Interleukin-2 gene methylation levels and interleukin-2 levels associated with environmental exposure as risk biomarkers for preterm birth.

AIM: To compare interleukin-2 levels (IL-2) and IL-2 gene site 1 methylation levels between preterm newborns (PN) and full-term newborns (FN) and investigate their association with the environmental exposure of their mothers during pregnancy. METHODS: IL-2 and IL-2 gene site 1 methylation levels were assessed in 50 PN and 56 FN. Newborns' mothers filled in questionnaires about their living and occupational environments, habits, diets, and hobbies. RESULTS: The mothers of PN were significantly more frequently agrarian/rural residents than the mothers of FN. PN had significantly higher IL-2 levels, and significantly lower methylation of IL-2 gene site 1 levels than FN. CONCLUSION: IL-2 levels, hypomethylation of the IL-2 gene site 1, and the mother's rural residence (probably due to pesticide exposure) were predictive biomarkers for preterm birth. For the first time, we present the reference values for the methylation of IL-2 gene site 1 in PN and FN, which can be used in the clinical setting and biomonitoring.

Expression of CD40 and CD192 in Classical Monocytes in Multiple Sclerosis Patients Assessed with Transcranial Magnetic Stimulation.

Expression of CD40 and CD192 markers in different monocyte subpopulations has been reported to be altered in people with MS (pwMS). Also, functional connectivity of the corticospinal motor system pathway alterations has been proved by transcranial magnetic stimulation (TMS). The study objective was to investigate the expression of CD40 and CD192 in classical (CD14++CD16-), intermediate CD14++CD16+ and non-classical (CD14+CD16++) blood monocyte subpopulations in pwMS, undergoing neurophysiological TMS assessment of the corticospinal tract integrity by recording motor-evoked potentials (MEPs). Radiological examination on lesion detection with MRI was performed for 23 patients with relapsing-remitting MS treated with teriflunomide. Then, immunological analysis was conducted on peripheral blood samples collected from the patients and 10 healthy controls (HC). The blood samples were incubated with anti-human CD14, CD16, CD40 and CD192 antibodies. Next, pwMS underwent neurological testing of functional disability (EDSS) and TMS assessment with recording MEPs from upper and lower extremity muscles. The results show that in comparison to HC subjects, both pwMS with normal and altered MEP findings (prolonged MEP latency or absent MEP response) had significantly decreased surface receptor expression measured (MFIs) of CD192 and increased CD40 MFI in classical monocytes, and significantly increased percentages of classical and total monocytes positive for CD40. Knowing CD40's pro-inflammatory action, and CD192 as a molecule that enables the passing of monocytes into the brain, decreased CD192 in classical monocytes could represent a beneficial anti-inflammatory parameter.

Hydrogen Bonding Drives Helical Chirality via 10-Membered Rings in Dipeptide Conjugates of Ferrocene-1,1'-Diamine.

Considering the enormous importance of protein turns as participants in various biological events, such as protein-protein interactions, great efforts have been made to develop their conformationally and proteolytically stable mimetics. Ferrocene-1,1'-diamine was previously shown to nucleate the stable turn structures in peptides prepared by conjugation with Ala (III) and Ala-Pro (VI). Here, we prepared the homochiral conjugates of ferrocene-1,1'-diamine with l-/d-Phe (32/35), l-/d-Val (33/36), and l-/d-Leu (34/37) to investigate (1) whether the organometallic template induces the turn structure upon conjugation with amino acids, and (2) whether the bulky or branched side chains of Phe, Val, and Leu affect hydrogen bonding. Detailed spectroscopic (IR, NMR, CD), X-ray, and DFT studies revealed the presence of two simultaneous 10-membered interstrand hydrogen bonds, i.e., two simultaneous ß-turns in goal compounds. A preliminary biological evaluation of d-Leu conjugate 37 showed its modest potential to induce cell cycle arrest in the G0/G1 phase in the HeLa cell line but these results need further investigation.

Medicinal signaling cells niche in stromal vascular fraction from lipoaspirate and microfragmented counterpart.

AIM: To expand our previous findings by increasing the number of patients in a study characterizing medicinal signaling cells (MSC) of stromal vascular fraction from lipoaspirate (SVF-LA) and from microfragmented lipoaspirate (SVF-MLA) applied for the treatment of osteoarthritis (OA). METHODS: Twenty OA patients, including 8 new patients, acquiring autologous microfragmented adipose tissue were enrolled. In-parallel immunophenotyping of SVF-LA and SVF-MLA was performed. The samples were incubated in a DuraClone SC prototype tube targeting the CD31, CD34, CD45, CD73, CD90, CD105, and CD146 surface markers, stained with the DRAQ7 cell nuclear dye and Live/Dead Yellow Fixable Stain, and analyzed by flow cytometry. RESULTS: The population phenotypes in SVF-LA and SVF-MLA samples included CD31+CD34+CD73±CD90±CD105±CD146± endothelial progenitors (EP), CD31+CD34-CD73±CD90±CD105-CD146± mature endothelial cells, CD31-CD34-CD73±CD90+CD105-CD146+ pericytes, CD31-CD34+CD73±CD90+CD105-CD146+ transitional pericytes, and CD31-CD34+CD73highCD90+CD105-CD146- supra-adventitial-adipose stromal cells. Compared with the autologous SVF-LA samples, the prevailing cell type in SVF-MLA were EP, which outnumbered leukocytes and supra-adventitial-adipose stromal cells (SA-ASC). The ratio of progenitor cells in SVF-MLA samples differed between female and male patients, showing a higher EP-pericyte and pericyte-SA-ASC ratio in men. CONCLUSION: Our results, hallmarked by EP-enriched anti-inflammatory features and indicating a possible sex-specific impact, contribute to defining the cellular composition of the clinically applied MSC serving as a regenerative cell therapy in OA.

Claudins: Beyond Tight Junctions in Human IBD and Murine Models.

Claudins are transmembrane proteins constituting one of three tight junction protein families. In patients with inflammatory bowel disease (IBD), disease activity-dependent changes in expression of certain claudins have been noted, thus making certain claudin family members potential therapy targets. A study was undertaken with the aim of exploring expression of claudins in human disease and two different animal models of IBD: dextrane sulfate sodium-induced colitis and adoptive transfer model of colitis. The expression of sealing claudin-1, claudin-3, claudin-4, and claudin-8, and pore-forming claudin-2 in humans and rodents has been evaluated by immunohistochemistry and quantitative polymerase chain reaction. Claudins were expressed by epithelial and cells of mesodermal origin and were found to be situated at the membrane, within the cytoplasm, or within the nuclei. Claudin expression by human mononuclear cells isolated from lamina propria has been confirmed by Western blot and flow cytometry. The claudin expression pattern in uninflamed and inflamed colon varied between species and murine strains. In IBD and both animal models, diverse alterations in claudin expression by epithelial and inflammatory cells were recorded. Tissue mRNA levels for each studied claudin reflected changes within cell lineage and, at the same time, mirrored the ratio between various cell types. Based on the results of the study, it can be concluded that 1) claudins are not expressed exclusively by epithelial cells, but by certain types of cells of mesodermal origin as well; 2) changes in the claudin mRNA level should be interpreted in the context of overall tissue alterations; and 3) both IBD animal models that were analyzed can be used for investigating claudins as a therapy target, respecting their similarities and differences highlighted in this study.

Polychromatic Flow Cytometric Analysis of Stromal Vascular Fraction from Lipoaspirate and Microfragmented Counterparts Reveals Sex-Related Immunophenotype Differences.

Mesenchymal stem/stromal cells or medicinal signaling cells (MSC)-based therapy holds promise as a beneficial strategy for treating knee OA (osteoarthritis), but there is no standardized protocols nor mechanistic understanding. In order to gain a better insight into the human MSC from adipose tissue applied for autologous OA treatment, we performed extensive comparative immunophenotyping of the stromal vascular fraction from lipoaspirate or microfragmented lipoaspirates by polychromatic flow cytometry and investigated the cellular components considered responsible for cartilage regeneration. We found an enrichment of the regenerative cellular niche of the clinically applied microfragmented stromal vascular fraction. Sex-related differences were observed in the MSC marker expression and the ratio of the progenitor cells from fresh lipoaspirate, which, in female patients, contained a higher expression of CD90 on the three progenitor cell types including pericytes, a higher expression of CD105 and CD146 on CD31highCD34high endothelial progenitors as well as of CD73 on supra-adventitialadipose stromal cells. Some of these MSC-expression differences were present after microfragmentation and indicated a differential phenotype pattern of the applied MSC mixture in female and male patients. Our results provide a better insight into the heterogeneity of the adipose MSC subpopulations serving as OA therapeutics, with an emphasis on interesting differences between women and men.

Mesenchymal Stromal Cells: Potential Option for COVID-19 Treatment.

The COVID-19 pandemic has significantly impacted the way of life worldwide and continues to bring high mortality rates to at-risk groups. Patients who develop severe COVID-19 pneumonia, often complicated with ARDS, are left with limited treatment options with no targeted therapy currently available. One of the features of COVID-19 is an overaggressive immune reaction that leads to multiorgan failure. Mesenchymal stromal cell (MSC) treatment has been in development for various clinical indications for over a decade, with a safe side effect profile and promising results in preclinical and clinical trials. Therefore, the use of MSCs in COVID-19-induced respiratory failure and ARDS was a logical step in order to find a potential treatment option for the most severe patients. In this review, the main characteristics of MSCs, their proposed mechanism of action in COVID-19 treatment and the effect of this therapy in published case reports and clinical trials are discussed.

Immunophenotyping of a Stromal Vascular Fraction from Microfragmented Lipoaspirate Used in Osteoarthritis Cartilage Treatment and Its Lipoaspirate Counterpart.

Osteoarthritis (OA) is a degenerative joint disease accompanied by pain and loss of function. Adipose tissue harbors mesenchymal stem/stromal cells (MSC), or medicinal signaling cells as suggested by Caplan (Caplan, 2017), used in autologous transplantation in many clinical settings. The aim of the study was to characterize a stromal vascular fraction from microfragmented lipoaspirate (SVF-MLA) applied for cartilage treatment in OA and compare it to that of autologous lipoaspirate (SVF-LA). Samples were first stained using a DuraClone SC prototype tube for the surface detection of CD31, CD34, CD45, CD73, CD90, CD105, CD146 and LIVE/DEAD Yellow Fixable Stain for dead cell detection, followed by DRAQ7 cell nuclear dye staining, and analyzed by flow cytometry. In SVF-LA and SVF-MLA samples, the following population phenotypes were identified within the CD45- fraction: CD31+CD34+CD73±CD90±CD105±CD146± endothelial progenitors (EP), CD31+CD34-CD73±CD90±CD105-CD146± mature endothelial cells, CD31-CD34-CD73±CD90+CD105-CD146+ pericytes, CD31-CD34+CD73±CD90+CD105-CD146+ transitional pericytes, and CD31-CD34+CD73highCD90+CD105-CD146- supra-adventitial-adipose stromal cells (SA-ASC). The immunophenotyping profile of SVF-MLA was dominated by a reduction of leukocytes and SA-ASC, and an increase in EP, evidencing a marked enrichment of this cell population in the course of adipose tissue microfragmentation. The role of EP in pericyte-primed MSC-mediated tissue healing, as well as the observed hormonal implication, is yet to be investigated.

Early results of intra-articular micro-fragmented lipoaspirate treatment in patients with late stages knee osteoarthritis: a prospective study.

AIM: To analyze clinical and functional effects of intra-articular injection of autologous micro-fragmented lipoaspirate (MLA) in patients with late stage knee osteoarthritis (KOA). Secondary aims included classifying cell types contributing to the treatment effect, performing detailed MRI-based classification of KOA, and elucidating the predictors for functional outcomes. METHODS: This prospective, non-randomized study was conducted from June 2016 to February 2018 and enrolled 20 patients with late stage symptomatic KOA (Kellgren Lawrence grade III, n=4; and IV, n=16) who received an intra-articular injection of autologous MLA in the index knee joint. At baseline radiological KOA grade and MRI were assessed in order to classify the morphology of KOA changes. Stromal vascular fraction cells obtained from MLA samples were stained with antibodies specific for cell surface markers. Patients were evaluated at baseline and 12-months after treatment with visual analog scale (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and Knee Injury and Osteoarthritis Outcome Score (KOOS). RESULTS: Three patients (15%) received a total knee replacement and were not followed up completely. Seventeen patients (85%) showed a substantial pattern of KOOS and WOMAC improvement, significant in all accounts. KOOS score improved from 46 to 176% when compared with baseline, WOMAC decreased from 40 to 45%, while VAS rating decreased from 54% to 82% (all P values were <0.001). MLA contained endothelial progenitor cells, pericytes, and supra-adventitial adipose stromal cells as most abundant cell phenotypes. CONCLUSION: This study is among the first to show a positive effect of MLA on patients with late stages KOA.

Discovery of 'click' 1,2,3-triazolium salts as potential anticancer drugs.

BACKGROUND: In order to increase the effectiveness of cancer treatment, new compounds with potential anticancer activities are synthesized and screened. Here we present the screening of a new class of compounds, 1-(2-picolyl)-, 4-(2-picolyl)-, 1-(2-pyridyl)-, and 4-(2-pyridyl)-3-methyl-1,2,3-triazolium salts and 'parent' 1,2,3-triazole precursors. METHODS: Cytotoxic activity of new compounds was determined by spectrophotometric MTT assay on several tumour and one normal cell line. Effect of the selected compound to bind double stranded DNA (ds DNA) was examined by testing its influence on thermal stability of calf thymus DNA while its influence on cell cycle was determined by flow cytometric analysis. Generation of reactive oxygen species (ROS) was determined by addition of specific substrate 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA). RESULTS: Parent triazoles were largely inactive, while some of the triazolium salts were highly cytotoxic for HeLa cells. Triazolium salts exhibited high cell-type dependent cytotoxicity against different tumour cells. Selected compound (4-(4-methoxyphenyl)-3-methyl-1-(2-picolyl)-1H-1,2,3-triazolium hexafluorophosphate(V) (2b) was significantly more cytotoxic against tumour cells than to normal cells, with very high therapeutic index 7.69 for large cell lung carcinoma H460 cells. Additionally, this compound was similarly cytotoxic against parent laryngeal carcinoma HEp-2 cells and their drug resistant 7T subline, suggesting the potential of this compound in treatment of drug resistant cancers. Compound 2b arrested cells in the G1 phase of the cell cycle. It did not bind ds DNA, but induced ROS in treated cells, which further triggered cell death. CONCLUSIONS: Our results suggest that the 'click' triazolium salts are worthy of further investigation as anti-cancer agents.

Discovery and optimization of an azetidine chemical series as a free fatty acid receptor 2 (FFA2) antagonist: from hit to clinic.

FFA2, also called GPR43, is a G-protein coupled receptor for short chain fatty acids which is involved in the mediation of inflammatory responses. A class of azetidines was developed as potent FFA2 antagonists. Multiparametric optimization of early hits with moderate potency and suboptimal ADME properties led to the identification of several compounds with nanomolar potency on the receptor combined with excellent pharmacokinetic (PK) parameters. The most advanced compound, 4-[[(R)-1-(benzo[b]thiophene-3-carbonyl)-2-methyl-azetidine-2-carbonyl]-(3-chloro-benzyl)-amino]-butyric acid 99 (GLPG0974), is able to inhibit acetate-induced neutrophil migration strongly in vitro and demonstrated ability to inhibit a neutrophil-based pharmacodynamic (PD) marker, CD11b activation-specific epitope [AE], in a human whole blood assay. All together, these data supported the progression of 99 toward next phases, becoming the first FFA2 antagonist to reach the clinic.

CD15s is a potential biomarker of serious bacterial infection in infants admitted to hospital.

Early recognition of serious bacterial infection (SBI) in children is essential for better treatment outcome. Flow cytometry analysis of neutrophil surface molecules has been more frequently utilized as a tool for diagnosis of infection. The infants (n = 105) under 6 months of age presenting to the pediatric emergency department with fever without apparent source who were hospitalized with suspicion of having SBI were enrolled in this prospective study. Sixty-nine infants were included into the training pool and were classified into bacterial or viral infection group. Validation pool consisted of 36 infants. The values of white blood cells counts, absolute neutrophil count (ANC), C-reactive protein (CRP), procalcitonin (PCT), neutrophil CD11b, CD15s and CD64 expression, and the percentage (%CD15s+) and absolute count (AC-CD15s+) of CD15s+ neutrophils were determined. In infants with SBI, %CD15s+ was 10.5 times more likely to be higher than the cut-off value. ANC, CRP, PCT, CD64, and AC-CD15s+ were also found as useful biomarkers for differentiation between bacterial and viral infection. The best fit multivariate logistic regression model included CRP, PCT, and %CD15s+ as strong predictors of SBI. The model's sensitivity (87 %) and specificity (83 %) indicated high model's accuracy. After validation on independent dataset, model's accuracy maintained high: 86 % sensitivity and 93 % specificity, confirming its reliability and supporting CRP, PCT, and %CD15s+ as real predictors. The findings of this study support assumption made in the literature on significance of CD15s in inflammation processes. Also, this study demonstrated for the first time that CD15s is potentially valuable biomarker of SBI in infants.

Impairment of lysosomal functions by azithromycin and chloroquine contributes to anti-inflammatory phenotype.

Azithromycin and chloroquine have been shown to exhibit anti-inflammatory activities in a number of cellular systems, but the mechanisms of these activities have still not been clarified unequivocally. Since both drugs are cationic, accumulate in acidic cellular compartments and bind to phospholipids with a consequent increase in lysosomal pH and induce phospholipidosis, we examined the relevance of these common properties to their anti-inflammatory activities. We compared also these effects with effects of concanamycin A, compound which inhibits acidification of lysosomes. All three compounds increased lysosomal pH, accumulation of autophagic vacuoles and ubiquitinated proteins and impaired recycling of TLR4 receptor with consequences in downstream signaling in LPS-stimulated J774A.1 cells. Azithromycin and chloroquine additionally inhibited arachidonic acid release and prostaglandin E2 synthesis. Therefore, impairment of lysosomal functions by azithromycin and chloroquine deregulate TLR4 recycling and signaling and phospholipases activation and lead to anti-inflammatory phenotype in LPS-stimulated J774A.1 cells.

Valosin containing protein (VCP) interacts with macrolide antibiotics without mediating their anti-inflammatory activities.

In addition to antibacterial activity, some macrolide antibiotics, such as azithromycin and clarithromycin, also exhibit anti-inflammatory properties in vitro and in vivo, although the targets and mechanism(s) of action remain unknown. The aim of the present study was to identify protein targets of azithromycin and clarithromycin which could potentially explain their anti-inflammatory effects. Using chemical proteomics approach, based on compound-immobilized affinity chromatography, valosin containing protein (VCP) was identified as a potential target of the macrolides. Validation studies confirmed the interaction of macrolides and VCP and gave some structural characteristics of this interaction. Cell based assays however, including the use of gene silencing and the study of VCP specific cellular functions in J774.A1 (murine macrophage) and IB3-1 (human cystic fibrotic epithelial) cell lines, failed to confirm an association between the binding of the macrolides to VCP and anti-inflammatory effects. These findings suggest the absence of an abundant high affinity protein target and the potential involvement of other biological molecules in the anti-inflammatory activity of macrolides.

Azithromycin drives in vitro GM-CSF/IL-4-induced differentiation of human blood monocytes toward dendritic-like cells with regulatory properties.

Azithromycin, a macrolide antibacterial, has been shown to modify the phenotype of macrophages. We have investigated whether azithromycin in vitro is able to modulate the differentiation of human blood monocytes to DCs. iA-DCs appear to have a unique phenotype, characterized by increased granularity, adherence, and a surface molecule expression profile similar to that of MDCs, namely, CD1a⁻CD14⁻CD71⁺CD209(high), as well as high CD86 and HLA-DR expression. The iA-DC phenotype is associated with increased IL-6 and IL-10 release, increased CCL2 and CCL18 expression and release, and M-CSF expression, as well as reduced CCL17 expression and release. Upon maturation with LPS, A-DCs and MDCs exhibit decreased expression of HLA-DR and costimulatory molecules, CD40 and CD83, as well as an increase in IL-10 and a decrease in CCL17 and CXCL11 secretion. These modulated responses of iA-DCs were associated with the ability to reduce a MLR, together with enhanced phagocytic and efferocytotic properties. Azithromycin, added 2 h before activation of iDCs with LPS, enhanced IL-10 release and inhibited IL-6, IL-12p40, CXCL10, CXCL11, and CCL22 release. In conclusion, azithromycin modulates the differentiation of blood monocyte-derived DCs to form iA-DCs with a distinct phenotype similar to that of iMDCs, accompanied by enhanced phagocytic and efferocytic capabilities. It also modifies LPS-induced DC maturation by decreasing surface molecule expression required for T cell activation, increasing IL-10 production, and inducing MLR-reducing properties.

Azithromycin inhibits macrophage interleukin-1ß production through inhibition of activator protein-1 in lipopolysaccharide-induced murine pulmonary neutrophilia.

Macrolide antibiotics, including azithromycin, also possess anti-inflammatory properties. However, the molecular mechanism(s) of activity as well as the target cells for their action have not been unambiguously identified as yet. In this study, the effects of azithromycin on lipopolysaccharide (LPS)-induced pulmonary neutrophilia were investigated in mice. Using immunohistochemistry, mRNA and specific protein assays, we confirmed that azithromycin ameliorates LPS-induced pulmonary neutrophilia by inhibiting interleukin-1ß (IL-1ß) expression and production selectively in alveolar macrophages as well as in LPS-stimulated J774.2 macrophage-derived cells in vitro. Inhibition by azithromycin of neutrophilia and IL-1ß was accompanied by prevention of nuclear expression of activator protein-1 (AP-1) in both alveolar macrophages and J774.2 cells. The macrolide did not alter nuclear factor kappa B (NF-κB) or extracellular signal-regulated kinase 1/2 (ERK1/2) expression, activation or localization in LPS-stimulated lungs or in J774.2 cells. In conclusion, we have shown that inhibition of LPS-induced pulmonary neutrophilia and IL-1ß concentrations in lung tissue following azithromycin treatment is mediated through effects on alveolar macrophages. In addition, we have shown for the first time, in an in vivo model, that azithromycin inhibits AP-1 activation in alveolar macrophages, an action confirmed on J774.2 cells in vitro.

Telomerase activity in HeLa cervical carcinoma cell line proliferation.

Normal human somatic cells in culture have a limited dividing potential. This is due to DNA end replication problem, whereby telomeres shorten with each subsequent cell division. When a critical telomere length is reached cells enter senescence. To overcome this problem, immortal HeLa cell line express telomerase, an enzyme that prevents telomere shortening. Although immortal, the existence of non-dividing cells that do not incorporate (3)H-thymidine over 24 h of growth has been well documented in this cell line. Using DiI labeling and high-speed cell sorting, we have separated and analyzed fractions of HeLa cells that divided vigorously as well as those that cease divisions over several days in culture. We also analyzed telomerase activity in separated fractions and surprisingly, found that the fraction of cells that divided 0-1 time over 6 days in culture have several times higher endogenous telomerase activity than the fastest dividing fraction. Additionally, the non-growing fraction regains an overall high labeling index and low SA-beta-Gal activity when subcultured again. This phenomenon should be considered if telomerase inhibition is to be used as an approach to cancer therapy. In this paper we also discuss possible molecular mechanisms that underlie the observed results.

Early-senescing human skin fibroblasts do not demonstrate accelerated telomere shortening.

Most normal mammalian cell lines demonstrate limited growth capacity due to the gradual accumulation of senescent cells in the culture. Senescent cells appear initially at a low incidence, but with increasing frequency as the culture accumulates more divisions. Because it has been suggested that senescence is regulated by telomere shortening in human cells, we compared the telomere lengths of the subpopulation of senescent cells, present in presenescent cultures, with those of young cells. Senescent cells were separated from young cycling cells by either bromodeoxyuridine (BrdU) incorporation followed by Hoechst dye and light treatment or DiI staining followed by separation on a high-speed cell sorter. Our results demonstrate that telomeres of early-senescing cells are the same length, and must shorten at the same rate, as cycling sister cells in the culture. Therefore, senescent cells in young mass cultures occur as a result of a stochastic, nontelomere-dependent process that we have described: sudden senescence syndrome.

Differing effects of two iron compounds on experimental arthritis, TNF-alpha levels and immune response in mice.

The effects of ferric-sorbitol-citrate and ferric-citrate on the severity of experimental arthritis, TNF-alpha secretion and the immune status were examined in mice. Arthritis was induced by footpad injection of methylated BSA and intraperitoneal injection of Bordetella pertussis. Joint and footpad swelling were measured weekly by a caliper. TNF-alpha serum levels were measured by ELISA. The immune status was determined by the response of mouse lymphocytes to ConA in vitro and by the antigen-presenting cell assay. Experimental arthritis was aggravated by ferric-citrate, whereas ferric-sorbitol-citrate did not promote it. If applied to normal (non-arthritic) mice three times a week for 4 weeks, ferric-sorbitol-citrate stimulated isolated splenocytes to increase production of TNF-alpha, the function of antigen-presenting cells and lymphocyte proliferation in response to ConA in vitro. TNF-alpha production by cultured splenocytes was also stimulated. In mice with antigen-induced arthritis, iron compounds did not additionally stimulate TNF-alpha production. Thus, we have shown that ferric-sorbitol-citrate stimulated TNF-alpha production, antigen-presenting cell activity and cellular immune response. Development of antigen-induced arthritis and TNF-alpha production in arthritic mice were not stimulated.