxidant Activities ◽  
Fold Increase ◽  
Anti Inflammatory

Microalgae contain a multitude of nutrients and can be grown sustainably. Fucoxanthin, a carotenoid from Phaeodactylum tricornutum, could have beneficial health effects. Therefore, we investigated the anti-inflammatory, antioxidative and antiproliferative effects of fucoxanthin derived from this diatom in vitro. The effects of purified fucoxanthin on metabolic activity were assessed in blood mononuclear cells and different cell lines. In cell lines, caspase 3/7 activity was also analyzed. Nitrogen monoxide release and mRNA-expression of proinflammatory cytokines were measured. For antioxidant assays, cell free assays were conducted. Additionally, the antioxidant effect in neutrophils was quantified and glutathione was determined in HeLa cells. The results show that neither did fucoxanthin have anti-inflammatory properties nor did it exert cytotoxic effects on mononuclear cells. However, the metabolic activity of cell lines was decreased up to 58% and fucoxanthin increased the caspase 3/7 activity up to 4.6-fold. Additionally, dose-dependent antioxidant effects were detected, resulting in a 63% decrease in chemiluminescence in blood neutrophils and a 3.3-fold increase in the ratio of reduced to oxidized glutathione. Our studies show that fucoxanthin possesses antiproliferative and antioxidant activities in vitro. Hence, this carotenoid or the whole microalgae P. tricornutum could be considered as a food or nutraceutical in human nutrition, showcasing beneficial health effects.

scholarly journals Injectable mechanical pillows for attenuation of load-induced post-traumatic osteoarthritis

2019 ◽  
Vol 6 (4) ◽  
pp. 211-219
Author(s):  
Derek T Holyoak ◽  
Tibra A Wheeler ◽  
Marjolein C H van der Meulen ◽  
Ankur Singh
Keyword(s):  
Knee Joint ◽  
Joint Damage ◽  
Mechanical Trauma ◽  
Protective Effects ◽  
Post Injury ◽  
On Demand ◽  
Post Traumatic ◽  
Anti Inflammatory

Abstract Osteoarthritis (OA) of the knee joint is a degenerative disease initiated by mechanical stress that affects millions of individuals. The disease manifests as joint damage and synovial inflammation. Post-traumatic osteoarthritis (PTOA) is a specific form of OA caused by mechanical trauma to the joint. The progression of PTOA is prevented by immediate post-injury therapeutic intervention. Intra-articular injection of anti-inflammatory therapeutics (e.g. corticosteroids) is a common treatment option for OA before end-stage surgical intervention. However, the efficacy of intra-articular injection is limited due to poor drug retention time in the joint space and the variable efficacy of corticosteroids. Here, we endeavored to characterize a four-arm maleimide-functionalized polyethylene glycol (PEG-4MAL) hydrogel system as a ‘mechanical pillow’ to cushion the load-bearing joint, withstand repetitive loading and improve the efficacy of intra-articular injections of nanoparticles containing dexamethasone, an anti-inflammatory agent. PEG-4MAL hydrogels maintained their mechanical properties after physiologically relevant cyclic compression and released therapeutic payload in an on-demand manner under in vitro inflammatory conditions. Importantly, the on-demand hydrogels did not release nanoparticles under repetitive mechanical loading as experienced by daily walking. Although dexamethasone had minimal protective effects on OA-like pathology in our studies, the PEG-4MAL hydrogel functioned as a mechanical pillow to protect the knee joint from cartilage degradation and inhibit osteophyte formation in an in vivo load-induced OA mouse model.

Regulation of LPS-mediated inflammation in vivo and in vitro by the thiol antioxidant Nacystelyn

2004 ◽  
Vol 286 (6) ◽  
pp. L1319-L1327 ◽  
Author(s):  
Frank Antonicelli ◽  
David Brown ◽  
Maryline Parmentier ◽  
Ellen M. Drost ◽  
Nik Hirani ◽  
...  
Keyword(s):  
Dna Binding ◽  
Transforming Growth Factor ◽  
Lavage Fluid ◽  
Human Macrophage ◽  
Antioxidant Agents ◽  
Thiol Antioxidant ◽  
Anti Inflammatory ◽  
Tgf Β1

Increased levels of proinflammatory cytokines are present in bronchoalveolar lavage fluid in various lung diseases. Redox-sensitive transcription factors such as NF-κB regulate gene transcription for these cytokines. We therefore studied the effect of a new thiol antioxidant compound, Nacystelyn (NAL), on IL-8 regulation in a human macrophage-derived cell line (THP-1). LPS (10 μg/ml) increased IL-8 release compared with control levels. This LPS activation was inhibited by coincubation with NAL (1 and 5 mM). Pretreatment with cycloheximide or okadaic acid, protein synthesis, and serine/threonine phosphatase inhibitors, respectively, did not modify inhibition of IL-8 release caused by NAL. NF-κB and C/EBP DNA binding were increased after LPS treatment compared with control, an effect inhibited by cotreatment with NAL. Activator protein (AP)-1 DNA binding was unaffected. The enhanced neutrophil chemotaxis produced by conditioned media from LPS-treated cells was inhibited when cells were cotreated with NAL. The selectivity of NAL inhibition upon IL-8 expression was studied. LPS-treated THP-1 cells also had higher levels of TNF-α, transforming growth factor (TGF)-β1 and -3, MIP-1α and -β, and RANTES gene expression. However, only LPS-induced IL-8 and TGF-β1 expressions were inhibited by NAL. An anti-inflammatory effect of NAL was confirmed in vivo as shown by a reduction in LPS-induced neutrophil recruitment to the lungs following instillation of NAL into the lungs. Our studies demonstrate that NAL has anti-inflammatory properties in vitro and in vivo, may therefore have a therapeutic role in lung inflammation, and has the advantage over other antioxidant agents in that it may be administrated by inhalation.

Association of β-catenin with P-Smad3 but not LEF-1 dissociates in vitro profibrotic from anti-inflammatory effects of TGF-β1

2012 ◽  
Vol 126 (1) ◽  
pp. 67-76 ◽  
Author(s):  
Xinrui Tian ◽  
Jianlin Zhang ◽  
Thian Kui Tan ◽  
J. Guy Lyons ◽  
Hong Zhao ◽  
...  
Keyword(s):  
Anti Inflammatory ◽  
Tgf Β1

Monosodium Urate Crystals Bind with Idiotype Protein and Promote Dendritic Cells to Induce Cytotoxic T Cells in Vitro

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4904-4904
Author(s):  
Ippei Sakamaki ◽  
Kunihiro Inai ◽  
Takanori Ueda ◽  
Hiroshi Tsutani
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Cytotoxic T Cells ◽  
Target Cells ◽  
Monosodium Urate ◽  
Cd4 Cells ◽  
Antigenic Protein ◽  
Positively Charged ◽  
Msu Crystals

Abstract Monosodium urate (MSU) crystals have been studied to act as a key substance in local immunoreactions. MSU released from damaged cells works as an endogenous danger signal to antigen-presenting cells. MSU crystals evoke specific cell immunity and work as an adjuvant in a mouse model. The crystals also have another unique characteristic to bind with positively charged proteins, which could help to deliver some antigens into human dendritic cells (DCs). We focused on the application of MSU crystals as a not only an adjuvant but also as a carrier of positively charged antigenic protein to induce human cytotoxic T cells (CTLs) efficiently in vitro. We confirmed that MSU crystals facilitated human DCs to express the maturation marker, CD83, deliver (Fab′)2 attaching to the crystals. In order to determine whether MSU crystals facilitate the T-cell proliferation activity of DCs, the proliferative effects of DCs on allogeneic CD4+ cells were investigated. DCs pulsed with MSU crystals significantly facilitated the proliferation of allogeneic CD4+ cells when compared to DCs alone. The stimulation index (SI) was 2.5 ± 0.1 and 1.7 ± 0.1, respectively. When using DCs pulsed with the Fab attached to MSU crystals, the proliferation of CD4+ cells was significantly greater than when using DCs pulsed with Fab alone. The SI was 2.6 ± 0.2 and 1.9 ± 0.1, respectively. No significant differences were seen in the proliferation of allogeneic CD4+ cells between DCs pulsed with the Fab attached to MSU and DCs pulsed with MSU alone. We selected the multiple myeloma IM-9 cell line and its product idiotype (Id) protein as an ideal pair of target cells and positively charged tumor-specific antigen, respectively. After sensitizing DCs derived from HLA-A matched volunteers pulsed with tumor-specific monoclonal IgG-Fab fragments (IM-9 Fab) attached to MSU crystals, the CD8+ T cells stimulated by the DCs killed significantly more target cells (38.5 ± 3.5%, n=4) than those stimulated by DCs pulsed with IM-9 Fab alone (3.5 ± 7.5%). These cytotoxic effects of CD8+ cells stimulated by the DCs pulsed with IM-9 Fab attached to MSU crystals were reduced (3.6 ± 1.7%) when MSU crystals were pre-coated with fetal bovine serum to block to bind with IM-9 Fab. For efficient induction of CTLs, it is necessary for Id proteins to attach to MSU crystals. MSU crystals have some advantages of a protein carrier binding with positively charged proteins and delivering antigenic protein into DCs, as well as an adjuvant promoting DC maturation and inducing CTLs.

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