Concentrations Of RANTES Research Articles

Thrombin generation and platelet activation related to subintimal percutaneous transluminal angioplasty

Objective: The purpose of this study was to measure the in vivo platelet activation and thrombin generation in arterial blood after passing a subintimal conduit. Methods: Subintimal percutaneous transluminal angioplasty (SPTA) is a technique where a subintimal channel is created, allowing recanalization of long peripheral arterial occlusion. From 10 patients with intermittent claudication, undergoing successful SPTA for femoropopliteal occlusive disease, we collected antecubital venous blood samples immediately before treatment, preprocedural arterial blood samples taken at the entry level proximal to the vessel occlusion, and subsequently at the reentry level after successful recanalization. Venous follow-up blood samples were taken after 24 hours. Plasma concentrations of β-thromboglobulin (β-TG), RANTES, and Prothrombin fragment (F1 + 2), were determined by immunoassay. Fibrinogen binding to platelets, leukocyte-platelet adhesion, and P-selectin were determined by flow cytometry. Results: We found a statistically significant transluminal increase in the plasma concentrations of RANTES, β-TG and F1 + 2 (p = 0.002, 0.001 and 0.001 respectively), which all normalized within 24 hours. Platelet-leukocyte aggregates significantly decreased after 24 hours compared with preprocedural and preentry levels (3.26% versus 5.26 %, p = 0.017). P-selectin expression on circulating platelets was statistically significantly increased in the blood sample taken at the re-entry level compared with the pre-procedural and pre-entry level (p = 0.007). After 24 hours there was no statistically significant difference to pre-procedural levels. There was no significant change in platelet fibrinogen binding at any levels. Conclusion: When passing a subintimal conduit, in vivo sampled blood demonstrated an extremely rapid and substantial uniform platelet activation and thrombin generation.

256 The JAK3-Inhibitor R348 Diminishes Acute and Chronic Rejection While Demonstrating Immune Cell Specifity

patients with CLAD (5 BOS and 3 RAS with no evidence of infection, median 24 months after bilateral lung transplantation) and 8 matched stable patients were included in this study. RAS was defined as CLAD (irreversible decline in forced expiratory volume in 1 second (FEV1 80% baseline)) with irreversible decline in total lung capacity (TLC 90% baseline). BOS was defined as CLAD without RAS. Cytokine expression was measured in BALF by Luminex-based multiplex cytokine array. Results: Median FEV1 and TLC to baseline were 98% and 100% in stable patients, 68% and 102% in BOS and 40% and 63% in RAS. CXCL1, 8, 9, 12 and HGF were upregulated in CLAD compared with stable patients. Strikingly, IL-6/IL-10 ratio and the concentration of RANTES was significantly elevated in RAS compared to BOS (p 0.025 in both), while lymphotoxinwas upregulated in BOS (p 0.025).

The high level of RANTES in the ectopic milieu recruits macrophages and induces their tolerance in progression of endometriosis

RANTES (C-C chemokine, regulated on activation, normal T cell expressed and secreted) is involved in progression of endometriosis, but the precise mechanism is understood inadequately. This study is to elucidate the roles of RANTES in macrophage recruitment and tolerance in the endometriotic milieu. The expression of RANTES was analyzed by immunohistochemistry. The cell co-cultures were applied to simulate the endometriotic milieu to investigate the regulation of RANTES secretion and its receptor CCR1 expression. Transwell migration assay was used for chemotaxis of U937 cells (macrophage line) to endometrial stromal cells (ESCs) and/or human pelvic mesothelial cells. The expression of CCR1 was analyzed by RT-PCR and qPCR in transcription and by western blot in translation respectively. Concentrations of RANTES, IL10, and IL12p70 were determined by ELISA. The phenotype of U937 cells and apoptosis of ESCs were analyzed by flow cytometry. We have found that the expression of RANTES is significantly higher in the endometriotic tissue and eutopic endometrium than that of the normal endometrium without endometriosis. The combination of 17β-estradiol and dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin increases significantly RANTES secretion in the endometriosis-associated cell co-culture which can recruit more macrophages, upregulate CCR1 expression, and induce tolerant phenotype, which inhibits the apoptosis of ESC in the milieu. In conclusion, the higher levels of RANTES in the ectopic milieu facilitate the onset and progression of endometriosis by macrophage recruitment and tolerance that in turn inhibits apoptosis and enhances growth of ESC.

RANTES in Exhaled Breath Condensate of Patients with Severe Persistent Allergic Asthma during Omalizumab Therapy

Background: Omalizumab is a humanized monoclonal anti-IgE antibody developed for the treatment of IgE-mediated diseases, including asthma. The aim of the study was to determine the effect of omalizumab treatment on changes in RANTES in exhaled breath condensate and other inflammatory markers in patients with persistent severe asthma. Methods: The study was conducted on a group of 19 patients with severe persistent allergic asthma treated with conventional therapy (according to GINA 2006) and with or without omalizumab (9 vs. 10 patients). Changes in inflammatory parameters [RANTES in exhaled breath condensate, exhaled nitric oxide, blood eosinophil count and serum eosinophil cationic protein (ECP)] were measured before and after 16 weeks of therapy. Results: Omalizumab-treated patients showed a statistically significant decrease in the concentrations of RANTES in exhaled breath condensate, exhaled nitric oxide (F_ENO), serum ECP, and blood eosinophil count compared with patients with conventional therapy after 16 weeks of treatment. In this group of patients, statistically significant correlations were revealed between the decrease in RANTES and a decrease in F_ENO and between the decrease in F_ENO and a decrease in ECP or blood eosinophil count after omalizumab therapy. Conclusions: Our results confirmed that during anti-immunoglobulin E therapy with omalizumab in patients with severe persistent allergic asthma, RANTES expression is decreased. This process in turn could lead to a limitation of airway inflammation and could be essential for the beneficial effect of anti-IgE therapy with omalizumab.

RANTES in Exhaled Breath Condensate of Allergic Asthma Patients with Exercise-Induced Bronchoconstriction

Background: The response of asthmatics to exercise differs from that of healthy subjects, and the mechanisms responsible for exercise-induced bronchoconstriction (EIB) remain to be elucidated. Objectives: The aim of this study was to evaluate changes in RANTES levels in exhaled breath condensate (EBC) following intensive exercise in allergic asthmatics. Methods: The study was conducted in a group of 19 asthmatics (11 with EIB and 8 without EIB) and 7 healthy volunteers. Changes in the concentrations of RANTES in EBC induced during the 24 h after intensive exercise were determined. Moreover, these measurements were tested for possible correlations with the results of other tests commonly associated with asthma as well as with changes in airway inflammation after exercise. Results: In contrast to asthmatic patients without EIB and healthy controls, in asthmatics with EIB RANTES concentrations were statistically significantly increased in EBC collected during the first 24 h after an exercise test. There was a statistically significant correlation between the maximum increase in RANTES concentrations in EBC after exercise and either baseline exhaled nitric oxide (F_ENO) or bronchial hyperreactivity to histamine and an increase in serum eosinophil cationic protein or F_ENO 24 h after exercise in the EIB asthmatics. Conclusions: The increase in RANTES in asthmatic airways, promoting the migration and activation of inflammatory cells including eosinophils, may play an important role in the upregulation of airway inflammation after EIB in asthmatic patients.

A distinct inflammatory marker pattern in patients with aromatase inhibitor (AI)-induced musculoskeletal symptoms.

Abstract Abstract #1129 Background. AI-associated musculoskeletal symptoms (AIMSS) have been reported in up to 50% of treated patients. Inflammatory processes can occur in cancer patients, and inflammatory mediators including interleukins, interferons, MMPs and immunoregulatory molecules have been associated with estrogen signaling. Ceramide and sphingosine-1-phosphate (S1P) are lipid mediators with roles in inflammation. We hypothesized that AIMSS may develop in the context of increased circulating inflammatory markers.
 Methods. Cases (n=24) were selected from 150 early stage ER-positive breast cancer patients who were enrolled on a prospective multicenter randomized trial of exemestane or letrozole. Changes in pain and function during therapy were assessed prospectively, and those patients whose symptoms exceeded a predefined threshold were referred for detailed rheumatologic evaluation. Controls (n=30), who did not develop AIMSS that exceeded the threshold, were matched for age, ethnicity, and prior chemotherapy. Serum samples collected at baseline and 1 and 6 mo following AI initiation were assayed in a blinded manner. The majority of inflammatory molecules were quantified in serum using an addressable laser bead multiplex assay (BMD, Paris) on a Luminex S100 analyzer (Austin, TX). Ceramide and S1P were alkaline chloroform/methanol-extracted from serum, 32P-labeled, and quantified using autoradiography. Statistical comparisons between cases and controls for each factor were performed using the non-parametric Mann-Whitney test or by log transforming the data and using unpaired two-tailed t tests.
 Results. The mean serum concentrations for the majority of assayed factors (IFNγ, IL1Rα, IL2/4/5/7/10/15/17, IL2R, IL12p40, IP10, MIP1α, MIG, bFGF, HGF, GCSF, and GMCSF) were significantly lower at baseline in cases compared to controls (p<0.05 for each factor); no differences in changes relative to baseline were evident at 1 and 6 mo of AI therapy. Baseline serum concentrations of RANTES, a chemokine, were higher for cases than controls (p=0.04); no differences relative to baseline were noted during therapy. At the 6 mo timepoint, serum concentrations of S1P were higher in cases and lower in controls (p=0.01); no differences were found at baseline or 1 month. No associations were found between serum concentrations of EGF, IL6, IL13, MCP1, MMP3/9/13, or ceramide and the development of AIMSS at any timepoint.
 Conclusions. Subjects who develop AIMSS appear to have an underlying anergic cytokine phenotype at baseline. During AI therapy, patients with AIMSS develop higher levels of S1P, a potential inflammatory mediator known to induce COX2 and NFκB. These hypothesis-generating results suggest a potential mechanism underlying the development of AIMSS, distinct from other rheumatologic conditions. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1129.

Augmentation of Angiogenic Structures by Umbilical Cord Blood-Derived Cells

The revascularization of patients with ischemic conditions is a major obstacle in medicine. Umbilical cord blood (UCB) holds revascularization potential as a source of stem cells and progenitor cells, particularly those designated by the surface markers CD133 and CD34. Human umbilical vein endothelial cells (HUVEC) have been used to study the formation of angiogenic structures. These structures result in networks characterized by nodes with two or more branch points. Our hypothesis is that the effector cells (UCB-derived CD133/34 pos cells) have a proangiogenic effect on the growth of HUVEC by a paracrine mechanism rather than integration into the network of angiogenic structures.When cultured on the gelatinous protein matrix Growth Factor Reduced Matrigel (GFR MG), HUVEC form a network of branching structures. Matrigel with reduced growth factors was used because we wanted to assess the role of growth factors released by effector cells. Growth factors in GFR MG include bFGF (0–0.1 pg/mL), EGF (<0.5 ng/mL), IGF-1 (5 ng/mL), PDGF (<5 pg/mL), NGF (<0.2 ng/mL), and TGF-b (1.7 ng/mL). A co-culture assay of HUVEC and CD133/34 pos cells on GFR MG allowed the measurement of the kinetics, magnitude, and persistence of structure growth. Because we wanted to mimic an ischemic bed, experiments were performed at 1% O2 in a PRO OX chamber (BioSpherix). To determine if cell-to-cell contact is required for augmentation of angiogenesis, Transwell experiments with CD133/34 pos cells suspended above HUVEC cultured on Matrigel were conducted. The pore size of 0.3 μm in the Tranwells did not permit cell penetration. In addition, levels of growth factors were measured by a Luminex assay for the factors hFGF, hHGF, hINF-g, hMIP-1a, hMIP-1a, hIL-8, hRANTES, hTNF-a, and hVEGF. Cells were cultured at 2 × 106 cells/mL for 24 hrs and media was collected for analysis.The number of branch points or nodes (a quantitative measurement of angiogenesis), was greater when magnetically-selected and fluorescence-activated cell sorted UCB-derived CD133/34 positive (pos) cells are added to the HUVEC culture on Matrigel than when HUVEC were cultured alone. When the CD133 negative (neg) cells from the cord blood are cultured with HUVEC, the number of nodes also increased at the first time at 16 ± 2 hours. The presence of both UCB-derived CD133/34 pos and CD133/34 neg cells also causes the nodes to persist longer than HUVECs alone, while the CD133/34 neg fraction allows the structures to persist to a greater degree than the CD133/34 pos fraction when followed for three days (Table).Day 1Day 2Day 3Node #% TotalNode #% TotalNode #% TotalHUVEC alone11.6 ± 4.91001.3 ± 1.159.20.33 ± 0.582.2HUVEC + HSC CD133/34 pos17.3 ± 9.21006.3 ± 4.535.34.7 ± 5.521.4HUVEC + HSC CD133/34 neg34 ± 13.210019.7 ± 2.561.610 ± 3.629.8Table: “Node #” refers to the mean node count for each day and culture type. “% Total” refers to the percentage of nodes remaining each day after the first count occurring on Day 1. Thus, all Day 1 totals are 100%.Importantly, in Transwell experiments, the number of nodes formed did not differ between co-cultures of HUVEC and CD133/34 pos cells compared to those with only HUVEC in the bottom chambers. Direct cell-to-cell contact between HUVEC and CD133 pos does not appear to be required for augmentation to occur but involves released factor (see Figure 1-Transwell Nodes).Two pro-angiogenic factors that are released by UCB-derived cells were identified from the Luminex assay that may be key players in the augmentation of the angiogenic structures—IL-8 and RANTES. Other measured factors were not produced at levels over the sensitivity of the assay. Measured cytokine concentrations for IL-8 were 3332 ± 498 pg/mL for CB CD133/34 pos cells and 7257 ± 1562 pg/mL for CB MNC. RANTES concentrations were 695 ± 183 pg/mL for CB CD133/34 pos cells and 1310 ± 75 pg/mL for CB MNC. Results of these assays demonstrate that the formation of angiogenic structures in a co-culture assay with HUVEC and UCB-derived cells is augmented by the effector cells releasing key factors, including IL-8 and RANTES. Although the CD133/34 neg (MNC) population holds potential in its greater cytokines production and greater ability to augment HUVEC structure formation, selected CD133/34 pos cells, known to illicit less immune responses, provide a significant level of augmentation of HUVEC structures. [Display omitted]

Changes in RANTES and β-Thromboglobulin after Intensive Exercise in Patients with Allergic Asthma

Background: There is increasing evidence that exercise-induced bronchoconstriction (EIB) is associated with eosinophilic airway inflammation. In the pathogenesis of EIB the role of chemokines – responsible for promoting the migration and activation of inflammatory cells – as well as blood platelets, a potential source of those chemokines, remains unclear. Methods: The study was conducted in a group of 19 asthmatics (11 with EIB, 8 without EIB) and 8 healthy volunteers. Changes in the plasma concentrations of RANTES and β-thromboglobulin (β-TG) induced by intensive exercise were determined. Moreover, the possible correlation of these measurements with the results of other tests used in the diagnosis of asthma as well as laboratory tests commonly associated with asthma were investigated. Results: A comparison of the concentrations of β-TG in all groups studied at rest did not reveal any significant differences. In all groups studied, 30 min after exercise elevated β-TG concentrations were observed; the most significant increase was revealed in asthmatics with EIB. The baseline concentrations of RANTES before exercise in both groups of asthmatics were significantly higher in comparison to the group of healthy volunteers. After exercise, in the group of patients with EIB, a significant increase in RANTES concentrations was observed. These changes correlated with an increase in other markers of airway inflammation 24 h after exercise. Conclusions: We suggest that platelet activation, resulting in elevated RANTES release, could be one of the factors responsible for the increase of airway inflammation observed in consequence of EIB in asthmatics.

RANTES in exhaled breath condensate of stable and unstable asthma patients

RANTES has been implicated in the allergic inflammation of asthma by promoting the migration and activation of the inflammatory cells, including eosinophils. The study was undertaken to evaluate RANTES levels in the exhaled breath condensate (EBC) of asthmatics with different degrees of asthma severity. EBC was collected from 33 patients with allergic asthma (11 with steroid-naïve mild asthma, 10 with ICS-treated, stable mild-to-moderate asthma, 12 with ICS-treated unstable, severe asthma) and seven healthy volunteers. In the three groups of asthmatics, RANTES concentrations in EBC were significantly higher compared with healthy volunteers. RANTES levels were significantly higher in patients with unstable asthma than in the two groups with stable disease. We observed statistically significant correlations between the concentrations of RANTES in EBC and F(ENO) in the three studied groups of asthmatics; notably, the correlation between the parameters described above was strong positive in the group of unstable and steroid-naïve stable asthmatics. We also discovered a significantly positive correlation between RANTES in EBC and the serum ECP or blood eosinophil count in the groups of asthmatics with severe, unstable asthma and between RANTES and serum ECP in the group of steroid-naïve stable asthmatics. Measurements of RANTES in EBC may provide another useful diagnostic tool for detecting and monitoring inflammation in patients with asthma.

Colifagina, a Novel Preparation of 8 Lysed Bacteria Ameliorates Experimental Colitis

Immune reactivity towards the bacterial intestinal flora plays an important part in the pathogenesis of inflammatory bowel disease. Administration of probiotic bacteria has beneficial effects on infectious and inflammatory diseases, principally in bowel disorders. However, little is known about the administration of soluble bacterial antigens in intestinal inflammation. We investigated the therapeutic effects of colifagina in experimental colitis. To assess this effect, C57BL/6 mice with dextran sulphate sodium-induced colitis were treated with colifagina, or with a placebo, for a period of 10 days. The mice were monitored, and inflammation was assessed by disease activity index (DAI). Analysis of fecal IgA concentration and measurement of IgA and inflammatory chemokine production in organ colonic culture was performed by ELISA. Clinically and histologically, bacterial-lysate-treated mice revealed significantly fewer DAI and a reduction of colonic histological inflammation. Treatment of healthy mice with colifagina significantly increased the fecal concentration of IgA and IgA production in organ culture. Colifagina administration in DSS-treated mice significantly increased the fecal concentration of IgA and IgA production in organ culture. MIP-1, MIP-2 and RANTES concentrations in colonic organ culture were significantly lower in colifagina-treated mice than in the placebo group. The use of colifagina is effective in amelioration of murine colitis.

Expression of AIF-1 and RANTES in Unexplained Spontaneous Abortion and Possible Association with Alloimmune Abortion

Objective To investigate the effects of allograft inflammatory factor-1 (AIF-1) and (RANTES) in sera and deciduas on unexplained early spontaneous abortion. Methods AIF-1 and RANTES were examined in sera and deciduas/endometria of 43 unexplained early spontaneous abortion women (group A), 40 healthy women with early pregnancy(group B) and 20 healthy women with no pregnancy (group C). Immunohistochemistry and enzyme linked immunosorbent assay (ELISA) were used in this study. Results AIF-1 protein was expressed both in deciduas of group A and in endometria of group C. In group A, H scores in the recurrent abortion deciduas specimens were significantly greater than those in the first abortion;in endometrium, expression of AIF-1 was greater in the secretory than in proliferative phase of group C. In group B, concentrations of RANTES in sera were higher in 7th-8th week of pregnancy than in 6th-7th and >8th week of pregnancy; expression of AIF-1 protein showed a negative correlation with RASNTES concentration; a significant increase of the RANTES levels in sera and tissue was observed in group B. Conclusion These results demonstrate, for the first time, that AIF-1 are expressed in deciduas of unexplained spontaneous abortion suggesting that AIF-1 involve in alloimmune abortion; RANTES might act as a novel blocking antibody;AIF-1 and RANTES might act as reliable markers for diagnosis of early alloimmune abortion.

Storage of buffy coat–derived platelets in additive solutions: in vitro effects of storage at 4°C

The aims of this in vitro study were to compare the storage of platelets (PLTs) at 4 degrees C with those stored at 22 degrees C and to determine the in vitro effects of preincubation at 37 degrees C for 1 hour before the analysis on the basis of the maintenance of PLT metabolic and cellular integrity. PLT concentrates (PCs) were prepared from pooled buffy coats (BCs) for paired studies (total eight pools from 160 BCs). Each pool was divided into four PCs and stored under different conditions: at 20 to 24 degrees C on a flatbed agitator, at 20 to 24 degrees C on a flatbed agitator and with incubation of the samples at 37 degrees C for 1 hour before the analysis, at 4 degrees C, and at 4 degrees C and with incubation of the samples at 37 degrees C for 1 hour before the analysis. Storage of PLTs at 4 degrees C resulted in reductions in the rate of glycolysis and better retention of pH after Day 10 than in PCs stored at 22 degrees C (Day 14, 7.003 +/- 0.047 vs. 7.201 +/- 0.146). Hypotonic shock response and extent of shape change were higher at 22 degrees C than at 4 degrees C and in preincubated PCs stored at 22 degrees C than in reference PCs stored at the same temperature (Day 5, 45.6 +/- 2.7 vs. 36.5 +/- 3.9 and 24.1 +/- 2.0 vs. 15.5 +/- 1.8). The concentration of RANTES was higher in PCs stored at 22 degrees C than at 4 degrees C (Day 7, 179 +/- 25 vs. 79 +/- 32). PLTs stored at 4 degrees C without agitation maintain metabolic and cellular characteristics to a great extent during 21 days of storage. These studies confirm the view that PLTs lose their discoid shape and that this loss with storage at 4 degrees C is associated with reductions in metabolic rate and in their release of alpha-granule content.

Transforming growth factor beta 1(TGF-β1) down-regulates TNFα-induced RANTES production in rheumatoid synovial fibroblasts through NF-κB-mediated transcriptional repression

Transforming growth factor (TGF)-β1 is a pleiotropic cytokine with many functions, including those related to growth modulation, immunosuppression, and pro-inflammation, in a wide variety of cell types. In this study, we investigated the ability of TGF-β1 to regulate RANTES production by activated rheumatoid synovial fibroblasts. Fibroblast-like synoviocytes (FLS) were cultured in the presence of TGF-β1 and IL-1β, IL-15, TNFα, or IL-17, and the secretion of RANTES into culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). Expression of RANTES encoded mRNA was determined by reverse transcription-polymerase chain reaction (RT-PCR), and NF-κB binding activity for RANTES transcription was determined by electrophoretic mobility shift assay (EMSA). We found that the concentrations of RANTES in synovial fluid (SF) from rheumatoid arthritis (RA) patients were lower than in SF from osteoarthritis (OA) patients, whereas the concentrations of TGF-β1 were higher in RA SF than in OA SF. TGF-β1 dose-dependently inhibited TNFα-induced production of RANTES protein and mRNA from RA FLS. Addition of RA SF with high-level TGF-β1 mimicked the effect of TGF-β1 on TNFα-induced RANTES production, which was inhibited by treatment with anti-TGF-β1 neutralizing antibody. TGF-β1 blocked the degradation of cytosolic IκB-α and the translocation of activated NF-κB to the nucleus. EMSA showed that the inhibitory effect of TGF-β1 was associated with decreased binding of NF-κB to the RANTES promoter. These results suggest that elevated TGF-β1 in rheumatoid synovial tissue may suppress joint inflammation by inhibiting RANTES secretion from synovial fibroblasts, thus blocking the infiltration of immune cells. These findings may provide an explanation for the mechanism by which TGF-β1 regulates immune function in RA.

HIV‐1 Infection Is Associated with Altered Innate Pulmonary Immunity

We obtained bronchoalveolar lavage (BAL) fluid from 45 Malawian adults, to measure the concentrations of innate pulmonary immune factors that are important in lung defense against infection. Increased concentrations of the beta -chemokine RANTES were found in BAL fluid from the human immunodeficiency virus (HIV)-1-infected subjects, compared with those in BAL fluid from the HIV-1-uninfected control subjects (mean, 86 pg/mL vs. 0 pg/mL; P<.001). Lysozyme concentrations were also elevated in the HIV-1-infected subjects, compared with those in the HIV-1-uninfected control subjects (1.9 mu g/mL vs. 1.1 mu g/mL; P=.03), but were not elevated in the HIV-1-infected subjects who had recently recovered from invasive pneumococcal disease. Concentrations of lactoferrin and secretory leukocyte protease inhibitor (SLPI) were not different when the subjects were compared by HIV-1 serostatus. Concentrations of RANTES (R2=0.68 and P<.0001) and SLPI (R2=0.29 and P=.001) correlated with BAL fluid HIV-1 load but not with plasma HIV-1 load.

Interleukin-11 and IL-17 and the pathogenesis of periodontal disease.

Interleukin (IL)-11 and IL-17 are cytokines that modulate the inflammatory process and have not been assessed within normal or inflamed gingival tissues. Our purpose was to compare concentrations of human IL-11 and IL-17 within healthy and diseased human gingiva to determine their possible role in the initiation or progression of periodontal diseases. Biopsies from healthy (non-hemorrhagic gingiva adjacent to a < or = 3 mm gingival sulcus) and diseased gingiva (hemorrhagic gingiva adjacent to a > or = 3 mm periodontal pocket) were studied. IL-11, IL-17, RANTES, and IL-6 concentrations were assessed within solubilized gingival biopsies by enzyme-linked immunosorbent assay. Data were compared by factorial analysis of variance and a post-hoc Tukey honestly significant difference (HSD) test. Regression analysis and partial correlation analysis (adjusted for sample weight) were also used to determine correlations between the variables. Interleukin-11 concentrations were highest within gingiva adjacent to 3 mm diseased pockets (P < 0.001), and IL-17 concentrations were highest at 4 to 5 mm sites compared to other sites (P < 0.001). Gingival concentrations of both cytokines were significantly lower in gingiva adjacent to a > or = 6 mm pocket. RANTES concentrations were significantly greater in gingiva adjacent to > or = 6 mm pockets than in tissues derived from other sites (P < 0.001). IL-11, IL-6, and RANTES concentrations were significantly correlated with sulcular depth. Gingival concentrations of IL-11 and IL-17 are different in diseased gingiva adjacent to 3, 4 to 5, and > or = 6 mm pockets, suggesting that their concentrations change as a consequence of the progression of gingivitis to periodontitis and that both cytokines could have a significant role in this progression. These data may be useful for the design of procedures for prevention of periodontal disease.

Storage of platelets in additive solutions: the effects of magnesium and potassium on the release of RANTES, beta-thromboglobulin, platelet factor 4 and interleukin-7, during storage.

Several studies have suggested that the accumulation of cytokines during storage of platelet concentrates may mediate non-haemolytic transfusion reactions. Prestorage leucodepletion can prevent the release of cytokines from white blood cells during storage, but not the release of platelet-derived cytokines. Therefore, we investigated whether the addition of magnesium and potassium to platelets stored in a platelet additive solution (PAS) would affect the generation of cytokines during platelet storage. Platelets were prepared from buffy coats using different suspension media: plasma; 70% PAS-III + 30% plasma; 70% PAS-III supplemented with magnesium and potassium +30% plasma; and 80% PAS-III supplemented with magnesium and potassium +20% plasma. The levels of certain cytokines--regulated on activation, normal, T-cell expressed, and secreted (RANTES), beta-thromboglobulin (beta-TG), platelet factor 4 (PF4) and interleukin-7 (IL-7)--were measured by enzyme-linked immunosorbent assay (ELISA) on days 1, 5 and 7. The concentrations of RANTES, beta-TG, PF4 and IL-7 increased, during storage, in all units. The increase was significantly greater in units stored in 70% PAS-III +30% plasma than in the other three suspension media. The storage of platelets in 70% PAS-III supplemented with magnesium and potassium +30% plasma significantly reduced the concentrations of platelet derived-cytokines during storage, as compared to platelets stored in 70% PAS-III + 30% plasma alone. The concentrations of platelet-derived cytokines increased, to a significantly greater extent, when platelets were stored in PAS-III than in plasma. However, when magnesium and potassium were added to PAS-III, the concentrations of platelet-derived cytokines obtained during storage were about the same as those produced by platelets stored in plasma.

CCR5 genotype and plasma beta-chemokine concentration of Brazilian HIV-infected individuals.

The 32-bp deletion in the HIV-1 co-receptor CCR5 confers a high degree of resistance to HIV-1 infection in homozygous individuals for the deleted allele and partial protection against HIV-1 during disease progression in heterozygotes. Natural ligands for CCR5, MIP-1alpha, MIP-1beta and RANTES, have been shown to inhibit HIV replication in CD4+ T cells. In the present study, we examined the CCR5 genotype by PCR and the plasma levels of RANTES and MIP-1alpha by ELISA among blood donors (N = 26) and among HIV-1-infected individuals (N = 129). The control group consisted of healthy adult volunteers and HIV-1-infected subjects were an asymptomatic and heterogeneous group of individuals with regard to immunologic and virologic markers of HIV-1 disease. The frequency of the CCR5 mutant allele (Delta32ccr5) in this population was 0.032; however, no Delta32ccr5 homozygote was detected. These results could be related to the intense ethnic admixture of the Brazilian population. There was no correlation between circulating beta-chemokines (MIP-1alpha, RANTES) and viral load in HIV-infected individuals. RANTES concentrations in plasma samples from HIV+ patients carrying the homozygous CCR5 allele (CCR5/CCR5) (28.23 ng/ml) were higher than in the control samples (16.07 ng/ml; P<0.05); however, this HIV+ patient group (mean 26.23 pg/ml) had significantly lower concentrations of MIP-1alpha than those observed in control samples (mean 31.20 pg/ml; P<0.05). Both HIV-1-infected and uninfected individuals heterozygous for the Delta32ccr5 allele had significantly lower concentrations of circulating RANTES (mean 16.07 and 6.11 ng/ml, respectively) than CCR5/CCR5 individuals (mean 28.23 and 16.07 ng/ml, respectively; P<0.05). These findings suggest that the CCR5 allele and beta-chemokine production may affect the immunopathogenesis of HIV-1.

P38 mitogen-activated protein kinase and c-Jun-NH2-terminal kinase regulate interleukin-8 and RANTES production in hyperosmolarity stimulated human bronchial epithelial cells.

We have previously shown that p38 mitogen-activated protein kinase (MAPK) regulates, at least in part, hyperosmolarity induced interleukin (IL)-8 expression in human bronchial epithelial cells (BEC). In the previous study, hyperosmolarity also activated c-Jun-NH2-terminal kinase (JNK); however, the role of the JNK signalling pathway has not been determined. In the present study, we examined the role of the JNK signalling pathway in hyperosmolarity induced IL-8 and RANTES production by BEC using the novel inhibitor of the JNK signalling pathway CEP 11004 in order to clarify these issues. Bronchial epithelial cells that had been pre-incubated with SB 203580, CEP 11004 or a combination of these were exposed to a hyperosmolar medium and then the p38 MAPK and JNK phosphorylation activity in these cells and IL-8 and RANTES concentrations in the culture supernatants were determined. The results showed that: (i) hyperosmolarity induced the threonine and tyrosine phosphorylation of p38 MAPK and JNK; (ii) SB 203580, as the specific inhibitor of p38 MAPK activity, and CEP 11004 attenuated hyperosmolarity induced p38 MAPK and JNK activity, respectively; (iii) SB 203580 and CEP 11004, but not PD 98059, partially attenuated IL-8 and RANTES production; and (iv) a combination of SB 203580 and CEP 11004 attenuated IL-8 and RANTES production in an additive fashion. These results indicate that p38 MAPK and the JNK pathway regulate hyperosmolarity induced IL-8 and RANTES production by BEC.

Inhalant corticosteroids inhibit mechanical strain-induced RANTES and eotaxin production by human airway smooth muscle cells

RANTES and eotaxin play an important role in the production of allergic inflammation of the airway through a chemotactic activity for eosinophils. Airway smooth muscle (ASM) cells are known to produce these cytokines in response to pro-inflammatory stimuli, such as tumor necrosis factor (TNF)-α; however, it has not been determined whether mechanical strain could induce cytokine production by ASM cells. In addition, in the present study we also examined the effect of inhalant corticosteroids, namely fluticasone propionate (FP) and budesonide (BUD), on RANTES and eotaxin production. To clarify these issues, human ASM cells cultured on silicone culture rubber dishes that had been incubated with or without inhalant corticosteroids were stretched or compressed. The ASM cells were simultaneously stimulated with TNF-α. The concentration of RANTES and eotaxin in the culture supernatants was determined. The results showed that: (i) mechanical strain, including stretch and compression, stimulated ASM cells to produce RANTES and eotaxin; and (ii) FP and BUD inhibited mechanical strain-induced and TNF-α-stimulated RANTES and eotaxin production by ASM cells. These results indicate that mechanical strain is capable of inducing RANTES and eotaxin production by ASM cells and that inhalant corticosteroids effectively inhibit RANTES and eotaxin production by mechanical strain-loaded ASM cells, as well as by TNF-α-stimulated cells. Mechanical strain and inflammatory stimuli are capable of inducing chemokine production; therefore, suppressing the contraction of, as well as chemokine production by, ASM cells is important for controlling the production and the progression of allergic inflammation.

Amantadine inhibits RANTES production by influenzavirus-infected human bronchial epithelial cells.

1. Amantadine can prevent and decrease airway inflammation by inhibiting influenza virus (IV) replication; however, the effect of amantadine on RANTES production by human bronchial epithelial cells (BEC) has not been determined. In the present study, we examined the effect of amantadine on RANTES production and also analysed p38 mitogen-activated protein (MAP) kinase and c-Jun-NH2-terminal kinase (JNK) activation to clarify the mechanism in the effect of amantadine on RANTES production, since we have previously shown that p38 MAP kinase and JNK regulate RANTES production by IV-infected BEC. 2. BEC that had been preincubated with amantadine were infected with IV and then p38 MAP kinase and JNK activation in the cells and RANTES concentrations in the culture supernatants were determined. 3. Amantadine-induced inhibition of virus replication resulted in a decrease in p38 MAP kinase and JNK activity and decreased expression of RANTES in IV-infected cells. 4. Amantadine did not inhibit p38 MAP kinase and JNK activation induced by tumour necrosis factor-alpha (TNF-alpha) as a non-viral stimulus. 5. These results indicate that amantadine inhibits IV infection-induced RANTES production by human BEC and that the inhibition by amantadine of RANTES production might result from an indirect inhibitory effect of amantadine on p38 MAP kinase and JNK activation via the inhibition of virus replication, and we emphasize that amantadine may produce a beneficial effect on controlling bronchial asthma exacerbation caused by IV infection.