Potential Use of Extracellular Vesicles Generated by Microbubble-Assisted Ultrasound as Drug Nanocarriers for Cancer Treatment.

Extracellular vesicles (EVs)-carrying biomolecules derived from parental cells have achieved substantial scientific interest for their potential use as drug nanocarriers. Ultrasound (US) in combination with microbubbles (MB) have been shown to trigger the release of EVs from cancer cells. In the current study, the use of microbubbles-assisted ultrasound (USMB) to generate EVs containing drug cargo was investigated. The model drug, CellTracker™ green fluorescent dye (CTG) or bovine serum albumin conjugated with fluorescein isothiocyanate (BSA FITC) was loaded into primary human endothelial cells in vitro using USMB. We found that USMB loaded CTG and BSA FITC into human endothelial cells (HUVECs) and triggered the release of EVs containing these compounds in the cell supernatant within 2 h after treatment. The amount of EV released seemed to be correlated with the increase of US acoustic pressure. Co-culturing these EVs resulted in uptake by the recipient tumour cells within 4 h. In conclusion, USMB was able to load the model drugs into endothelial cells and simultaneously trigger the release of EVs-carrying model drugs, highlighting the potential of EVs as drug nanocarriers for future drug delivery in cancer.

Tumor Drug Distribution after Local Drug Delivery by Hyperthermia, In Vivo.

Tumor drug distribution and concentration are important factors for effective tumor treatment. A promising method to enhance the distribution and the concentration of the drug in the tumor is to encapsulate the drug in a temperature sensitive liposome. The aim of this study was to investigate the tumor drug distribution after treatment with various injected doses of different liposomal formulations of doxorubicin, ThermoDox (temperature sensitive liposomes) and DOXIL (non-temperature sensitive liposomes), and free doxorubicin at macroscopic and microscopic levels. Only ThermoDox treatment was combined with hyperthermia. Experiments were performed in mice bearing a human fibrosarcoma. At low and intermediate doses, the largest growth delay was obtained with ThermoDox, and at the largest dose, the largest growth delay was obtained with DOXIL. On histology, tumor areas with increased doxorubicin concentration correlated with decreased cell proliferation, and substantial variations in doxorubicin heterogeneity were observed. ThermoDox treatment resulted in higher tissue drug levels than DOXIL and free doxorubicin for the same dose. A relation with the distance to the vasculature was shown, but vessel perfusion was not always sufficient to determine doxorubicin delivery. Our results indicate that tumor drug distribution is an important factor for effective tumor treatment and that its dependence on delivery formulation merits further systemic investigation.

Effect of Bronchoscopy on Gas Exchange and Respiratory Mechanics in Critically Ill Patients With Atelectasis: An Observational Cohort Study.

Background: Atelectasis frequently develops in critically ill patients and may result in impaired gas exchange among other complications. The long-term effects of bronchoscopy on gas exchange and the effects on respiratory mechanics are largely unknown. Objective: To evaluate the effect of bronchoscopy on gas exchange and respiratory mechanics in intensive care unit (ICU) patients with atelectasis. Methods: A retrospective, single-center cohort study of patients with clinical indication for bronchoscopy because of atelectasis diagnosed on chest X-ray (CXR). Results: In total, 101 bronchoscopies were performed in 88 ICU patients. Bronchoscopy improved oxygenation (defined as an increase of PaO2/FiO2 ratio > 20 mmHg) and ventilation (defined as a decrease of > 2 mmHg in partial pressure of CO2 in arterial blood) in 76 and 59% of procedures, respectively, for at least 24 h. Patients with a low baseline value of PaO2/FiO2 ratio and a high baseline value of PaCO2 were most likely to benefit from bronchoscopy. In addition, in intubated and pressure control ventilated patients, respiratory mechanics improved after bronchoscopy for up to 24 h. Mild complications, and in particular desaturation between 80 and 90%, were reported in 13% of the patients. Conclusions: In selected critically ill patients with atelectasis, bronchoscopy improves oxygenation, ventilation, and respiratory mechanics for at least 24 h.

Stemmed DNA nanostructure for the selective delivery of therapeutics.

DNA has emerged as a biocompatible biomaterial that may be considered for various applications. Here, we report tumor cell-specific aptamer-modified DNA nanostructures for the specific recognition and delivery of therapeutic chemicals to cancer cells. Protein tyrosine kinase (PTK)7-specific DNA aptamer sequences were linked to 15 consecutive guanines. The resulting aptamer-modified product, AptG15, self-assembled into a Y-shaped structure. The presence of a G-quadruplex at AptG15 was confirmed by circular dichroism and Raman spectroscopy. The utility of AptG15 as a nanocarrier of therapeutics was tested by loading the photosensitizer, methylene blue (MB), to the G-quadruplex as a model drug. The generated MB-loaded AptG15 (MB/AptG15) showed specific and enhanced uptake to CCRF-CEM cells, which overexpress PTK7, compared with Ramos cells, which lack PTK7, or CCRF-CEM cells treated with a PTK7-specific siRNA. The therapeutic activity of MB/AptG15 was tested by triggering its photodynamic effects. Upon 660 nm light irradiation, MB/AptG15 showed greater reactive oxygen species generation and anticancer activity in PTK7-overexpressing cells compared to cells treated with MB alone, those treated with AptG15, and other comparison groups. AptG15 stemmed DNA nanostructures have significant potential for the cell-type-specific delivery of therapeutics, and possibly for the molecular imaging of target cells.

Urinary Tissue Inhibitor of Metalloproteinase and Insulin-like Growth Factor-7 as Early Biomarkers of Delayed Graft Function After Kidney Transplantation.

BACKGROUND: Recently, urinary tissue inhibitor of metalloproteinase-2 (TIMP-2) and insulin-like growth factor-7 (IGFBP-7), markers for G1 cell cycle arrest, have been identified and validated in predicting the development of acute kidney injury in critically ill patients. It is unknown, however, whether these two biomarkers could predict the development of delayed graft function (DGF) after kidney transplantation (KT). METHODS: This is a single-center, prospective, observational study. We enrolled 74 patients who underwent KT between August 2013 and December 2016. Urine sample were collected immediately after the operation. The primary outcome was development of DGF as defined by need for dialysis of more than 1 session within 7 days of KT. RESULTS: Twenty-three patients (31%) were diagnosed with DGF. In univariate analysis, kidneys from expanded criteria donors, higher donor serum creatinine, lower donor estimated glomerular filtration rate, antithymoglobulin exposure, neutrophil gelatinase associated lipocalin, and urinary [TIMP-2]·[IGFBP7] were significantly different between early graft function and DGF. However, in multivariate analysis adjusting other factors, deceased donor and urinary [TIMP-2]·[IGFBP7] at 0 hours post-transplantation could predict the development of DGF. The receiver operating characteristic curve for prediction of DGF showed an area under the curve of 0.867 (sensitivity 0.86, specificity 0.71) for a cutoff value of 1.39. CONCLUSIONS: Our results indicate that urine [TIMP-2]·[IGFBP7] immediately after transplantation could be an early, predictive biomarker of DGF in kidney transplantation.

Immune-camouflaged graphene oxide nanosheets for negative regulation of phagocytosis by macrophages.

Signal regulatory protein alpha (SIRPα) is highly expressed in macrophages of the reticuloendothelial system and in tumor-associated macrophages, whereas tumor cells express the surface membrane protein, CD47, which interacts with SIRPα to negatively regulate phagocytosis. In this study, we modified the surfaces of graphene oxide (GO) nanosheets with a CD47-like SIRPα-binding peptide (SP). The presence of SP on GO nanosheets reduced the macrophage uptake to a greater extent than the PEGylation of such nanosheets. This reduced uptake was found to be mediated by the activation of Src homology region 2 domain-containing phosphatase 1 (SHP-1) and the downstream inhibition of myosin assembly, which is necessary for phagosome formation. Unlike SP-coated GO nanosheets, PEGylated GO nanosheets did not affect myosin assembly or phagocytosis. After in vivo systemic administration, the clearance of SP-coated GO nanosheets was slower than that of PEGylated GO nanosheets, and this difference increased with repeated administration. Finally, SP-coated GO nanosheets showed a higher distribution to tumor tissues than PEGylated GO nanosheets or a physical mixture of SP and GO nanosheets. Our findings indicate that immune-camouflaged GO nanosheets with natural CD47-like SIRPα-binding molecules can reduce the nonspecific loss of such nanosheets through macrophage uptake, thereby enhancing their blood circulation and tumor delivery after multiple injections.

Simultaneous determination of water-soluble whitening ingredients and adenosine in different cosmetic formulations by high-performance liquid chromatography coupled with photodiode array detection.

OBJECTIVE: The Korean Cosmetic Act regulates the use of functional cosmetics) by the law. Four functional cosmetic groups, whitening, anti-wrinkle, UV protection and combination of whitening and anti-wrinkle, were categorized according to the Korean Cosmetic Act and Functional Cosmetics Codex. In this study, high-performance liquid chromatography (HPLC) coupled with photodiode array detection (DAD) was employed for the simultaneous detection of arbutin (and its decomposition product, hydroquinone), niacinamide, ascorbyl glucoside, ethyl ascorbyl ether and adenosine in functional cosmetic products such as creams, emulsions and lotions. METHODS: Separation by HPLC-DAD was conducted using a C18 column with a gradient elution of 5 mm KH2PO4 buffer (containing 0.1% phosphoric acid) and methanol (containing 0.1% phosphoric acid). The wavelengths for the detection of arbutin, hydroquinone, niacinamide, adenosine, ascorbyl glucoside and ethyl ascorbyl ether were 283, 289, 261, 257, 238 and 245 nm, respectively. RESULTS: This method exhibited good linearity (R(2) ≥ 0.999), precision (expressed as relative standard deviation (RSD) < 2%) and mean recoveries (89.42-104.89%). The results obtained by monitoring 100 market samples showed that the detected levels of the tested materials are within the acceptable authorized concentration. CONCLUSION: The method developed herein is simple and can be used for market survey and quality control of functional cosmetics.

Simultaneous pancreas-kidney transplantation from living donor using hand-assisted laparoscopic donor surgery: single-center experience.

BACKGROUND: Simultaneous pancreas-kidney (SPK) transplantation has been the fundamental treatment and has shown significant results in selected patients diagnosed with type 1 diabetes with renal insufficiency. Most pancreas transplantations are dependent on deceased donors, yet the waiting time for SPK transplantation from deceased donors is significantly long in Asian countries. METHODS: In 3 cases, living-donor SPK transplantation was performed with the use of hand-assisted laparoscopic donor surgery (HALS). Three cases of patients who underwent SPK transplantation from living donors (LDSPK) with the use of HALS at Korea University Anam Hospital from 2012 to 2013 were retrospectively reviewed regarding patient characteristics and clinical outcomes of donors and recipients. For the donors, the pancreas and renal function had been well preserved postoperatively. RESULTS: One donor had a pancreatic fistula, which was controlled with conservative management. Of the 3 cases of recipient operation, 1 case was performed by ABO incompatibility donor. The levels of creatinine, serum insulin, and C-peptide of recipients were normalized and remained stable at the last follow-up. CONCLUSIONS: LDSPK can be an efficient alternative in cases in which the deceased donor is not present at the proper time, depending on the degree of completion in the operator's skill.

Pegfilgrastim vs filgrastim in PBSC mobilization for autologous hematopoietic SCT: a systematic review and meta-analysis.

Trial outcomes comparing cytokine agents for PBSC mobilization in autologous hematopoietic transplant patients have been controversial. We performed a systematic review and meta-analysis of evidence available on pegfilgrastim vs filgrastim in chemo-cytokine mobilization. Electronic literature searches of PubMed, EMBASE and CENTRAL identified nine articles eligible for qualitative analysis with one randomized controlled trial. Eight articles involving 719 patients were included in the meta-analysis. Results showed similar CD34+ cell collection yields for pegfilgrastim and filgrastim (SDM -0.08, 95% CI: -0.388 to 0.228). On comparison with filgrastim, pegfilgrastim showed a significantly earlier apheresis onset time (SDM: -0.512, 95% CI: -0.973 to -0.050) and reduction in required apheresis procedures (SDM -0.260, 95% CI: -0.466 to -0.054). Times to leukocyte (⩾1.0 × 10(9)/L) and platelet (⩾20 × 10(9)/L) recovery were similar between groups (SDM: 0.015, 95% CI: -0.41 to 0.44 and SDM: 0.309, 95% CI: -0.11 to 0.72, respectively). Both agents were well tolerated and mild bone pain was the most frequently reported adverse event. Pegfilgrastim may be a convenient alternative to filgrastim in PBSC mobilization for multiple myeloma and lymphoma patients, but further studies are required to clarify effects of cytokine dosage and previous cytotoxic exposure in specific subpopulations.

Comparison of steroid administration methods in patients with idiopathic sudden sensorineural hearing loss: a retrospective observational study.

OBJECTIVES: To compare the recovery rates of patients with idiopathic sudden sensorineural hearing loss (ISSHL) treated with oral systemic steroids (PO) or intratympanic steroid injection (IT) or both. DESIGN: A retrospective observational study. SETTING: Tertiary referral centre. PARTICIPANTS: Eight hundred and forty-four patients diagnosed with ISSHL within 14 days of the onset of symptoms. The patients were divided into three groups by treatment modality. MAIN OUTCOME MEASURES: Threshold of pure-tone tests, age, accompanying symptoms and underlying diseases were compared. The level of final hearing recovery was evaluated by the application of the results of the pure-tone test that was performed at least 3 months after the completion of each treatment. RESULTS: Final hearing recovery rate differed significantly by the type of treatment (P = 0.031). Recovery rates in the PO and combined groups were significantly higher in patients with mild (85.1% and 88.6%, respectively) than with profound (52.8% and 69.0%, respectively) hearing loss (P < 0.05). In contrast, severity and recovery rate were not significantly correlated in the IT group (P > 0.05). Combined treatment yielded significantly higher recovery rates than other treatment modalities in patients without hypertension (HTN) and diabetes mellitus (DM) (P = 0.021). CONCLUSION: In the group treated with combined therapy, better hearing improvement was obtained than in the groups treated with systemic steroid only or with intratympanic steroid injection only without complications. These findings suggest that the combination of systemic administration and intratympanic injection may improve patient prognosis.

MicroRNA-29c functions as a tumor suppressor by direct targeting oncogenic SIRT1 in hepatocellular carcinoma.

Mammalian sirtuin 1 (SIRT1) has connected to an ever widening circle of activities that encompass cellular stress resistance, energy metabolism and tumorigenesis. However, underlying mechanisms leading to oncogenic SIRT1 overexpression are less understood. In this study, we identified SIRT1 regulatory microRNA (miRNA) and its function in hepatocellular carcinoma (HCC). Aberrant SIRT1 overexpression was demonstrated in a subset of human HCCs. SIRT1 knockdown suppressed HCC cell growth by transcriptional deregulation of cell cycle proteins. This led to hypophosphorylation of pRb, which inactivated E2F/DP1 target gene transcription, and thereby caused significant increase of HCC cells to remain in the G1/S phase. A comprehensive miRNA profiling analysis indentified five putative endogenous miRNAs that are significantly downregulated in HCC. Ectopic expression of miRNA mimics evidenced miR-29c to suppress SIRT1 in HCC cells. Notably, ectopic miR-29c expression repressed cancer cell growth and proliferation, and it recapitulated SIRT1 knockdown effects in HCC cells. In addition, miR-29c expression was downregulated in a large cohort of HCC patients, and low expression of miR-29c was significantly associated with poor prognosis of HCC patients. Taken together, we demonstrated that miR-29c suppresses oncogenic SIRT1 by way of binding to 3'-untranslated region of SIRT1 mRNA causing translational inhibition in liver cancer cells. The loss or suppression of miR-29c may cause aberrant SIRT1 overexpression and promotes liver tumorigenesis. Overall, we suggest that miR-29c functions as a tumor suppressor by regulating abnormal SIRT1 activity in liver.

Clinical outcomes in kidney transplantation patients from deceased donors with acute kidney injury.

BACKGROUND: This single-center study sought to examine the clinical outcomes of kidney transplant recipients from donors displaying acute kidney injury (AKI). METHODS: We analyzed retrospectively the medical records of the donors and recipients of 54 deceased-donor kidney transplantations performed in our center between March 2009 and March 2012. RESULTS: Among the 54 deceased donors, 36 (66.7%) experienced AKI as determined by the final mean serum creatinine levels measured before graft harvest of 2.66 ± 1.62 mg/dL versus 0.82 ± 0.28 mg/dL among non-AKI donors. The risks of delayed graft function and slow graft function were increased among the AKI versus non-AKI groups in the early post-transplantation period. However, the renal function status of recipients at 3, 6, and 12 months after transplantation was not significantly different between the two groups. Moreover, rejection-free survival rates during the study period were similar. Multivariate analysis revealed an acute rejection episodes (P = .047) and a lower body mass index in the donor relative to the recipient (P = .011) to be independent risk factors predicting poor graft function defined as a 1-year estimated glomerular filtration rate less than 50 mL/min/l.73 m(2). Donor AKI with either a high level (>4.0 mg/dL), an increasing trend of creatinine, or greater severity by the Risk, Injury, Failure, Loss, and End-stage kidney disease (RIFLE) classification was not a significant risk factor. CONCLUSION: Transplantation of kidneys from the AKI donors, namely, patients with severely decreased renal function, displayed excellent short-term outcomes. Accordingly, kidney transplantations from deceased donors with AKI should be considered more actively to expand the donor pool in Korea.

Differential lineage specification of thymic epithelial cells from bipotent precursors revealed by TSCOT promoter activities.

Mechanism of thymic compartmentalization was studied in the transgenic system using the promoter of thymic stromal cotransporter (TSCOT), a cortical thymic epithelium-specific gene. The transgenic 3.1 kb TSCOT (3.1T) and 4.4 kb TSCOT (4.4T) promoters recapitulated the thymic organ and the cortical epithelial cell-specific expression at the newborn stage. However, the 3.1T driving enhanced green fluorescent protein (EGFP; 3.1T-EGFP) or Cre-recombinase (3.1T-CreE) redistributed the expression into the medulla at the adult stages. Two Cre-transgenic lines (3.1T-CreE and 4.4T-CreE), when crossed with the ROSA LacZ or EGFP lines, showed the reporter expression in both the cortex and the medulla. TSCOT promoter activities were also verified in the transient thymic epithelial cell (TEC) population expressing keratin 5 and keratin 8. These indicate that the TSCOT promoter is turned on in the bipotent TEC precursors and regulated in a compartment-specific, developmentally regulated fashion. These transgenic lines provide the useful systems for delineating the specific pathways for TEC lineage development and function.

Clinical efficacy of leflunomide in primary Sjogren's syndrome is associated with regulation of T-cell activity and upregulation of IL-7 receptor α expression.

OBJECTIVES: To investigate whether the immunomodulatory capacities of leflunomide are associated with clinical efficacy in the treatment of primary Sjögren's syndrome (SS) in a phase II pilot study. METHODS: Peripheral blood mononuclear cells from 13 primary SS patients were obtained at baseline and after 24 weeks of leflunomide treatment. Ex-vivo production of interleukin (IL) 1ß and tumour necrosis factor α (TNFα) and of interferon (IFN), IL-4, as well as TNFα ELISA measured production on T-cell and monocyte stimulation. In addition, the authors investigated the ability of leflunomide to influence systemic levels of inflammatory cytokines, as well as T-cell activation markers and the expression of IL-7 receptor α by flow cytometry. Correlations between changes in cytokine levels and changes in clinical response parameters were studied. RESULTS: Ex-vivo production of IL-1ß and TNFα was decreased at 24 weeks in the whole patient group, whereas IFN and IL-4 production were not significantly changed. However, a significant decrease in T-cell-stimulated IFN and TNFα production was observed in clinical responders, but not in non-responders. Moreover, significant correlations were found between increased sialometry values and decreased IFN and TNFα production. In addition, leflunomide reduced levels of inflammatory serum cytokines and CD40L expression, whereas it upregulated IL-7Rα expression on CD4 T cells with persistent serum IL-7 concentrations. CONCLUSIONS: Leflunomide treatment suppressed cytokine release from circulating immune cells. Inhibition of T-helper 1 cell cytokine production was related to clinical efficacy. This suggests that selective T-cell targeting might be a relevant therapeutic strategy in primary SS, possibly enhancing clinical efficacy and safety.

Management of patients on the waiting list for deceased donor kidney transplantation.

BACKGROUND: Although the number of wait-listed patients for deceased donor kidney transplantation has been continuously increasing in Korea, no standard guidelines exist for their management. METHODS: We retrospectively analyzed the medical records of our 1,231 wait-listed patients between 2000 and 2010. RESULTS: The time to transplantation of the 201 recipients was 51.9 ± 31.2 months. Ninety-seven patients died while waiting. Diabetic or older patients have increased among new registrants; however, <50% of them have undergone regular screening for malignancy or cardiovascular diseases. Patients with regular screening were more likely to get a chance to receive a transplant (P = .016). Malignancy was newly diagnosed in 26 patients (2.1%) and reversible cardiac ischemia was detected in 9.7%. The presence of anti-HLA antibodies was strongly associated with a lower transplantation rate, whereas blood type O was not. Although use of expanded criteria donor (ECD) kidneys increased, many patients avoided them. CONCLUSION: It is necessary to improve management programs for wait-listed patients by establishing comorbidity screening and ECD education.

Analysis of prognostic factors for postoperative bleeding after tonsillectomy.

Postoperative bleeding is the most frequent surgical complication after tonsillectomy and may be associated with increased mortality rate. We have, therefore, analyzed factors associated with and prognostic for bleeding after tonsillectomy. The 2,254 patients who underwent tonsillectomy under general anesthesia at our institution from January 2005 to December 2009 were divided into bleeding and non-bleeding groups, and their demographic and clinical characteristics were compared. Age, administration of steroid immediately after general anesthesia, absence of administration of non-steroidal anti-inflammatory drugs, and the surgeon's experience were significantly associated with bleeding. In contrast, gender, chief complaints, performance of associated surgery, and type of anesthetic were not associated with postoperative bleeding. Hemorrhage after tonsillectomy was associated with the administration of steroids and with the non-administration of non-steroidal anti-inflammatory drugs.

A pre- and postoperative bacteriological study of chronic suppurative otitis media.

BACKGROUND: Although many bacteriological studies on preoperative otorrhea in patients with chronic suppurative otitis media (CSOM) have been performed, there are few studies on postoperative otorrhea. In this study, we analyzed the pathogenic microorganisms, changes in the bacterial species before and after surgery, and the antibiotic sensitivity on preoperative and postoperative cultures. METHODS: This was a retrospective study of 87 postoperative otorrhea patients who were part of a sample of 1,754 patients with CSOM who underwent tympanomastoidectomy; preoperative and postoperative otorrhea samples were obtained from January 2002 to April 2009. We analyzed patients with postoperative otorrhea divided into two groups: those with early onset (<3 months after surgery, n = 45) and those with late onset (>3 months after surgery, n = 42) otorrhea. RESULTS: Four species of organisms, methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), Pseudomonas, and coagulase-negative Staphylococcus (CNS), showed higher prevalence than others on both the preoperative and postoperative cultures. When we compared the early and late onset otorrhea groups, we found that 'no growth' was significantly higher in the early onset group (n = 19 vs. n = 5), whereas MSSA was significantly higher in the late onset group (n = 1 vs. n = 12). Of the 67 patients with positive preoperative cultures, 15 (22.4%) had the same bacteria after surgery, 34 (50.8%) had other bacteria, 2 (3.0%) had fungi, and 16 (23.8%) showed no growth on postoperative bacteriological testing. MSSA (9%) and MRSA (16.7%) were rarely recultured after surgery, whereas Pseudomonas was recultured frequently (61.5%). CONCLUSION: Unlike MSSA and MRSA, ciprofloxacin-resistant P. aeruginosa (CRP) occasionally causes early onset postoperative otorrhea due to the lack of highly potent antibiotics against this species. The success rate of infection control by surgery and antibiotics was low for CRP.

Elevated expression of interleukin-7 receptor in inflamed joints mediates interleukin-7-induced immune activation in rheumatoid arthritis.

OBJECTIVE: To evaluate the expression and functional ability of the high-affinity interleukin-7 receptor (IL-7Ralpha) in patients with rheumatoid arthritis (RA). METHODS: Expression of IL-7Ralpha and IL-7 was determined in synovial tissue from RA patients and was compared with that in synovial tissue from patients with undifferentiated arthritis (UA) and osteoarthritis (OA). IL-7Ralpha expression on CD4 T cells, CD19 B cells, and CD14 monocyte/macrophages from RA synovial tissue, synovial fluid, and peripheral blood was also assessed. The proliferative capacity of IL-7Ralpha(bright) and IL-7Ralpha(dim/-) T cells was measured. In addition, we examined IL-7R blockade with soluble human IL-7Ralpha (hIL-7Ralpha) in the prevention of immune activation of peripheral blood mononuclear cells. RESULTS: We found significantly higher IL-7Ralpha expression in RA and UA synovial tissue than in OA synovial tissue, and the level of IL-7Ralpha expression correlated significantly with the levels of CD3 and IL-7 expression. CD4 T cells from RA synovial fluid and synovial tissue strongly expressed IL-7Ralpha. A substantial percentage of B cells and macrophages from RA synovial fluid and synovial tissue also expressed IL-7Ralpha, although less prominently than T cells. We found that peripheral blood IL-7Ralpha(bright) T cells that did not express FoxP3 were highly proliferative as compared with IL-7Ralpha(dim/-) T cells that did express high levels of FoxP3. Soluble hIL-7Ralpha inhibited IL-7-induced proliferation and interferon-gamma production by mononuclear cells from RA patients. CONCLUSION: Our data suggest that enhanced expression of IL-7Ralpha and IL-7 in RA patients contributes significantly to the joint inflammation by activating T cells, B cells, and macrophages. The inhibition of IL-7R-mediated immune activation by soluble hIL-7Ralpha further indicates an important role of IL-7Ralpha in inflammatory responses in RA, suggesting IL-7Ralpha as a therapeutic target for immunotherapy in RA.

Proinflammatory mediator-induced reversal of CD4+,CD25+ regulatory T cell-mediated suppression in rheumatoid arthritis.

OBJECTIVE: We previously demonstrated that CD4+,CD25+ regulatory T (Treg) cells are present in increased numbers in the synovial fluid (SF) of rheumatoid arthritis (RA) patients and display enhanced suppressive activity as compared with their peripheral blood (PB) counterparts. Despite the presence of these immunoregulatory cells in RA, chronic inflammation persists. The purpose of the present study was to investigate whether particular proinflammatory mediators that are associated with RA could abrogate CD4+,CD25+ Treg-mediated suppression. METHODS: Monocyte phenotype was determined by flow cytometry and cytokine levels by enzyme-linked immunosorbent assay. Magnetically sorted CD4+,CD25- and CD4+,CD25+ T cells derived from the PB and SF obtained from RA patients were stimulated alone or in coculture with anti-CD3 monoclonal antibody (mAb) and autologous antigen-presenting cells, in the absence or presence of anti-CD28 mAb or the proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha), or IL-7. RESULTS: Monocytes from the SF of RA patients displayed increased expression of HLA class II molecules, CD80, CD86, and CD40 as compared with PB-derived monocytes, indicating their activated status. Mimicking this increased costimulatory potential, addition of anti-CD28 mAb to cocultures of CD4+,CD25- and CD4+,CD25+ T cells resulted in reduced CD4+,CD25+ Treg-mediated suppression in both PB and SF. Furthermore, IL-7 and, to a limited extent, TNFalpha, both of which are produced by activated monocytes and were detected in SF, abrogated the CD4+,CD25+ Treg-mediated suppression. In contrast, IL-6 did not influence Treg-mediated suppression. CONCLUSION: Our findings suggest that the interaction of CD4+,CD25+ Treg cells with activated monocytes in the joint might lead to diminished suppressive activity of CD4+,CD25+ Treg cells in vivo, thus contributing to the chronic inflammation in RA.

Persistence of interleukin 7 activity and levels on tumour necrosis factor alpha blockade in patients with rheumatoid arthritis.

OBJECTIVES: To identify the mechanism of interleukin (IL)7-stimulated tumour necrosis factor alpha (TNFalpha) production and to determine the relationship between intra-articular IL7 and TNFalpha expression levels in patients with rheumatoid arthritis (RA). In addition, the effect of TNFalpha blockade on IL7 activity and on IL7 levels was studied. METHODS: The effect of IL7 on isolated CD4 T cells and CD14 monocytes/macrophages was studied. IL7 and TNFalpha levels were measured in the synovial fluid of patients with RA. In RA synovial tissue, IL7 and TNFalpha expression was assessed in addition to IL1beta, numbers of inflammatory cells and adhesion molecule expression. The extent to which TNFalpha blockade could prevent IL7-induced lymphocyte responses was studied in vitro. In addition, regulation of serum IL7 levels on anti-TNFalpha therapy (adalimumab) was studied. RESULTS: IL7 induced cell contact-dependent TNFalpha production by cocultures of T cells and monocytes, but not by T cells and monocytes cultured separately. IL7 and TNFalpha levels in RA synovial fluid and synovial tissue significantly correlated. IL7-stimulated lymphocyte responses were not inhibited by TNFalpha blockade. Circulating IL7 levels were significantly reduced in patients who successfully responded to anti-TNFalpha treatment. However, IL7 levels persisted in non-responders. CONCLUSION: The present data suggest that IL7 is an important inducer of T cell-dependent TNFalpha production in RA joints. This may contribute to the correlation of intra-articular IL7 and TNFalpha in these joints. Furthermore, the persistence of IL7-induced inflammatory activity on TNFalpha blockade in vitro and persistence of IL7 levels and disease activity in anti-TNFalpha non-responders suggest that IL7 might additionally promote TNFalpha-independent inflammation.