Extracellular vesicle miRNA predict FDG-PET status in patients with classical Hodgkin Lymphoma.

Minimally-invasive tools to assess tumour presence and burden may improve clinical management. FDG-PET (metabolic) imaging is the current gold standard for interim response assessment in patients with classical Hodgkin Lymphoma (cHL), but this technique cannot be repeated frequently. Here we show that microRNAs (miRNA) associated with tumour-secreted extracellular vesicles (EVs) in the circulation of cHL patients may improve response assessment. Small RNA sequencing and qRT-PCR reveal that the relative abundance of cHL-expressed miRNAs, miR-127-3p, miR-155-5p, miR-21-5p, miR-24-3p and let-7a-5p is up to hundred-fold increased in plasma EVs of cHL patients pre-treatment when compared to complete metabolic responders (CMR). Notably, in partial responders (PR) or treatment-refractory cases (n = 10) the EV-miRNA levels remain elevated. In comparison, tumour specific copy number variations (CNV) were detected in cell-free DNA of 8 out of 10 newly diagnosed cHL patients but not in patients with PR. Combining EV-miR-127-3p and/or EV-let-7a-5p levels, with serum TARC (a validated protein cHL biomarker), increases the accuracy for predicting PET-status (n = 129) to an area under the curve of 0.93 (CI: 0.87-0.99), 93.5% sensitivity, 83.8/85.0% specificity and a negative predictive value of 96%. Thus the level of tumour-associated miRNAs in plasma EVs is predictive of metabolic tumour activity in cHL patients. Our findings suggest that plasma EV-miRNA are useful for detection of small residual lesions and may be applied as serial response prediction tool.

Gradual tapering TNF inhibitors versus conventional synthetic DMARDs after achieving controlled disease in patients with rheumatoid arthritis: first-year results of the randomised controlled TARA study.

OBJECTIVES: The aim of this study is to evaluate the effectiveness of two tapering strategies after achieving controlled disease in patients with rheumatoid arthritis (RA), during 1 year of follow-up. METHODS: In this multicentre single-blinded (research nurses) randomised controlled trial, patients with RA were included who achieved controlled disease, defined as a Disease Activity Score (DAS) ≤ 2.4 and a Swollen Joint Count (SJC) ≤ 1, treated with both a conventional synthetic disease-modifying antirheumatic drugs (csDMARD) and a TNF inhibitor. Eligible patients were randomised into gradual tapering csDMARDs or TNF inhibitors. Medication was tapered if the RA was still under control, by cutting the dosage into half, a quarter and thereafter it was stopped. Primary outcome was proportion of patients with a disease flare, defined as DAS > 2.4 and/or SJC > 1. Secondary outcomes were DAS, European Quality of Life-5 Dimensions (EQ5D) and functional ability (Health Assessment Questionnaire Disability Index [HAQ-DI]) after 1 year and over time. RESULTS: A total of 189 patients were randomly assigned to tapering csDMARDs (n = 94) or tapering anti-TNF (n = 95). The cumulative flare rates in the csDMARD and anti-TNF tapering group were, respectively, 33 % (95% CI,24% to 43 %) and 43 % (95% CI, 33% to 53 % (p = 0.17). Mean DAS, HAQ-DI and EQ-5D did not differ between tapering groups after 1 year and over time. CONCLUSION: Up to 9 months, flare rates of tapering csDMARDs or TNF inhibitors were similar. After 1 year, a non-significant difference was found of 10 % favouring csDMARD tapering. Tapering TNF inhibitors was, therefore, not superior to tapering csDMARDs. From a societal perspective, it would be sensible to taper the TNF inhibitor first, because of possible cost reductions and less long-term side effects. TRIAL REGISTRATION NUMBER: NTR2754.

Long-Term Outcomes of Patients With Recent-Onset Rheumatoid Arthritis After 10 Years of Tight Controlled Treatment: A Randomized Trial.

BACKGROUND: Treat-to-target therapy is effective for patients with rheumatoid arthritis (RA), but long-term results of continued targeted treatment are lacking. OBJECTIVE: To evaluate long-term outcomes in patients with early RA after 10 years of targeted treatment in 4 treatment strategies. DESIGN: Randomized trial. (Nederlands Trial Register: NTR262 and NTR265). SETTING: The Netherlands. PATIENTS: 508 patients with early active RA. INTERVENTION: Sequential monotherapy (strategy 1), step-up combination therapy (strategy 2), or initial combination therapy with prednisone (strategy 3) or with infliximab (strategy 4), all followed by targeted treatment aiming at low disease activity. MEASUREMENTS: Functional ability (Health Assessment Questionnaire [HAQ] score) and radiographic progression (Sharp-van der Heijde score) were primary end points. Survival in the study population was compared with the general population using the standardized mortality ratio. RESULTS: 195 of 508 of patients (38%) dropped out of the study (28% in strategy 4 vs. 40% to 45% in strategies 1 to 3, respectively). At year 10, mean HAQ score (SD) was 0.57 (0.56); 53% and 14% of patients were in remission and drug-free remission, respectively, without differences among the strategies. Over 10 years, mean HAQ scores were 0.69, 0.72, 0.64, and 0.58 in strategies 1 to 4, respectively (differences not clinically relevant). Radiographic damage was limited for all strategies, with mean Sharp-van der Heijde estimates during follow-up of 11, 8, 8, and 6 in strategies 1 to 4, respectively (P = 0.15). Standardized mortality ratio was 1.16 (95% CI, 0.92 to 1.46) based on 72 observed and 62 expected deaths, with similar survival among the 4 strategies (P = 0.81). LIMITATION: Dropout rate varied by strategy. CONCLUSION: In patients with early RA, initial (temporary) combination therapy results in faster clinical improvement and targeted treatment determines long-term outcomes. Drug-free remission, with prevention of functional deterioration and clinically relevant radiographic damage, and normalized survival are realistic outcomes. PRIMARY FUNDING SOURCE: Dutch College of Health Insurance Companies, Schering-Plough, and Janssen.

The association of treatment response and joint damage with ACPA-status in recent-onset RA: a subanalysis of the 8-year follow-up of the BeSt study.

OBJECTIVE: Anticitrullinated protein antibodies (ACPAs) are suggested to identify different subsets of patients with rheumatoid arthritis (RA). The authors compared the clinical and radiological responses to Disease Activity Score (DAS)-steered treatment in patients with RA positive or RA negative for ACPA. METHODS: In the BehandelStrategieën (BeSt) study, 508 patients with recent-onset RA were randomised to four treatment strategies aimed at a DAS ≤2.4. Risks of damage progression and (drug-free) remission in 8 years were compared for ACPA-positive and ACPA-negative patients using logistic regression analysis. Functional ability and DAS components over time were compared using linear mixed models. RESULTS: DAS reduction was achieved similarly in ACPA-positive and ACPA-negative patients in all treatment strategy groups, with a similar need to adjust treatment because of inadequate response. Functional ability and remission rates were not different for ACPA-positive and ACPA-negative patients. ACPA-positive patients had more radiological damage progression, especially after initial monotherapy. They had a lower chance of achieving (persistent) drug-free remission. CONCLUSION: Clinical response to treatment was similar in ACPA-positive and ACPA-negative patients. However, more ACPA-positive patients, especially those treated with initial monotherapy, had significant radiological damage progression, indicating that methotrexate monotherapy and DAS- (≤2.4) steered treatment might be insufficient to adequately suppress joint damage progression in these patients.

Medical-grade honey enriched with antimicrobial peptides has enhanced activity against antibiotic-resistant pathogens.

Honey has potent activity against both antibiotic-sensitive and -resistant bacteria, and is an interesting agent for topical antimicrobial application to wounds. As honey is diluted by wound exudate, rapid bactericidal activity up to high dilution is a prerequisite for its successful application. We investigated the kinetics of the killing of antibiotic-resistant bacteria by RS honey, the source for the production of Revamil® medical-grade honey, and we aimed to enhance the rapid bactericidal activity of RS honey by enrichment with its endogenous compounds or the addition of antimicrobial peptides (AMPs). RS honey killed antibiotic-resistant isolates of Pseudomonas aeruginosa, Staphylococcus epidermidis, Enterococcus faecium, and Burkholderia cepacia within 2 h, but lacked such rapid activity against methicillin-resistant S. aureus (MRSA) and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. It was not feasible to enhance the rapid activity of RS honey by enrichment with endogenous compounds, but RS honey enriched with 75 µM of the synthetic peptide Bactericidal Peptide 2 (BP2) showed rapid bactericidal activity against all species tested, including MRSA and ESBL E. coli, at up to 10-20-fold dilution. RS honey enriched with BP2 rapidly killed all bacteria tested and had a broader spectrum of bactericidal activity than either BP2 or honey alone.

Clinical synovitis in a particular joint is associated with progression of erosions and joint space narrowing in that same joint, but not in patients initially treated with infliximab.

OBJECTIVES: To assess the relationship between joint tenderness, swelling and joint damage progression in individual joints and to evaluate the influence of treatment on these relationships. METHODS: First-year data of the Behandel Strategieën (BeSt) study were used, in which patients recently diagnosed as having rheumatoid arthritis (RA) were randomly assigned into four different treatment strategies. Baseline and 1-year x-rays of the hands and feet were assessed using the Sharp-van der Heijde score (SHS). With generalised estimating equations, 3-monthly assessments of tender and swollen joints of year 1 were related to erosion progression, joint space narrowing (JSN) progression and total SHS progression at the individual joint level (definition > 0.5 SHS units) in year 1, corrected for potential confounders and within-patient correlation for multiple joints per patient. RESULTS: During year 1, 59% of all 13 959 joints analysed were ever tender and 45% ever swollen, 2.1% showed erosion progression, 1.9% JSN progression and 3.6% SHS progression. Swelling and tenderness were both independently associated with erosion and JSN progression with comparable OR, although with higher OR in the hands than in the feet. Local swelling and tenderness were not associated with local damage progression in patients initially treated with infliximab. CONCLUSION: Clinical signs of synovitis are associated with erosion and JSN progression in individual joints after 1 year in RA. A disconnect between synovitis and joint damage progression was observed at joint level in patients who were treated with methotrexate and infliximab as initial treatment, confirming the disconnect between synovitis and the development of joint damage in tumour necrosis factor blockers seen at patient level.

Identification of an internalising peptide in differentiated Calu-3 cells by phage display technology; application to gene delivery to the airways.

Differentiated, human submucosal-gland carcinoma, Calu-3 cell monolayers were used as in vitro model for the airway epithelium. Internalised phage were selected from a recombinant pComb8 phage library by repetitive cycles of bio-panning on Calu-3 monolayers, protease K degradation, cell-lysis and amplification. After four selection rounds, sequence analysis of 15 enriched phage colonies revealed two clones of 73 and 27% abundancy, named IB1 and IB2, respectively. The IB2 sequence was eliminated due to a frame shift. IB1-phage internalisation at 4 degrees C was significantly lower (P < 0.05) than at 37 degrees C, suggesting involvement of a receptor-mediated endocytosis pathway. The IB1 peptide was synthesised, biotinylated and complexed to streptavidin. IB1/streptavidin-complexes co-administrated with PEI/DNA-polyplexes, enhanced polyplex transfection efficiency, dose dependently, by 6- and 4-fold in Calu-3 cells. IB1/Alexa488-streptavidin complexes were used for confocal laser-scanning microscopy (CLSM) visualisation and showed basolateral localisation in membrane associated and internalising vesicles. This study demonstrates the potential of phage display technology for identification of internalising peptide-epitopes that can enhance gene delivery efficiency in differentiated airway epithelial cells.

NEC1, a novel gene, highly expressed in nectary tissue of Petunia hybrida.

To study the molecular regulation of nectary development, we cloned NEC1, a gene predominantly expressed in the nectaries of Petunia hybrida, by using the differential display RT-PCR technique. The secondary structure of the putative NEC1 protein is reminiscent of a transmembrane protein, indicating that the protein is incorporated into the cell membrane or the cytoplast membrane. Immunolocalization revealed that NEC1 protein is present in the nectaries. Northern blot analyses showed that NEC1 is highly expressed in nectary tissue and weakly in the stamen. GUS expression driven by the NEC1 promoter revealed GUS activity in the outer nectary parenchyma cells, the upper part of the filament and the anther stomium. The same expression pattern was observed in Brassica napus. GUS expression was observed as blue spots on the surface of very young nectaries that do not secrete nectar and do accumulate starch. GUS expression was highest in open flowers in which active secretion of nectar and starch hydrolysis had taken place. Ectopic expression of NEC1 resulted in transgenic plants that displayed a phenotype with leaves having 3-4 times more phloem bundles in mid-veins than the wild-type Petunia. The possible role of NEC1 gene in sugar metabolism and nectar secretion is discussed.

Effect of simulated American, Bulgarian, and Japanese human diets and of selenium supplementation on the incidence of virally induced mammary tumors in female mice.

In attempts to simulate the effects of diet on human breast cancer development groups of female C3H mice infected with mammary tumor virus (MMTV-) were maintained on diets formulated to resemble the typical American, Bulgarian, and Japanese human diets. The incidence of mammary tumors was the highest (84%) in group of mice receiving the simulated meat- and fat-rich American diet, which was also low in selenium (Se content: 0.15 ppm). The appearance of mammary tumors was delayed in the mice maintained on the simulated Bulgarian diet, and the final tumor incidence (27%) paralleled the correspondingly lower Bulgarian breast cancer incidence. The simulated Bulgarian diet contained more Se (0.25 ppm), and was lower in fat, meat, and sugar and higher in complex carbohydrates (cereals) than the simulated American diet. In the mice fed the simulated Japanese diet, the appearance of mammary tumors was also delayed, and the tumor incidence was diminished to 47%. In this diet, fish meal was a major source of Se, which is known to have low bioavailability. Additional supplementation of the Japanese-type diet with bioavailable Se (1 ppm) lowered the tumor incidence to 10%. Based on these studies, recommendations are made for breast cancer risk reduction by dietary means.

Selenium in the blood of Japanese and American women with and without breast cancer and fibrocystic disease.

Selenium concentrations in whole blood of Japanese and American women with and without breast cancer and benign fibrocystic breast disease were determined. The observed blood Se levels of healthy Japanese women (0.286 +/- 0.021 micrograms/ml) were similar to previously reported values for healthy Japanese adults. The Japanese patients with benign breast disease and with breast cancer exhibited blood selenium concentrations of 0.200 +/- 0.045 and 0.195 +/- 0.057 micrograms/ml, respectively. The mean blood Se concentration of Japanese breast cancer patients with recurrence was 0.188 +/- 0.061 micrograms/ml. The mean blood Se concentrations of healthy American women from San Diego, Calif., were 0.191 +/- 0.023 micrograms/ml; of women with benign fibrocystic disease, 0.142 +/- 0.010 micrograms/ml; and of breast cancer patients, 0.167 +/- 0.032 micrograms/ml. The higher blood Se concentrations of Japanese healthy subjects as compared to healthy Americans can be attributed to differences in the dietary Se intakes; low blood Se concentration may be indicative of increased breast cancer risk.