Whole Blood Cell Cultures Research Articles

Metabolic and immune/inflammatory alterations induced by a triathlon under extreme conditions.

PurposeTo investigate the effects of triathlon racing under extreme conditions on metabolic and immune/inflammatory responses.MethodsThirteen amateur athletes participated in an extreme triathlon competition (swim – 3.8 km; cycling – 180 km; running – 4 2 km; with a 3,700 m accumulated altitude). Blood samples were collected on three different occasions: pre-competition (baseline), immediately post-competition (IM), and 12 h post-competition (12 h) to evaluate glycemic and lipid profiles, leukocytes count, and cytokines levels in plasma and in whole-blood cell culture supernatant stimulated or not with LPS.ResultsDecreased glucose and triglycerides levels, increased LDL, and a significant leukocytosis were observed at IM and 12 h compared to baseline. In addition, higher serum levels of IL-6, IL-8, and IL-10 were found at IM than in baseline and post-12 h. Whereas increased IL-12p40 levels were observed for 12 h compared to baseline. At baseline, in LPS-stimulated cell culture, IL-6, IL-8, and IL-12p70 were higher, while IL-12p40 levels were lower than non-stimulated cell culture. At IM, IL-12p40 levels were unchanged, while higher levels of other cytokines were found in LPS-stimulated cell culture compared to non-stimulated cell culture. The 12 h results showed higher levels of IL-6, IL-8, and IL-10 in LPS-stimulated cell culture than in non-stimulated cell culture. Additionally, a significant negative correlation between circulating glucose levels and IL-6 was found.ConclusionThe triathlon competition's performance under extreme conditions has remarkable impacts on the lipid profile and systemic immune/inflammatory responses. For the first time, significant alterations in the cytokine responses of whole blood cell culture to LPS-stimulation in baseline, IM, and 12h were demonstrated.

Interferon-Beta Treatment Differentially Alters TLR2 and TLR4-Dependent Cytokine Production in Multiple Sclerosis Patients

Objective: Multiple sclerosis (MS) is a multifactorial chronic disease that affects the central nervous system (CNS). Toll-like receptors (TLRs) play a central role in cytokine production after pathogen- and danger-associated molecular patterns (PAMPs and DAMPs) and contribute to CNS damage in MS patients. Here, we evaluated the effects of interferon (IFN)-β treatment in TLR2 and TLR4-dependent cytokine production and mRNA expression in whole-blood cell cultures from MS patients. Methods: We evaluated cytokine production by ELISA from whole-blood cell culture supernatants and mRNA expression by real-time polymerase chain reaction in peripheral blood mononuclear cells (PBMCs). Results: In patients treated with IFN-β, tumor necrosis factor (TNF)-α production after exposure to TLR2 agonist (Pam₃Cys) was lower than in healthy controls and untreated MS patients. However, IFN-β treatment had no significant effect on TNF-α production after TLR4 agonist (LPS) stimulation. On the other hand, interleukin (IL)-10 production was increased in TLR4- but not in TLR2-stimulated whole-blood cell culture from MS patients under IFN-β treatment when compared to the controls. No differences in TNF-α or IL-10 mRNA expression in PBMCs from healthy controls and untreated or treated MS patients were detected, although PBMCs from treated patients presented higher levels of IL-32γ mRNA than those from controls. Conclusions: Our data suggest that IFN-β treatment alters the TLR-dependent immune response of PBMCs from MS patients. This may contribute to the beneficial effects of IFN-β treatment.

The effects of silver nanoparticles on RAW 264.7. Macrophages and human whole blood cell cultures.

Silver nanoparticles (AgNPs) are commonly found in consumer products due to their antimicrobial properties. This study evaluated the effects of AgNPs on the murine macrophage cell line RAW 264.7 and human whole blood cell cultures (WBCs). Effects of AgNPs on RAW cells were assessed in the presence or absence of lipopolysaccharide (LPS). Effects of AgNPs on WBCs were monitored under basal conditions and in the presence of either LPS or phytohaemmagglutinin (PHA). AgNPs were cytotoxic to WBCs at 250 μg/ml. Under basal conditions, RAW cells ≥ 62.5. μg/ml and WBCs > 25 μg/ml AgNPs induced biomarkers associated with inflammation. Under LPS stimulated conditions, 250 μg/ml AgNP inhibited biomarkers associated with inflammation for both cultures. Under basal conditions, and in the presence of 250 μg/ml AgNP, WBCs produced acquired immune system cytokines IL-10 and IFNγ. IL-10 synthesis by WBCs was partially inhibited by 250 μg/ml AgNP in the presence of PHA. Proteome profiles of RAW cell supernatants show that AgNPs modulate biomarkers associated with inflammation. WBCs proteome analysis shows modulation of biomarkers associated with anti-inflammatory effects.

Both Very Low- and Very High In Vitro Cytokine Responses Were Associated with Infant Death in Low-Birth-Weight Children from Guinea Bissau

BackgroundThe mechanisms behind heterologous immunity and non-specific effects of vaccines on mortality are not well understood. We examined associations between cytokine responses and subsequent mortality in low-birth-weight infants in Guinea-Bissau.MethodsA low-birth-weight trial randomized children to Bacille Calmette-Guérin (BCG) at birth or later according to local policy. Blood samples were obtained from a sub-group at age 6 weeks. Interleukin (IL)-5, IL-10, IL-13, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α were measured in whole-blood cell cultures stimulated with lipopolysaccharide (LPS), phytohaemagglutinin (PHA), or purified protein derivative (PPD). The outcome was mortality between bleeding and 1 year of age. Non-linear associations between cytokine responses and mortality were examined.ResultsCytokine measurements were available from 390 children. The mortality rate (MR) was high (6.8/100 person-years-observation (PYO)). Both low and high cytokine responses to LPS and PHA were associated with high mortality (MR up to 25/100 PYO in the lowest 10% and 9.2/100 PYO in the highest 10%). In BCG-vaccinated children, higher IFN-γ responses to PPD were associated with better survival (MR ratio = 0.43 (0.24–0.77)).ConclusionsData presented a rare opportunity to explore associations between cytokine responses and mortality. Both low and high cytokine responses were associated with high mortality; a balanced response to invading pathogens seems preferable.

Whole‐Blood Cultures From Patients With Chronic Periodontitis Respond Differently to Porphyromonas gingivalis but not Escherichia coli Lipopolysaccharide

Porphyromonas gingivalis lipid A heterogeneity modulates cytokine expression in human cells. This study investigates the effects of two lipid A isoforms of P. gingivalis, lipopolysaccharide (LPS)1435/1449 and LPS1690, on the secretion of proinflammatory and regulatory cytokines in total blood cultures from patients with and without chronic periodontitis (CP). A cross-sectional study was conducted in 38 systemically healthy individuals divided in two groups: 1) the CP group (n = 19), in which patients were diagnosed with CP; and 2) the no periodontitis (NP) group (n = 19), which included control patients without CP. Blood samples were collected from all patients, and whole-blood cell cultures (WBCCs) were stimulated for 48 hours with P. gingivalis LPS1435/1449 and LPS1690 and Escherichia coli LPS. Unstimulated WBCCs served as negative controls. The secretion of interferon-γ (IFN-γ), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) was detected in WBCC supernatants by enzyme-linked immunosorbent assays. E. coli LPS significantly increased the expression of all cytokines in WBCCs from both the NP and CP groups when compared to non-stimulated cells (control treatment). P. gingivalis LPS preparations increased IFN-γ levels in the CP group but not in the NP group when compared with controls (P <0.05). P. gingivalis LPS preparations also increased IL-10 and TGF-β levels in both CP and NP groups, but P. gingivalis LPS1690 showed a three-fold increase on IL-10 production in the NP group (P <0.05) when compared to P. gingivalis LPS1435/144. These data demonstrate that WBCC cell populations obtained from healthy individuals and patients with CP may differ in the cytokine response to P. gingivalis but not E. coli LPS. This is consistent with the notion that CP alters the systemic WBCC response and that this can be detected by the different P. gingivalis LPS structures.

Peripheral blood monocyte responses in periodontitis

Periodontitis results from the interaction of bacteria on the tooth surfaces and the host immune response. Although periodontal pathogens are essential for the initiation and progression of the disease, the tissue damage in periodontitis is primarily mediated by the host immune response. Differences in the susceptibility to the disease and in the clinal outcome of the therapy seem to be less dependent on genetics but more on lifestyle factors, like smoking, overweight, stress and nutrition. It has been shown that these lifestyle factors may modulate the immune response and therefore influence the initiation and progression of the disease. To study the host immune response, whole blood cell cultures (WBCC) stimulated with lipopolysaccharide (LPS) have been widely used and they specifically reflect the behaviour of monocytes. It has been shown that peripheral blood monocytes in LPS-stimulated WBCC from non-smoking periodontitis patients display a T-helper 2 (Th2)-promoting phenotype in comparison with controls. After periodontal therapy, this phenotype reversed and was comparable with controls. However, in smoking but treated patients, the Th2-promoting phenotype of monocytes still remained. Therefore, the aberrant phenotype of monocytes in the peripheral blood from periodontitis patients is likely to be a systemic response to exogenous and endogenous danger molecules released or induced by the periodontal infection or by smoking. It can be concluded that periodontal therapy in non-smoking periodontitis patients has beneficial health effects and that smoking cessation should be an integral part of the therapy as well for general health reasons as for the clinical outcome.

Exercise training increases endothelial progenitor cells and decreases asymmetric dimethylarginine in peripheral arterial disease: A randomized controlled trial

BackgroundSupervised exercise training (SET) is recommended as initial treatment to improve walking capacity in peripheral arterial disease (PAD) patients with intermittent claudication. Various mechanisms by which SET yields beneficial effects are postulated, however data regarding its influence on angiogenesis are scarce. Thus, we designed a prospective randomized controlled trial to study the impact of SET on markers of angiogenesis and endothelial function in PAD. MethodsForty PAD patients were randomized to SET on top of best medical treatment (SET+BMT) for 6 months versus best medical treatment (BMT) only. Endothelial progenitor cells (EPC) were assessed by whole-blood flow cytometry (co-expression of CD34+ CD133+ KDR+) and cell culture assays (endothelial cell-colony forming units, circulating angiogenic cells, migration assay) at baseline, 3, 6 and 12-months after inclusion. Changes of plasma levels of asymmetric dimethylarginine (ADMA), vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1) and maximum walking distance were determined. ResultsEPC – measured by flow cytometric and cell culture techniques – increased significantly upon training paralleled by a significant decrease of ADMA when compared to the BMT group (p<0.05). Six months after training cessation, the beneficial effect of SET on EPC diminished, but maximum walking distance was significantly improved compared to baseline and controls (p<0.05). No significant changes were observed for VEGF and SDF-1 plasma levels in time course. ConclusionsSET increases circulating EPC counts and decreases ADMA levels reflecting enhanced angiogenesis and improved endothelial function, which might contribute to cardiovascular risk reduction.

Monozygotic twins are discordant for chronic periodontitis: white blood cell counts and cytokine production after ex vivo stimulation

The aim of this study was to investigate the extent of concordance in the number of leucocytes and their cytokine secretion after ex vivo stimulation in a twin population discordant for the amount of periodontal breakdown. Venous blood was collected from 18 adult twin pairs (10 monozygotic and eight dizygotic twins). Each twin pair consisted of a diseased twin (proband) and his/her co-twin. In venous blood, leucocytes were counted. The cytokines interleukin (IL)-1beta, IL-6, IL-8, IL-10 and IL-12p40 were assessed after stimulation of monocytic cells, while IL-13 and interferon (IFN)-gamma were determined after lymphocytic stimulation. In the study population as a whole, probands showed higher total numbers of leucocytes and lower IL-12p40 levels compared with their co-twins. In monozygotic twins, no difference was found in the leucocyte counts, but probands secreted more IL-6 than their co-twins; an opposite trend was found for IL-12p40. The results suggest that the observed discordance in periodontal breakdown in the studied monozygotic twin population may be related to the relatively high levels of IL-6 and the low levels of IL-12p40 secretion after ex vivo stimulation of whole-blood cell cultures. This cytokine secretion profile may be regarded as a risk indicator of periodontitis.

Abstract 5088: Exercise Training Increases Endothelial Progenitor Cells and Decreases Asymmetrical Dimethylarginine in Patients With Intermittent Claudication

Background - Exercise training is a well-established, non-invasive method to improve peripheral arterial perfusion and walking capacity in PAD patients. Hypothesis - We designed a prospective randomized controlled trial to test the effect of training on laboratory markers of angiogenesis. Methods - Thirty patients with intermittent claudication were randomized to supervised training twice a week for 6 months or standard medical therapy. Endothelial progenitor cells (EPC) were assessed by whole-blood flow cytometry (co-expression of CD34+CD133+KDR+) and cell culture assays (colony forming units/CFU and circulating angiogenic cells/CAC) at baseline, 3-months and 6-months after training as well as 12-months after study begin. Plasma ADMA was determined by immunoassay. In addition, changes in maximum walking distance were recorded. Results - EPC measured by flow cytometry increased significantly three and six months after training paralleled by a significant decrease of ADMA (asymmetric dimethylarginine), a major endogenous inhibitor of NO synthase (see table ). The number of CFUs and CACs were significantly higher after 6-months compared to baseline upon exercise (P&lt;0.05). No differences were observed in the standard group in timecourse. However, 6-months after training cessation the number of EPC and ADMA levels were similar to baseline. Exercise training showed a benefit on treadmill maximum walking distance with a trendwise increase at 6-months (training: baseline 116.6m±12.8 (mean±SEM) vs. 6-months 149.1m±17.8, P=0.08; standard: baseline 125.2m±23.9 vs. 6-months 108m±19.9, P=0.3). The benefit was sustained 6 months after training cessation in the exercise group (training: 12-months 157.5m±18.4, P=0.07; standard: 12-months 129.5m±26.9). Conclusions - The beneficial effect of exercise training in PAD patients might partly be due to EPC mobilization mediated by improved NO synthesis as we observed reduced ADMA levels. EPC and ADMA levels in timecourse

Effects of smoking on the ex vivo cytokine production in periodontitis

Smoking is associated with increased severity of periodontitis. The underlying mechanisms of this phenomenon are not well understood. The purpose of the present study was to compare the monocyte-derived T cell directing (Th1/Th2) response and pro-inflammatory cytokine production in ex vivo whole blood cell cultures (WBCC) of smoking and non-smoking chronic periodontitis patients. Venous blood was collected from 29 periodontitis patients (18 non-smokers and 11 smokers) receiving supportive periodontal treatment, and diluted 10-fold for WBCC. The WBCC were stimulated for 18 h with Neisseria meningitidis lipo-oligosaccharide (LOS) or Porphyromonas gingivalis sonic extract (Pg-SE). The production of the T cell directing cytokines interleukin (IL)-12 p40 and IL-10, as well as the pro-inflammatory cytokines IL-1beta, IL-6 and IL-8, was measured in the culture supernatants. After LOS stimulation of WBCC, smokers showed a lower IL-12 p40/IL-10 ratio than non-smokers (P < 0.05). Interleukin-1beta production was significantly lower in smokers compared with non-smokers after stimulation with either LOS or Pg-SE (P < 0.05). Interleukin-6 and IL-8 production was similar in WBCC from both smokers and non-smokers, for both LOS and Pg-SE. A more pronounced Th2 response in smoking periodontitis patients may be related to increased severity of the disease.

Whole-blood culture is a valid low-cost method to measure monocytic cytokines — A comparison of cytokine production in cultures of human whole-blood, mononuclear cells and monocytes

Whole-blood and peripheral blood mononuclear cell (PBMC) cultures are used as non-validated surrogate measures of monocytic cytokine production. The aim of this investigation was to compare ex vivo cytokine production from human whole-blood and PBMC with that from isolated monocytes. We also assessed the intra- and inter-individual variation in cytokine production. In 64 healthy men (age 19–40 years) IL-6, TNF and IL-10 were measured by enzyme-linked immunosorbent assay in supernatants from whole-blood, PBMC and monocytes cultured 24 h with lipopolysaccharide (LPS) or UV-killed L. acidophilus. Cytokines produced from whole-blood was found to be more strongly correlated with monocytic cytokines than cytokines from PBMC, particularly after LPS-stimulation: r=0.57, P<0.001 versus r=0.33, P=0.01 for IL-6 and r=0.43, P<0.001 versus r=0.30, P=0.02 for TNF-α. Adjustment for a preceding 8-week dietary fatty acid-intervention did not change any of the associations. Based on measurements at three time-points 8 weeks apart the intra-individual variation was ≥50% smaller than the inter-individual variation (P<0.05) in most whole-blood cytokine responses and LPS-stimulated IL-6 from PBMC. We conclude that whole-blood cultures are well-suited low-cost proxy-measures of monocytic cytokine production. Moreover, large inter-individual variation in cytokine production was demonstrated whereas the individual responses in whole-blood were reproducible even over long time-periods.

Possible involvement of heparin-binding protein in transfusion-related acute lung injury

In antibody-mediated nonhemolytic transfusion reactions, transfusion-related acute lung injury (TRALI) tends to occur typically within 2 hours after a blood transfusion. White cell antibodies or immune complexes have been frequently shown to be associated with the syndrome, although the mechanisms by which they induce TRALI are poorly understood. The aim of this study was to characterize soluble mediators that are released from cells at an early stage after immune stimulation. To explore the mechanism of TRALI, an in vitro whole-blood cell culture assay was established in which cells were stimulated by human antibodies and the activation of neutrophils was monitored by a cell surface marker (Mac-1) with flow cytometry and by measurement of the release of soluble factors, including perforin, interleukin-6, tumor necrosis factor-alpha, and heparin-binding protein (HBP) with enzyme-linked immunosorbent assays. In addition, the involvement of two neutrophil FcgammaRs (FcgammaRIIIb and FcgammaRIIa, also known as CD16 and CD32, respectively) was examined during antibody-induced cell activation with anti-FcgammaR blocking antibodies. Substantial amounts of HBP were released within 30 minutes of stimulation by human antibodies, although other soluble mediators were not released within the same period. Furthermore, the release of HBP was mediated via signals through both FcgammaRIIIb and FcgammaRIIa. HBP appears to be one of the primary effector molecules of antibody-mediated nonhemolytic transfusion reactions including TRALI.

Superantigen-induced cytokine release from whole-blood cell culture as a functional measure of drug efficacy after oral dosing in nonhuman primates

Evaluation of drug efficacy for human diseases is routinely performed in animal models for efficiency and in accordance with FDA regulations. Rhesus macaques have been used as models for various lethal diseases and correlates of immunity, as nonhuman primates (NHP) closely resemble humans. We examined the ex vivo cytokine response of superantigen-stimulated whole-blood cells as a first step to therapeutic efficacy testing for bacterial superantigen-induced shock in NHP after oral dosing of pentoxifylline. Doses of 120 mg/kg of pentoxifylline effectively attenuated staphylococcal enterotoxin B-induced tumor necrosis factor α (TNFα), gamma interferon (IFNγ) and interleukin 2 (IL-2) in ex vivo culture of NHP whole-blood cells by 88%, 81%, and 76%, respectively, whereas lower doses of 48 or 72 mg/kg had no inhibitory effect. Thus cytokine release of stimulated peripheral blood cells provides a convenient biological measurement of the anti-inflammatory potency of pentoxifylline and has the advantage of assessing functional responses to a specific biotoxin of interest.

Validation of a new highly standardised, lab-independent whole-blood leukocyte function assay for clinical trials (ILCS ®)

Physical stress induced in healthy volunteers by the Loughborough intermittent shuttle test (LIST) was used to validate a newly developed whole-blood cell culture system (Instant leukocyte culture system (ILCS ®)). Exercise induced immune modulation was investigated through measurement of cytokine levels after activating leukocytes in peripheral blood ex vivo using the physiologic stimulant lipopolysaccharide (LPS). LPS induced production of three different cytokines, interferon gamma (IFNγ), interleukin-6 (IL-6), and interleukin-10 (IL-10). IFNγ levels were significantly decreased ( P=0.02 and P=0.001) and IL-10 levels significantly increased ( P=0.04 and 0.03) after exercise. LPS induced IL-6 production was only marginally further increased by exercise. In conclusion, the ILCS ® system provided a reliable ex vivo method, showing common as well as subject specific features in the time course of the immune modulation caused by the LIST protocol. This system will be useful for studies of the elderly, where cytokine standardisation is notoriously difficult.

Monocyte-derived RANTES is intrinsically elevated in periodontal disease while MCP-1 levels are related to inflammation and are inversely correlated with IL-12 levels.

Bacteria colonizing tooth surfaces are essential in the induction of an inflammatory response in the periodontal tissues, but do not cause periodontitis in everyone, implicating differences in the host immune response. These possible differences were studied using lipopolysaccharide (LPS)-stimulated whole blood cell cultures (WBCC), which revealed a down regulation of monocyte derived interleukin-12 (IL-12p70) in untreated periodontitis patients and an up regulation after therapy. IL-12p70 is a crucial factor in the differentiation of Th1 cell responses. Since CC chemokines are able to influence the T cell differentiation via cytokine secretion in antigen-presenting cells, the production of CC chemokines in periodontitis was evaluated. Therefore WBCC were stimulated with LPS from Escherichia coli for 18 h and the levels of IL-12p70 and CC chemokines were measured in the supernatants by ELISA. Untreated periodontitis patients released 2 fold more RANTES (regulated on activation normal T cell expressed and secreted) (P = 0.01) and lower levels of IL-12p70 in comparison to controls (P < 0.05). A trend towards higher levels of macrophage chemoattractant protein-1 (MCP-1) (P = 0.07) was also seen in untreated periodontitis patients; while similar levels of monocyte derived chemokine (MDC) and macrophage inflammatory proteins-1 alpha and -1 beta (MIP-1 alpha and -1 beta) were found. After periodontal therapy no changes were seen with regard to MDC, MIP-1 alpha, MIP-1 beta and RANTES, whereas the MCP-1 levels decreased (P < 0.05) and the IL-12p70 levels strongly increased (P < 0.01). The data showed a consistent inverse correlation between the levels of MCP-1 and IL-12p70, and their proportional changes after therapy correlated with the clinical inflammatory response after therapy. This indicates that the disease state regulates the release of IL-12p70 and MCP-1 in E. coli LPS-stimulated WBCC. In contrast, the persistent augmented levels of RANTES after therapy are suggestive for an intrinsic behaviour.

A type 2 response in lipopolysaccharide (LPS)-stimulated whole blood cell cultures from periodontitis patients

It is acknowledged that periodontitis results from the interaction of the host immune response with bacteria accumulating on the tooth surfaces. Although bacteria are essential, they are insufficient to cause the disease. Despite this knowledge it remains unclear why certain individuals are more susceptible to periodontitis than others. Therefore the present study investigated whether differences exist in the actual immune response between periodontitis patients and controls after stimulation of peripheral blood cells. Whole blood cell cultures (WBCC) were stimulated with LPS from Escherichia coli during 18 h and the release of prostaglandin E2 (PGE2), IL-1beta, IL-6, IL-8, IL-10, IL-12p40, IL-12p70 and tumour necrosis factor-alpha (TNF-alpha) was measured. The levels of PGE2 were two-fold higher in the WBCC from periodontitis patients than from controls. In contrast, the levels of IL-12p70 in WBCC from patients were two-fold lower. Furthermore, WBCC from patients secreted lower levels of IL-1beta and higher levels of IL-8 when compared with WBCC from controls. No differences were observed with respect to IL-6, IL-10, IL-12p40 and TNF-alpha production. It is known from the literature that LPS-stimulated WBCC reflect specifically the behaviour of the monocytes and that monocytes are peripheral precursors of antigen-presenting cells (APC). Therefore it is concluded that the monocytes in the present WBCC from periodontitis patients are responsible for the higher levels of PGE2 and lower levels of IL-12p70. Since it is has been shown that APC-derived IL-12p70 induces type (Th1) cells that promote cellular immunity, while APC-derived PGE2 induces type 2-helper (Th2) cells that promote humoral immunity, it is postulated that APC from periodontitis patients may have a bias in directing Th2 responses and thereby promoting the humoral immunity in periodontitis.

Rheumatoid factors, anti-filaggrin antibodies and low in vitro interleukin-2 and interferon-γ production are useful immunological markers for early diagnosis of community cases of rheumatoid arthritis. A preliminary study

Objective. To determine whether measurements of different autoantibodies (Ab) and cytokines are useful to distinguish very early rheumatoid arthritis (RA) from other inflammatory rheumatisms. Methods. From a population-based recruitment, 32 patients with very early polyarthritis (median duration: 4 months) were studied. Evaluations at entry (M0), and at 6 (M6) and 12 months (M12). Ab tested: rheumatoid factors (RF) by agglutination methods and ELISA, antiperinuclear factor (APF), antikeratin Ab (AKA), anti-Sa and antinuclear Ab. Cytokine production (TNFα, IL2, IFNγ, IL1β, IL10) in whole blood cell culture (WBCC) was determined at M0. At M12, patients were classified as having RA ( N = 15) or other rheumatic diseases. Results. At M0, AKA/APF and anti-Sa Ab frequencies were low, 13% and 7%, respectively. While most Ab detected at MO persisted, others appeared during follow-up, particularly APF, which rose from 13 to 40% at M12. At M6, IgM-RF was detected in two RA patients exclusively by ELISA. AKA/APF were found to be highly specific markers for RA (100% specificity). At some time during follow-up, two RF-negative RA patients were AKA-positive. In two patients, AKA and APF were present at M0 before they satisfied ACR criteria. IL2 and IFNγ production was significantly lower ( P < 0.05) for RA patients. Conclusion . AKA/APF and anti-Sa Ab were detected in community cases of very early RA. AKA/APF and RF detected by ELISA might contribute to an earlier diagnosis of RA. Low production of IFNγ and IL2 in WBCC constituted a distinct immunopathological feature in very early RA patients.

Influence of AFP, CEA and PSA on the in vitro Production of Cytokines

Several studies have demonstrated a decreased cytokine production in patients with cancer. Likewise, there is some evidence showing that tumor markers may play a role in immunoregulation. In this work, we have studied the in vitro production of IL-1β, IL-6 and TNF-α in whole-blood cell cultures of 10 healthy subjects after polyclonal activation with lipopolysaccharide of Salmonella enteridis and phytohemagglutinin in the presence or absence of three markers, AFP, CEA and PSA. Each sample was incubated for 48 h at 37°C in a humidified atmosphere of 5% CO₂. Subsequently, cytokine levels in the supernatant were determined. AFP did not significantly affect the production of the three cytokines compared to the basal value obtained on adding PBS. In contrast, CEA significantly increased the production of IL-6 (p <0.001) and TNF-α (p = 0.002), while PSA significantly decreased IL-1β (p <0.001), IL-6 (p = 0.031) and TNF-α (p <0.0001) production. These results suggest a possible role of CEA and PSA in the production of these cytokines.

Tumor necrosis factor alpha decreases, and interleukin-10 increases, the sensitivity of human monocytes to dexamethasone: potential regulation of the glucocorticoid receptor.

Resistance to glucocorticoid therapy has been observed in patients with autoimmune/inflammatory diseases and may be related to the inflammatory process itself. The aim of this study was to examine the ability of tumor necrosis factor alpha (TNFalpha, a proinflammatory cytokine) and interleukin (IL)-10 (an anti-inflammatory cytokine) to differentially regulate the sensitivity of human monocytes/macrophages to glucocorticoids. To accomplish this, we first analyzed the pattern of TNFalpha and IL-10 inhibition by dexamethasone in LPS-stimulated whole-blood cell cultures. Second, we studied the modulation of the sensitivity of these cells to dexamethasone by preincubation with TNFalpha or IL-10 and measurement of LPS-stimulated IL-6 secretion. In addition, we evaluated the effect of dexamethasone on phorbolmyristate-acetate-stimulated IL-1 receptor antagonist secretion by the human monocytic cell line U937. Finally, we investigated whether the modulation of corticosensitivity in TNFalpha- and IL-10-pretreated U937 cells was related to a change of the glucocorticoid receptor concentration and affinity. Dexamethasone had different effects on LPS-induced TNFalpha and IL-10 secretion; whereas it suppressed TNFalpha in a dose-dependent fashion, its effect on IL-10 secretion was biphasic, producing stimulation at lower, and inhibition at higher doses. The concentration of LPS employed influenced the effect of dexamethasone on IL-10 secretion (P < 0.001). Pretreatment with TNFalpha diminished, and with IL-10 improved, the ability of dexamethasone to suppress IL-6 secretion in whole-blood cell cultures (P < 0.01 for both) and to enhance IL-1 receptor antagonist secretion by U937 cells (P < 0.05 for both). TNFalpha decreased (P < 0.001), while IL-10 increased (P < 0.001), the concentration of dexamethasone binding sites in these cells, with no discernible effect on their binding affinity. We conclude that glucocorticoids differentially modulate TNFalpha and IL-10 secretion by human monocytes in a LPS dose-dependent fashion and that the sensitivity of these cells to glucocorticoids is altered by TNFalpha or IL-10 pretreatment; TNFalpha blocks their effects, whereas IL-10 acts synergistically with glucocorticoids. This is accompanied by opposite glucocorticoid receptor changes, respectively opposing and favoring glucocorticoid actions. This study suggests that the pattern of pro-/antiinflammatory cytokine secretion may alter the response of patients to glucocorticoid therapy.

Effects of tumour necrosis factor alpha and melphalan on the cytokine production of circulating T cells in patients with cancer.

The objective of the present study was to investigate the effects of isolated limb perfusion (ILP) with tumour necrosis factor alpha (TNF-alpha) and melphalan on circulating T cells from cancer patients using two different methods. Eight patients undergoing an ILP entered the study. At first, the number of T cells at several time points was determined using FACScan. Subsequently, production of interferon gamma (IFN-gamma) (T-helper 1) and interleukin (IL) 4 (T-helper 2) was measured at the intracytoplasmic level after stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. IFN-gamma, IL-4 and IL-2 (also T-helper 1) production in the whole-blood cell culture system was then determined after stimulation with a combination of anti-CD3/anti-CD28 monoclonal antibodies. An enormous decrease in the number of circulating T cells was observed. In the remaining T-cell population cytokine production (IL-2, IL-4, IFN-gamma) was depressed, showing the same pattern in both methods. No difference could be detected between the effect of TNF-alpha and melphalan on Th1 cells and Th2 cells. The results demonstrate that TNF-alpha and melphalan reduce the number of circulating T cells and at the single-cell level decrease cytokine production in the remaining circulating T cells. No selective effect of TNF-alpha on Th1 or Th2 cells could be detected. If the impaired T-cell function is representative of all T cells remaining in the systemic circulation, this could help to explain the tolerability of high TNF concentrations after ILP, perhaps by decreasing the synthesis and production of T-cell-derived cytokines.