IL-7 and IL-15 Levels Reflect the Degree of T Cell Depletion during Lymphopenia and Are Associated with an Expansion of Effector Memory T Cells after Pediatric Hematopoietic Stem Cell Transplantation.

Differentially and functionally distinct T cell subsets are involved in the development of complications after allogeneic hematopoietic stem cell transplantation (HSCT), but little is known about factors regulating their recovery after HSCT. In this study, we investigated associations between immune-regulating cytokines, T cell differentiation, and clinical outcomes. We included 80 children undergoing allogeneic HSCT for acute leukemia using bone marrow or peripheral blood stem cells grafted from a matched sibling or unrelated donor. Cytokines (IL-7, IL-15, IL-18, SCF, IL-6, IL-2, and TNF-α) and active anti-thymocyte globulin (ATG) levels were longitudinally measured along with extended T cell phenotyping. The cytokine profiles showed a temporary rise in IL-7 and IL-15 during lymphopenia, which was strongly dependent on exposure to active ATG. High levels of IL-7 and IL-15 from graft infusion to day +30 were predictive of slower T cell recovery during the first 2 mo post-HSCT; however, because of a major expansion of memory T cell stages, only naive T cells remained decreased after 3 mo (p < 0.05). No differential effect was seen on polarization of CD4+ T cells into Th1, Th2, or Th17 cells or regulatory T cells. Low levels of IL-7 and IL-15 at day +14 were associated with acute graft-versus-host disease grades II-IV in ATG-treated patients (p = 0.0004 and p = 0.0002, respectively). Children with IL-7 levels comparable to healthy controls at day +14 post-HSCT were less likely to develop EBV reactivation posttransplant. These findings suggest that quantification of IL-7 and IL-15 may be useful as biomarkers in assessing the overall T cell depletion and suggest a potential for predicting complications after HSCT.

Local and Systemic IL-7 Concentration in Gastrointestinal-Tract Cancers.

BACKGROUND AND OBJECTIVES: Interleukin-7 (IL-7) is exploited in cancer immunotherapies although its status in solid tumors is largely unknown. We aimed to determine its systemic and local concentrations in esophageal (EC), gastric (GC), and colorectal (CRC) cancers. MATERIALS AND METHODS: IL-7 was immunoenzymatically measured in paired surgical specimens of tumors and tumor-adjacent tissue (n = 48), and in the sera of 170 individuals (54 controls and 116 cancer patients). Results: IL-7 was higher in tumors as compared to noncancerous tissue in all cancers (mean difference: 29.5 pg/g). The expression ratio (tumor to normal) was 4.4-fold in GC, 2.2-fold in EC, and 1.7-fold in CRC. However, when absolute concentrations were compared, the highest IL-7 concentrations were in CRC, both when tumor and noncancerous tissue were analyzed. In CRC tumors, IL-7 was 2 and 1.5 times higher than in EC and GC tumors. In noncancerous CRC tissue, IL-7 was 2.3- and 2.8-fold higher than in EC and GC. IL-7 overexpression was more pronounced in Stage 3/4 and N1 cancers as a result of decreased cytokine expression in noncancerous tissue. Tumor location was a key factor in determining both local and systemic IL-7 concentrations. Serum IL-7 in CRC and EC was higher than in controls, GC, and patients with adenocarcinoma of gastric cardia (CC), but no significant correlation with the disease advancement could be observed. Conclusions: IL-7 protein is overexpressed in EC, GC, and CRC, but concentrations differ both in tumor and tumor-adjacent tissue with respect to tumor location. More advanced cancers have lower IL-7 concentrations in the immediate environment of the tumor. At the systemic level, IL-7 is elevated in CRC and EC, but not CC or GC. IL-7 dependence on the location of the primary tumor should be taken into account in future IL-7-based immunotherapies. Functional studies explaining a role of IL-7 in gastrointestinal cancers are needed.

Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by aligners: a pilot study.

INTRODUCTION: The search for more aesthetic and comfortable orthodontic devices has led to an increase in the use of clear aligners. OBJECTIVE: To increase knowledge on biological mechanisms of orthodontic tooth movement using Invisalign aligners. METHODS: This study included 11 patients with a mean age of 23.6 ± 4.8 years. Cases planning included alignment and leveling of lower incisors using Invisalign aligners. Gingival crevicular fluid samples were collected from the lower incisors on the day of delivery of aligner number 1 (T0) and after 1 (T24h), 7 (T7d), and 21 (T21d) days. During the observation period of the study, the patients used only the aligner number 1. Levels of nine cytokines were quantified using Luminex's multi-analysis technology. Non-parametric tests were used for comparisons between cytokine expression levels over time. RESULTS: Cytokine expression levels remained constant after 21 days of orthodontic activation, except those of MIP-1ß, which presented a statistical difference between T24h and T21d with a decrease in the concentration levels. IL-8, GM-CSF, IL-1ß, MIP-1ß, and TNF-α showed the highest concentrations over time. CONCLUSIONS: The different behavior in the levels of the investigated cytokines indicates a role of these biomarkers in the tissue remodeling induced by Invisalign.

Expression of cytokines in gingival crevicular fluid associated with tooth movement induced by aligners: a pilot study

ABSTRACT Introduction: The search for more aesthetic and comfortable orthodontic devices has led to an increase in the use of clear aligners. Objective: To increase knowledge on biological mechanisms of orthodontic tooth movement using Invisalign aligners. Methods: This study included 11 patients with a mean age of 23.6 ± 4.8 years. Cases planning included alignment and leveling of lower incisors using Invisalign aligners. Gingival crevicular fluid samples were collected from the lower incisors on the day of delivery of aligner number 1 (T0) and after 1 (T24h), 7 (T7d), and 21 (T21d) days. During the observation period of the study, the patients used only the aligner number 1. Levels of nine cytokines were quantified using Luminex's multi-analysis technology. Non-parametric tests were used for comparisons between cytokine expression levels over time. Results: Cytokine expression levels remained constant after 21 days of orthodontic activation, except those of MIP-1β, which presented a statistical difference between T24h and T21d with a decrease in the concentration levels. IL-8, GM-CSF, IL-1β, MIP-1β, and TNF-α showed the highest concentrations over time. Conclusions: The different behavior in the levels of the investigated cytokines indicates a role of these biomarkers in the tissue remodeling induced by Invisalign.

RESUMO Introdução: a busca por dispositivos ortodônticos mais estéticos e confortáveis gerou um aumento no uso de alinhadores transparentes. Objetivo: ampliar o conhecimento sobre os mecanismos biológicos associados ao movimento dentário ortodôntico promovido por alinhadores Invisalign®. Métodos: a amostra foi constituída por 11 pacientes, com idade média de 23,6 ± 4,8 anos. O planejamento dos casos incluiu alinhamento e nivelamento de incisivos inferiores usando os alinhadores. O fluido gengival crevicular foi coletado na superfície vestibular de incisivos inferiores no dia da entrega do alinhador número 1 (T0) e após 1 (T24h), 7 (T7d) e 21 (T21d) dias. Durante o período de observação do estudo, os pacientes utilizaram apenas o alinhador número 1. Os níveis de nove citocinas foram quantificados por meio do sistema Luminex de multianálise. Testes não paramétricos foram realizados para comparações entre os níveis de expressão de citocinas ao longo do tempo. Resultados: a concentração das citocinas manteve-se constante após 21 dias de ativação ortodôntica, exceto a MIP-1β, que apresentou uma redução estatisticamente significativa entre os tempos T24h e T21d. As IL-8, GM-CSF, IL-1β, MIP-1β e TNF-α apresentaram as maiores concentrações ao longo do tempo. Conclusão: a constância na expressão dos níveis das citocinas parece estar compatível com o estímulo mecânico induzido por alinhadores.

A preliminary investigation of short-term cytokine expression in gingival crevicular fluid secondary to high-level orthodontic forces and the associated root resorption: case series analytical study.

BACKGROUND: Orthodontically induced iatrogenic root resorption (OIIRR) is an unavoidable inflammatory process. Several factors claimed to be related to the severity of OIIRR. Orthodontic forces cause micro-trauma to the periodontal ligament and activate a cascade of cellular events associated with local periodontal inflammation. The purpose of this split-mouth study were (1) to investigate the changes in cytokine profile in the gingival crevicular fluid (GCF) secondary to heavy orthodontic forces and (2) to compare the cytokine expression between participants showing high and low root resorption. METHODS: Eight participants requiring maxillary first premolar extractions involved in this study. The teeth on the tested side (TS) received 225 g of controlled buccal tipping force for 28 days, while the contralateral teeth act as a control (CS). GCF was collected from both TS and CS teeth at 0 h (prior to application of force) and 3 h, 1 day, 3 days, 7 days and 28 days after the application of force, and analysed with multiplex bead immunoassay to determine the cytokine levels. RESULTS: Statistically significant temporal increase was found in the TS teeth for tumour necrosis factor alpha (TNF-α) at 3 h and 28 days (p = 0.01). Interleukin 7 (IL-7) significantly peaked at the 28th day. Comparing cytokine profile for participants with high and low root resorption (>0.35 and <0.15 mm3, respectively), the levels of GM-CSF was significantly greater in low root resorption cases (p < 0.05). The amounts of root resorption which craters on mesial, distal surfaces and middle third region were significant in the TS teeth (p < 0.05). CONCLUSIONS: IL-7 and TNF-α (pro-resorptive cytokine) increased significantly secondary to a high-level of orthodontic force application. Significantly high levels of granulocyte macrophage colony-stimulating factor (anti-resorptive cytokine) were detected in mild root resorption cases secondary to high-level orthodontic force application. A future long-term randomised clinical trial with larger sample taking in consideration gender, age and growth pattern distribution would be recommended.

Influence of type 2 diabetes on local production of inflammatory molecules in adults with and without chronic periodontitis: a cross-sectional study.

BACKGROUND: Pathological changes in periodontal tissues are mediated by the interaction between microorganisms and the host immune-inflammatory response. Hyperglycemia may interfere with this process. The aim of this study was to compare the levels of 27 inflammatory molecules in the gingival crevicular fluid (GCF) of patients with type 2 diabetes, with and without chronic periodontitis, and of chronic periodontitis subjects without diabetes. A putative correlation between glycated haemoglobin (HbA1c) and levels of the inflammatory molecules was also investigated. METHODS: The study population comprised a total of 108 individuals, stratified into: 54 with type 2 diabetes and chronic periodontitis (DM + CP), 30 with chronic periodontitis (CP) and 24 with type 2 diabetes (DM). Participants were interviewed with the aid of structured questionnaire. Periodontal parameters (dental plaque, bleeding on probing and periodontal pocket depth) were recorded. The GCF levels of the 27 inflammatory molecules were measured using multiplex micro-bead immunoassay. A glycated haemoglobin (HbA1c) test was performed for patients with diabetes by boronate affinity chromatography. RESULTS: After adjustment for potential confounders, the DM + CP group had higher levels of IL-8 and MIP-1ß, and lower levels of TNF-α, IL-4, INF-γ, RANTES and IL-7 compared to the CP group. Moreover, the DM + CP group had lower levels of IL-6, IL-7 and G-CSF compared to the DM group. The DM group had higher levels of IL-10, VEGF, and G-CSF compared to the CP group. The levels of MIP-1α and FGF were lower in diabetes patients (regardless of their periodontal status) than in chronic periodontitis subjects without diabetes. Diabetes patients (DM + CP and DM) had higher Th-2/Th-1 ratio compared to the CP group. HbA1c correlated positively with the pro-inflammatory cytokines (Pearson correlation coefficient = 0.27, P value: 0.02). CONCLUSION: Type 2 diabetes and chronic periodontitis may influence the GCF levels of inflammatory molecules synergistically as well as independently. Type 2 diabetes was associated with high Th-2/Th-1 ratio, and modulated the local expression of molecules involved in the anti-inflammatory and healing processes.

STAT5 programs a distinct subset of GM-CSF-producing T helper cells that is essential for autoimmune neuroinflammation.

T helper (TH)-cell subsets, such as TH1 and TH17, mediate inflammation in both peripheral tissues and central nervous system. Here we show that STAT5 is required for T helper-cell pathogenicity in autoimmune neuroinflammation but not in experimental colitis. Although STAT5 promotes regulatory T cell generation and immune suppression, loss of STAT5 in CD4+ T cells resulted in diminished development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Our results showed that loss of encephalitogenic activity of STAT5-deficient autoreactive CD4+ T cells was independent of IFN-γ or interleukin 17 (IL-17) production, but was due to the impaired expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), a crucial mediator of T-cell pathogenicity. We further showed that IL-7-activated STAT5 promotes the generation of GM-CSF-producing CD4+ T cells, which were preferentially able to induce more severe EAE than TH17 or TH1 cells. Consistent with GM-CSF-producing cells being a distinct subset of TH cells, the differentiation program of these cells was distinct from that of TH17 or TH1 cells. We further found that IL-3 was secreted in a similar pattern as GM-CSF in this subset of TH cells. In conclusion, the IL-7-STAT5 axis promotes the generation of GM-CSF/IL-3-producing TH cells. These cells display a distinct transcriptional profile and may represent a novel subset of T helper cells which we designate as TH-GM.

Effects of interim acrylic resins on the expression of cytokines from epithelial cells and on collagen degradation.

STATEMENT OF PROBLEM: Interim acrylic resins release agents that alter cytokine expression in the surrounding tissues, which could alter extracellular matrix degradation. PURPOSE: The purpose of the study was to evaluate the responses of human epidermal keratinocytes to eluates of interim acrylic resins in regards to cytokine expression and cell-mediated collagen degradation. MATERIAL AND METHODS: Specimens of 4 different interim acrylic resins (HI-I, Jet Acrylic, SNAP acrylic, and Protemp Plus) were placed in Epilife medium for 48 hours and the eluates collected. The cells were incubated for 72 hours in nontoxic concentrations of the eluates. Cytotoxicity was evaluated with lactate dehydrogenase assays and cytokine expression with cytokine antibody arrays. Collagen degradation was determined with a collagen type I assay. The experiments were performed 3 times. Data were analyzed with 1-way and mixed-model ANOVA (α=.05). RESULTS: None of the eluates were cytotoxic. Cytokine expression from the heat-activated polymethyl methacrylate resin group was significantly less for interleukin-3, but significantly greater for interlukin-7. Expression for the chemically activated polymethyl methacrylate resin group was significantly less for growth-regulated oncogene-α, interleukin-1α, and interleukin-3. Expression for the chemically activated polyethyl methacrylate resin group was significantly less for interleukin-1α and interleukin-3, but significantly greater for interleukin-13 and monocytes chemoattractant protein-3. The cytokine expression induced by chemically activated bis-acryl composite resin was significantly greater for granulocyte-macrophage colony stimulating factor, interleukin-7, and monocytes chemoattractant protein-3, but significantly less for growth-regulated oncogene-α. Collagen degradation was not significantly different in any of the groups. CONCLUSIONS: The eluates used were not cytotoxic and did not induce cell-mediated collagen degradation. Some significant changes in cytokine expression were noted.

Effects of resolvin D1 on cell survival and cytokine expression of human gingival fibroblasts.

BACKGROUND: Tissue breakdown in periodontitis is initiated by bacteria, such as Porphyromonas gingivalis, and is caused largely by host responses. Resolvins protect the host against acute inflammation by blocking the migration of polymorphonuclear neutrophils to initiate resolution. The effects of resolvins on human gingival fibroblasts (HGFs) are unknown. This study examines the effects of resolvin D1 on HGF survival and cytokine expression when treated with or without P. gingivalis supernatant. METHODS: Cytotoxicity of resolvin D1 on HGFs with or without a toxic level of P. gingivalis supernatant was measured with lactate dehydrogenase assays. Cytokine arrays were performed on HGF-conditioned media treated with or without resolvin D1 and with or without P. gingivalis supernatant. RESULTS: Resolvin D1 had no cytotoxic effects on HGFs at concentrations between 1 and 1,000 nM (all P > 0.05). Resolvin D1 (1,000 nM) significantly inhibited the toxic effects of 13.5% (v/v) P. gingivalis supernatant on HGFs (P = 0.002). Resolvin D1 significantly reduced the expression of interleukin (IL)-6 (P = 0.010) and monocyte chemoattractant protein (MCP)-1 (P = 0.04) in untreated fibroblasts. P. gingivalis (10%) supernatant significantly increased the expression levels of granulocyte-macrophage colony-stimulating factor (CSF), granulocyte CSF, growth-regulated oncogene (GRO), IL-5, IL-6, IL-7, IL-8, IL-10, MCP-1, MCP-2, MCP-3, and monokine induced by γ-interferon. Resolvin D1 significantly reduced the expression of GRO (P = 0.04), marginally reduced the levels of MCP-1 (P = 0.10), and marginally increased the levels of transforming growth factor (TGF)-ß1 (P = 0.07) from HGFs treated with P. gingivalis supernatant. CONCLUSIONS: Resolvin D1 altered the cytotoxicity of P. gingivalis supernatant on HGFs. Resolvin D1 significantly reduced GRO, marginally reduced MCP-1, and marginally increased TGF-ß1 from P. gingivalis-treated HGFs, which could alter the ability of P. gingivalis to induce inflammation.

Bacteriological, biochemical, and immunological modifications in human colostrum after Holder pasteurisation.

OBJECTIVE: The objective of this work was to evaluate the effect of Holder pasteurisation of human colostrum on a variety of microbiological, biochemical, and immunological parameters. METHODS: Colostrum samples from 10 donors, and 8 samples of mature milk used as controls, were heated at 62.5°C for 30 minutes. Bacterial counts and the concentration of furosine, lactose, myoinositol, glucose, lactulose, cytokines, and immunoglobulins were determined before and after the heat treatment. RESULTS: Mean bacterial counts in nonpasteurised colostrum samples oscillated between 2.72 and 4.13 log10 colony-forming units per millilitre in the agar media tested. Holder pasteurisation led to the destruction of the bacteria originally present in the samples. Furosine was detected in all samples before pasteurisation and increased significantly after the heat treatment (from 6.60 to 20.59 mg/100 g protein). Lactulose content was below the detection limit in nonpasteurised colostrum, but it was detected in all samples and quantified in 7 of them (from 10.68 to 38.02 mg/L) after Holder pasteurisation. Lactose, glucose, and myoinositol concentrations did not change after Holder pasteurisation. The concentrations of most cytokines and immunoglobulins were significantly higher in colostrum than in mature milk samples. Immunoglobulin content, both in colostrum and in milk samples, was reduced during pasteurisation, whereas, among cytokines, only macrophage inflammatory protein-1ß, interleukin-7, and granulocyte-macrophage-colony-stimulating factor concentrations were affected by this heat treatment. CONCLUSIONS: Lactulose and furosine content could be used as heat treatment indicators in colostrum samples. Holder pasteurisation modified the immunological profile of both colostrum and mature milk.

High rhinovirus burden in lower airways of children with cystic fibrosis.

BACKGROUND: Rhinovirus (RV)-induced pulmonary exacerbations are common in cystic fibrosis (CF) and have been associated with impaired virus clearance by the CF airway epithelium in vitro. Here, we assess in vivo the association of RV prevalence and load with antiviral defense mechanisms, airway inflammation, and lung function parameters in children with CF compared with a control group and children with other chronic respiratory diseases. METHODS: RV presence and load were measured by real-time reverse transcription-polymerase chain reaction in BAL samples and were related to antiviral and inflammatory mediators measured in BAL and to clinical parameters. RESULTS: BAL samples were obtained from children with CF (n = 195), non-CF bronchiectasis (n = 40), or asthma (n = 29) and from a control group (n = 35) at a median (interquartile range [IQR]) age of 8.2 (4.0-11.7) years. RV was detected in 73 samples (24.4%). RV prevalence was similar among groups. RV load (median [IQR] x 10(3) copies/mL) was higher in children with CF (143.0 [13.1-1530.0]), especially during pulmonary exacerbations, compared with children with asthma (3.0 [1.3-25.8], P = .006) and the control group (0.5 [0.3-0.5], P < .001), but similar to patients with non-CF bronchiectasis (122.1 [2.7-4423.5], P = not significant). In children with CF, RV load was negatively associated with interferon (IFN)- b and IFN- l , IL-1ra levels, and FEV 1 , and positively with levels of the cytokines CXCL8 and CXCL10. CONCLUSIONS: RV load in CF BAL is high, especially during exacerbated lung disease. Impaired production of antiviral mediators may lead to the high RV burden in the lower airways of children with CF. Whether high RV load is a cause or a consequence of inflammation needs further investigation in longitudinal studies.

Association of the CagA gene positive Helicobacter pylori and tissue levels of interleukin-17 and interleukin-8 in gastric ulcer patients.

It has been reported that CagA gene positive Helicobacter pylori (CagA+ H. pylon) induces severe gastric mucosal inflammation. On the other hand, Interleukin (IL)-17 is known to stimulate IL-8 release by the gastric epithelial cells which facilitates chemotaxis of neutrophils through an IL-8-dependent mechanism. The aim of the study is to determine the role of IL-17 and IL-8 in the development of gastritis and gastric ulcer in H. pylori infected patients. Mucosal biopsy samples were obtained from the ulcer site of gastric mucosa of 28 patients with gastric ulcer (GU), 27 with gastritis and 8 controls subjects without gastritis or ulcers. Infection with H. pylori of patients and controls was assessed by a rapid urease test, histological examination and culture. Measurement of the tissue levels of IL-17 and IL-8 were assayed by ELISA. H. pylori cagA gene was assessed by polymerase chain reaction (PCR). Out of the 28 patients with GU, 18 (64.2%) patients were positive for H. pylori infection, while 13 (48.1%) patients with gastritis and none of the controls were positive for H. pylori infection The CagA gene was detected in 12 (66.6%) in H. pylori GU patients, and 7 (53.8%) H. pylori positive gastritis. IL-17 was significantly higher in GU-CagA+ve H. pylori compared to GU-CagA- H. pylori (P <0.05), while IL-8 showed no significant difference between groups. The mean levels of IL-8 in gastritis-CagA+ H. pylori) was significantly higher compared to gastritis--CagA- H. pylori- (P <0.05). IL-17 showed significant association with the number of neutrophils in both GU and gastritis (r = 0.689, P < 0.05 & r = 0.618, P < 0.05). Also, IL-8 showed significant association with the number of neutrophils in both GU and gastritis n (r = 0.468, P < 0.05 & r = 0.727, P < 0.05). It is concluded that the Cag+ve H. pylori is associated with induction of mucosal injury. Also, IL-8 and IL-17 plays a role in the development of GU and gastritis especially in CagA+ H. pylori.

Elevated number of recently activated T cells in bone marrow of patients with rheumatoid arthritis: a role for interleukin 15?

OBJECTIVES: (1) To compare the absolute T-cell numbers in bone marrow (BM) isolated from patients with rheumatoid arthritis (RA) and osteoarthritis (OA); (2) to measure the levels of soluble interleukin 15 (IL-15) and IL-7; (3) to analyse the expression of activation markers on T cells; (4) to analyse influence of IL-15 stimulation on T-cell proliferation. METHODS: BM samples were obtained from patients undergoing joint replacement surgery. Concentrations of IL-15 and IL-7 were measured using specific ELISAs. The absolute number of T lymphocytes, their activation status and proliferation were evaluated by flow cytometry. RESULTS: BM from patients with RA contained double the number of CD3 T cells in comparison with OA (6.1 vs 2.7 × 10(6) cells/ml, p<0.008). Ratio CD3CD4:CD3CD8 was increased in RA BM, clearly indicating accumulation of CD3CD4 cells. T cells obtained from patients with RA expressed higher level of early activation markers than from OA. Elevated levels of IL-15 were found in BM plasma from patients with RA in comparison with patients with OA (1304.5±956.3 pg/ml and 760±238.7 pg/ml respectively, p<0.01). These data were confirmed by immunohistochemistry of RA BM from regions proximal and distal to the joint. Although both CD3CD4 and CD3CD8 cells proliferated after IL-15 stimulation in vitro, CD3CD4 cells from patients with RA proliferated more vigorously than those from patients with OA, reflecting the composition of T-cell subsets in BM. CONCLUSION: These results suggest that locally overproduced IL-15 may be responsible for the activation and proliferation of T cells in situ, reflected by significantly increased number of activated T cells in RA BM, possibly contributing to the pathogenesis of RA.

A multiplex immunoassay demonstrates reductions in gingival crevicular fluid cytokines following initial periodontal therapy.

BACKGROUND AND OBJECTIVE: Cytokines and chemokines play an important role in the pathogenesis of periodontal diseases. The objective of this study was to quantitatively assess the effect of initial periodontal therapy on gingival crevicular fluid levels of a comprehensive panel of cytokines and chemokines, including several less extensively studied mediators. MATERIAL AND METHODS: Clinical examinations were performed and gingival crevicular fluid samples obtained from six subjects with generalized severe chronic periodontitis prior to initial periodontal therapy and at re-evaluation (6-8 weeks). Four diseased and two healthy sites were sampled in each subject. Twenty-two gingival crevicular fluid mediators were examined using a multiplex antibody capture and detection platform. Statistical analyses were performed by fitting mixed effects linear models to log-transformed gingival crevicular fluid values. RESULTS: Gingival crevicular fluid interleukin (IL)-1alpha and IL-1beta were the only cytokines to differ in initially diseased vs. initially healthy sites. Following initial therapy, 13 of the 16 detectable cytokines and chemokines decreased significantly in diseased sites, including IL-1alpha, IL-1beta, IL-2, IL-3, IL-6, IL-7, IL-8, IL-12 (p40), CCL5/regulated on activation, normally T cell expressed and secreted (RANTES), eotaxin, macrophage chemotactic protein-1, macrophage inflammatory protein-1alpha and interferon-gamma. At healthy sites, only three of the 16 mediators were significantly altered following therapy. CONCLUSION: This is the first study, to our knowledge, to evaluate such an extensive panel of gingival crevicular fluid mediators within the same sample prior to and following initial therapy. The results confirm that periodontal therapy effectively reduces pro-inflammatory cytokines and chemokines, including less well-described mediators that may be important in initiation and progression of periodontitis. The multiplex assay will prove useful for future gingival crevicular fluid studies.

Effects of in utero arsenic exposure on child immunity and morbidity in rural Bangladesh.

Chronic exposure to arsenic, a potent carcinogen and toxicant, via drinking water is a worldwide public health problem. Because little is known about early-life effects of arsenic on immunity, we evaluated the impact of in utero exposure on infant immune parameters and morbidity in a pilot study. Pregnant women were enrolled at 6-10 weeks of gestation in Matlab, a rural area of Bangladesh, extensively affected by arsenic contamination of tubewell water. Women (n=140) delivering at local clinics were included in the study. Anthropometry and morbidity data of the pregnant women and their children, as well as infant thymic size by sonography were collected. Maternal urine and breast milk were collected for immune marker and arsenic assessment. Maternal urinary arsenic during pregnancy showed significant negative correlation with interleukin-7 (IL-7) and lactoferrin (Ltf) in breast milk and child thymic index (TI). Urinary arsenic was also positively associated with fever and diarrhea during pregnancy and acute respiratory infections (ARI) in the infants. The effect of arsenic exposure on ARI was only evident in male children. The findings suggest that in utero arsenic exposure impaired child thymic development and enhanced morbidity, probably via immunosuppression. The effect seemed to be partially gender dependent. Arsenic exposure also affected breast milk content of trophic factors and maternal morbidity.

Profiling of inflammatory cytokines produced by gingival fibroblasts after human cytomegalovirus infection.

INTRODUCTION: The purpose of this study was to determine the profile of inflammatory cytokines that are produced after in vitro infection of gingival fibroblasts with human cytomegalovirus (HCMV). MATERIALS AND METHODS: Gingival fibroblasts were infected with the Towne strain of HCMV and the cytokine profile in the supernatant was studied using a human inflammation antibody array. Expression of messenger RNA (mRNA) using reverse transcription-polymerase chain reaction for interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) was also analyzed in infected gingival fibroblasts and gingival specimens from subjects with and without periodontitis according to HCMV detection. HCMV was determined in subgingival samples by nested polymerase chain reaction. RESULTS: Gingival fibroblasts produced mainly IL-1alpha, IL-12p40, IL-12p70, IL-6, TNF-alpha, and IL-1beta after HCMV infection. Expression of mRNA for IL-1beta and TNF-alpha was increased after HCMV infection. Production of IL-1beta and TNF-alpha was increased in HCMV-positive periodontitis specimens. In addition, infected gingival fibroblasts produced more IL-8, monocyte chemoattractant protein 1, macrophage inflammatory proteins 1alpha, and 1beta over time postinfection in comparison to baseline. The lowest production of all cytokines studied corresponded to IL-2, IL-4, IL-13, and interferon-gamma. A decreasing production pattern was observed for granulocyte-macrophage colony-stimulating factor, IL-7, and IL-17 while IL-11 and macrophage colony-stimulating factor were increased at 72 h postinfection. CONCLUSIONS: HCMV infection in gingival fibroblasts upregulated the production of proinflammatory-related cytokines and chemokines. The expression of IL-1beta and TNF-alpha was increased both in vitro and in specimens from HCMV-positive subjects with periodontitis. The overproduction of proinflammatory cytokines and chemokines as a result of viral infection should be considered an important pathogenic mechanism linking HCMV to periodontitis in vivo.

Lymphocytes and synovial fluid fibroblasts support osteoclastogenesis through RANKL, TNFalpha, and IL-7 in an in vitro model derived from human psoriatic arthritis.

Psoriatic arthritis (PsA) is an inflammatory joint disease, characterized by extensive bone resorption, whose mechanisms have not been fully elucidated. Thus, in the present study we investigated the involvement of RANKL, TNFalpha, and IL-7 in the osteoclastogenesis of PsA patients. In vitro osteoclastogenesis models, consisting of unfractionated and T-cell-depleted mononuclear cells from peripheral blood (PBMCs) and synovial fluid (SFMCs) of 20 PsA patients as well as from healthy donors were studied. Freshly isolated T and B cells from PBMCs and T cells and fibroblasts from SFMCs of PsA patients were subjected to RT-PCR to detect the levels of RANKL, TNFalpha, and IL-7. Osteoclastogenesis was studied in the presence of RANK-Fc, anti-TNFalpha, and anti IL-7 functional antibodies. We demonstrate that lymphocytes and fibroblasts support osteoclast (OC) formation in PsA patients through the production of osteoclastogenic cytokines. In particular, OC formation was completely abolished in unstimulated T cell-depleted PBMC cultures, and reduced by approximately 70% in unstimulated T cell-depleted SFMC cultures. Freshly isolated T cells from PBMCs and SFMCs of PsA patients overexpressed RANKL and TNFalpha, while fibroblasts from synovial fluid produced only RANKL. We show that the presence of RANK-Fc and/or anti-TNFalpha functional antibodies reduced OC formation. Moreover, T and B cells from PBMCs as well as T cells and fibroblasts from SFMCs expressed IL-7 mRNA. Finally, the anti-IL-7 functional antibody significantly reduced osteoclastogenesis. Our results suggest that fibroblasts, B and T lymphocytes support OC formation by producing RANKL, TNFalpha, and IL-7, contributing to the aggressive bone resorption in PsA patients.

[Immunogenic reactivity of CTLs induced by electrofusion cells of human dendritic cells and gastric cancer cells].

In tumor immunotherapy, there were several reports of attempts to induce anti-tumor immunity by fusion hybrid cells generated with dendritic and tumor cells. One of them reported that vaccination of hybrid cells resulted in a remarkable reduction of tumor cells in a lab mouse experiment. In our study, fusion cells were generated successfully with human matured dendritic and human gastric cancer cells by electrofusion technique and employed to induce CTLs. The evaluated fusion rate was 47.8% by FACS analysis. We tried to induce CTLs by co culture of effector and stimulator cells in the presence of IL-2, IL-7 and IL-12 for 4 weeks. Although it was not statistically significant in tumor cytotoxic assay, effector cells induced by the fusion cells as stimulator cells showed a few cytotoxic responses in an immunological tumor specific manner. Our data suggest that fusion hybrid cells may facilitate stimulation and expansion of tumor-specific T cells, but further investigation is required for clinical application of fusion cells in adoptive immunotherapy.

IL-7-dependent homeostatic proliferation in the presence of a large number of T cells in gld mice.

In gld mice, CD4 and 8-double-negative (DN) T cells as well as naive and memory-phenotype T cells accumulate in the peripheral lymphoid organs. Although Fas ligand (L) defect accounts for the progressive accumulation of abnormal DN T cells, the existence of other mechanisms which may be involved in the defective homeostasis in gld mice has been unclear. In this study, we analyze T-cell homeostasis in gld mice using adoptive transfer systems. It was shown that a gld, but not C57BL/6 (B6), environment led to augmented proliferation of B6 T cells transferred without up-regulation of CD69. Thus, the augmented T-cell proliferation seemed to result from mal-homeostatic proliferation even in the presence of a large number of recipient T cells. T cells from lpr mice showed no significant proliferation in the B6 environment, suggesting that the absence of Fas-Fas L interaction was not responsible for the mal-homeostatic proliferation. Although similar levels of IL-7 mRNA were detected in gld and B6 spleens, the intensity of CD127 and the proportion of CD127+ cells in the T cells were significantly lower in gld mice than in B6 mice, suggesting that IL-7 excess in a gld environment is responsible for the abnormal proliferation of transferred T cells. The administration of anti-CD127 antibody inhibited the proliferation of transferred lymphocytes. Thus, IL-7-dependent proliferation seems to be involved in the abnormal proliferation of lymphocytes in gld recipients.