A super-SILAC based proteomics analysis of diffuse large B-cell lymphoma-NOS patient samples to identify new proteins that discriminate GCB and non-GCB lymphomas.

Diffuse large B-cell lymphoma-not otherwise specified (DLBCL-NOS) is a large and heterogeneous subgroup of non-Hodgkin lymphoma. DLBCL can be subdivided into germinal centre B-cell like (GCB) and activated B-cell like (ABC or non-GCB) using a gene-expression based or an immunohistochemical approach. In this study we aimed to identify additional proteins that are differentially expressed between GCB and non-GCB DLBCL. A reference super-SILAC mix, including proteins of eight B-cell lymphoma cell lines, was mixed with proteins isolated from seven non-GCB DLBCL and five GCB DLBCL patient tissue samples to quantify protein levels. Protein identification and quantification was performed by LC-MS. We identified a total of 4289 proteins, with a four-fold significant difference in expression between non-GCB and GCB DLBCL for 37 proteins. Four proteins were selected for validation in the same cases and replication in an independent cohort of 47 DLBCL patients by immunohistochemistry. In the validation cohort, we observed a non-significant trend towards the same differential expression pattern as observed in the proteomics. The replication study showed significant and consistent differences for two of the proteins: expression of glomulin (GLMN) was higher in GCB DLBCL, while expression of ribosomal protein L23 (RPL23) was higher in non-GCB DLBCL. These proteins are functionally linked to important pathways involving MYC, p53 and angiogenesis. In summary, we showed increased expression of RPL23 and decreased expression of GLMN in non-GCB compared to GCB DLBCL on purified primary DLBCL patient samples and replicated these results in an independent patient cohort.

The microenvironment of classical Hodgkin lymphoma: heterogeneity by Epstein-Barr virus presence and location within the tumor.

This corrects the article DOI: 10.1038/bcj.2016.26.

Allergen inhalation decreases adenosine receptor expression in sputum and blood of asthma patients.

BACKGROUND: Adenosine is a signalling nucleoside that has been proposed to contribute to the pathogenesis of asthma. Adenosine is produced in inflammatory environments and acts via adenosine receptors (A(1)R, A(2A)R, A(2B)R, and A(3)R) expressed by a wide variety of cells, resulting in pro- and anti-inflammatory effects. OBJECTIVE: To compare AR expression in asthma patients and healthy subjects, and to assess the effect of allergen challenge on AR expression of inflammatory cells and on cytokines in peripheral blood and sputum in asthma. METHODS: Asthma patients underwent an allergen challenge, and blood and induced sputum samples were taken before and 24 h after allergen challenge to study inflammatory cells numbers, AR expression and cytokine production. Blood and sputum were investigated at one time point in healthy subjects. AR expression was measured by flow cytometry (blood) or on cytospins using immunocytochemistry (sputum). Cytokines (luminex, ELISA) and adenosine (HPLC) were measured in sputum supernatant. RESULTS: The percentage of A(2B)R expressing neutrophils in sputum was lower in asthma patients than in healthy subjects (P = 0.016). Allergen challenge decreased A(1)R and A(2A)R expression on neutrophils and A(1)R expression on T cells in peripheral blood (all P < 0.05). Allergen challenge increased IL-8 levels and eosinophil numbers (P < 0.05), whereas it decreased thymic stromal lymphopoietin levels and the percentage of A(1)R expressing macrophages in induced sputum (P < 0.05). CONCLUSIONS: Allergen challenge has a down-regulatory effect on AR expression in asthma, suggesting a contribution of adenosine-related effector mechanisms in the pathophysiology.

Systemic and local interferon-gamma production following Mycobacterium ulcerans infection.

Buruli ulcer disease (BUD) is an emerging predominantly tropical disease caused by Mycobacterium ulcerans. The initial pre-ulcerative skin lesion often breaks down into an ulcer with undermined edges. Healing is common but may require considerable time, and scarring often results in functional limitations. Considerable evidence has now emerged that patients with early BUD cannot mount a sufficient protective T helper 1 (Th1) cell response to M. ulcerans, but uncertainty remains as to whether immune protection is restored over time. This study investigates the Th1 cell response of patients with various stages of BUD on mycobacterial antigens. We measured interferon (IFN)-gamma levels after ex vivo whole blood stimulation with tuberculin purified protein derivative (PPD), and compared the Th1 cell response of individuals with pre-ulcerative, ulcerative and healed BUD as well as healthy controls. Moreover, the systemic Th1 cell response was related to histopathological features in the various stages of surgically resected BUD lesions. We show that patients with ulcerative and healed BUD produce significantly higher IFN-gamma levels after mycobacterial ex vivo whole blood stimulation than healthy controls, and that patients with a granulomatous tissue response produce higher IFN-gamma levels than individuals without. We therefore suggest that the mounted Th1 cell response in ulcerative BUD patients might be related to their histopathological tissue response.

Effect of 1-year smoking cessation on airway inflammation in COPD and asymptomatic smokers.

Smoking cessation is the only treatment in patients with chronic obstructive pulmonary disease (COPD) effective in slowing down disease progression. Its effect on airway inflammation in COPD is unknown, although cross-sectional studies suggest ongoing inflammation in ex-smokers. In order to elucidate the effect of smoking cessation on airway inflammation, 28 smokers with COPD (mean age: 55 yrs; forced expiratory volume in one second: 71% predicted) and 25 asymptomatic smokers with normal lung function (aged 50 yrs) were included in a 1-yr smoking cessation programme. Effects of smoking cessation on airway inflammation were investigated in bronchial biopsies (baseline, 12 months) and sputum samples (baseline, 2, 6 and 12 months). In the 12 candidates with COPD who successfully ceased smoking, airway inflammation persisted in bronchial biopsies, while the number of sputum neutrophils, lymphocytes, interleukin (IL)-8 and eosinophilic-cationic-protein levels significantly increased at 12 months. In the 16 asymptomatic smokers who successfully quitted, inflammation significantly reduced (i.e. number of sputum macrophages, percentage of eosinophils and IL-8 levels) or did not change. The current authors suggest that the observed persistent airway inflammation in patients with chronic obstructive pulmonary disease is related to repair of tissue damage in the airways. It remains to be elucidated whether this reflects a beneficial or detrimental effect.

Smoking cessation improves both direct and indirect airway hyperresponsiveness in COPD.

Smoking induces chronic obstructive pulmonary disease (COPD) and is associated with airway inflammation and airway hyperresponsiveness (AHR). It has not been studied in COPD whether direct (methacholine) and indirect (adenosine-5'-monophosphate (AMP)) stimuli are associated with airway inflammation and neither whether smoking cessation improves these features. The current authors cross-sectionally investigated the relationship of AHR to methacholine and AMP with lung function and inflammatory cells in the sputum of 33 smokers with COPD. In addition, changes in these parameters were prospectively assessed in 14 smokers who successfully quit smoking for 1 yr. The presence of AHR to both methacholine and AMP was associated with lower lung function, but not with sputum inflammation. AHR to methacholine and AMP improved significantly after a 1-yr smoking cessation, yet this was unrelated to changes in sputum cell counts. The numbers of neutrophils and epithelial cells significantly increased with smoking cessation. Both direct and indirect airway hyperresponsiveness are associated with lower lung function, but not with sputum inflammation in chronic obstructive pulmonary disease. Interestingly, 1-yr smoking cessation improved both direct and indirect airway hyperresponsiveness, yet without a significant association with changes in lung function or sputum inflammation. Thus, other factors are likely to induce these improvements, e.g. a reduction in stimulation of irritant receptors, airway wall changes or mucus hypersecretion.

Vascular adhesion molecules in nocturnal asthma: a possible role for VCAM-1 in ongoing airway wall inflammation.

BACKGROUND: Increased airway inflammation at night is thought to be one of the underlying mechanisms in nocturnal asthma. Vascular adhesion molecules may be important for the recruitment of inflammatory cells in the process of asthmatic airway inflammation. OBJECTIVE: To determine the possible role of vascular adhesion molecules in increased airway inflammation at night in subjects with nocturnal asthma. METHODS: Bronchial biopsies were obtained at 16.00 h and 04.00 h from 13 healthy controls, 15 asthmatic patients with PEF variation < or = 15% and 10 asthmatic patients with PEF variation > 15%. Biopsies were snap-frozen and double-immunostained for CD31 in combination with P-selectin, E-selectin, ICAM-1 or VCAM-1. RESULTS: No significant day-night differences in expression of adhesion molecules were found in any of the three groups. The percentage of VCAM-1 positive vessels in biopsies of asthmatic patients was higher than in biopsies of healthy controls: 5.8 vs 2.5% (P < 0.05) at 16.00h and 11 vs 0% (P<0.05) at 04.00 h. In asthma, VCAM-1 expression was correlated with the number of EG2 positive cells: at 16.00 h (rho = 0.57, P < 0.01) as well as at 04.00 h (rho = 0.64, P< 0.01). Moreover, VCAM-1 expression was correlated with the number of CD25 positive cells at 16.00 h (rho = 0.43, P < 0.05) and at 04.00 h (rho = 0.41, P < 0.05). CONCLUSION: Increased nocturnal airway obstruction in asthma is not associated with an increased nocturnal expression of vascular E-selectin, P-selectin, ICAM-1 or VCAM-1. The relationship between vascular VCAM-1 expression and sub-mucosal EG2 and CD25 positive cells, both at 16.00 h and 04.00 h, suggests a role for VCAM-1 in the ongoing airway wall inflammation of asthma.

Techniques in human airway inflammation: differences in plastic-embedded and snap-frozen sections for CD3, CD4, and CD8 immunostaining of bronchial biopsy specimens.

Today, the quantification of inflammatory cells in human airway biopsies might be facilitated by better morphologic resolution provided by special resin (plastic)-embedding techniques. The present study compares the numbers of CD3-, CD4-, and CD8-positive cells in glycolmethacrylate-embedded versus snap-frozen biopsy specimens of normal bronchial mucosa in 10 patients with various pulmonary diseases. In general, larger numbers of CD3-, CD4-, and CD8-positive cells were counted in snap-frozen specimens than in plastic-embedded ones. Loss of antigenic properties during storage of plastic-embedded tissue (blocks) might have contributed to the weak correlation between both methods. An additional study showed that the number of CD3-, CD4-, and CD8-positive cells decreased significantly within a few months after embedding in glycolmethacrylate. Therefore, we recommend processing glycolmethacrylate-embedded specimens as soon as possible. For standard evaluation of established inflammatory cell parameters such as CD3, CD4, CD8, and EG2, frozen tissue is preferable because of the ease of the method and its reliable cell counting. Because glycolmethacrylate-embedded tissue shows superior morphologic resolution, under strict rules, this method seems attractive for the study, in particular, of cell-cell and cell-matrix relationships.

Interleukin-4, interferon-gamma and interleukin-5 in peripheral blood of children with moderate atopic asthma.

BACKGROUND: In asthmatic inflammation, TH2 cells play an important role. TH2 cells specifically secrete cytokines like IL-4 and IL-5. IL-4 stimulates IgE production and IL-5 is involved in hemopoiesis, chemotaxis, priming and activation of eosinophils. IFNgamma, produced by TH1 cells, has an inhibitory action on IgE production. OBJECTIVES: To investigate the TH1/TH2-cell pattern in the cytokine production of peripheral blood of asthmatic children. We determined IL-4, IFNgamma and IL-5 in serum and in supernatants of unstimulated and stimulated (24 h with Concanavaline A) cultures of peripheral blood mononuclear cells (PBMCs) in 22 children with moderate asthma (mean age 9.3 years) and in 17 healthy controls (mean age 10.3 years). All children visited the out-patient department (OPD) where history taking, physical examination and blood sampling took place. Children younger than 8 years of age performed symptom and peak flow registration during 1 week after the visit to the OPD. RESULTS: The number of eosinophils were significantly higher in children with asthma, compared with healthy controls. The concentration of IFNgamma in supernatants of cultures of stimulated PBMCs was significantly lower and the ratio of IL-4/IFNgamma was significantly higher in children with asthma compared with healthy controls. The FEV1 was directly and IgE was inversely related to the concentration of IFNgamma in supernatants of cultures of stimulated PBMCs. CONCLUSION: IFNgamma may play an important role in the pathophysiology of childhood atopic asthma.

Inflammatory cell number and mediators in bronchoalveolar lavage fluid and peripheral blood in subjects with asthma with increased nocturnal airways narrowing.

BACKGROUND: Increased nocturnal airways narrowing (NAN) in asthma is thought to occur as the result of intensification of inflammatory processes in the airways. In this study we investigated the presence of inflammatory cells and mediators in bronchoalveolar lavage (BAL) fluid and peripheral blood (PB) and assessed their relationship with the occurrence of increased NAN. METHODS: BAL fluid and PB samples were assessed at 16:00 and 04:00 hours, separated by 7 days or more, in eight nonatopic healthy subjects (group 1) and 17 atopic subjects with asthma who were using inhaled bronchodilators only. The latter subjects were prospectively assigned to groups with and without NAN, as defined by a mean circadian peak expiratory flow variation of less than 15% (group 2) and 15% or more (group 3), respectively. RESULTS: Significantly higher eosinophil numbers and inflammatory activation products (eosinophil cationic protein, eosinophil-derived neurotoxin, histamine) were found in BAL fluid and PB from subjects with asthma in comparison with control subjects. However, increased NAN was not generally associated with a circadian fluctuation in cell number and inflammatory mediators in BAL fluid and PB. No differences in inflammatory cell numbers existed that distinguished between groups 2 and 3. However, in group 3 significantly higher BAL prostaglandin D2 levels (70 vs 24 pg/ml; range, 28 to 102 vs 11 to 90 pg/ml; p = 0.04) and serum eosinophil cationic protein levels (17.6 vs 16.1 ng/ml; range, 6.3 to 17.5 vs 6.3 to 60.3 ng/ml; p = 0.03) at 16:00 hours were detected compared with group 2. CONCLUSIONS: Our findings suggest that increased NAN is more likely to occur in subjects with asthma with ongoing increased cellular activation during the day.

Lymphocyte and macrophage activation in bronchoalveolar lavage fluid in nocturnal asthma.

Increased nocturnal airway narrowing is thought to occur as a consequence of an intensification of inflammatory processes at night. Lymphocyte and alveolar macrophage (AM) activation are thought to be associated with the clinical expression of asthma, and may be important in the occurrence of nocturnal asthma as well. The expression of CD25 and HLA-DR receptors on lymphocytes from bronchoalveolar lavage (BAL) and peripheral blood (PB) CD4+, as well as of CD14, IgG Fc, and CD11/CD18 leukocyte adhesion receptors on AM in BAL fluid and monocytes in PB, were determined at 16.00 and 04.00 h by flow cytometry. Their relationship with the occurrence of nocturnal asthma was investigated in eight nonatopic controls (Group 1) and 17 atopic asthmatic subjects, prospectively assigned to groups with a mean circadian peak expiratory flow (PEF) variation < 15% (Group 2) and > or = 15% (Group 3). The occurrence of an increased circadian variation in PEF in asthmatic subjects was on the whole not associated with a day-night fluctuation in lymphocyte numbers and subsets in PB or BAL fluid, nor with day-night changes in receptor expression on AM from BAL or monocytes from PB. The only exception was the presence of a greater day-night change in the proportion of HLA-DR-expressing CD4+ lymphocytes in the BAL fluid along with an increasing circadian PEF rhythm in asthmatic subjects (r = 0.68, p = 0.03). A further finding was that a lower number of BAL CD4+ lymphocytes at daytime was significantly related to a higher circadian PEF variation in asthmatic subjects (r = -0.66, p = 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

[Studies on intersucking in dairy cattle (author's transl)]. / Een onderzoek naar melkzuigen bij runderen.

Intersucking occurred on nearly 50 per cent of the farms in the Province of Utrecht. The incidence was not affected by housing systems, the size of the herd and the surface area available to each cow in cubicle stalls. It is unlikely that the animals imitate intersucking by others. Intersucking often occurs prior to evening milking, the sucking animal usually forming a steady pair with the suckled cow. Of four A.I. bulls studied, one had a significantly higher proportion of intersucking heifers among his offspring than the others. Some other factors possibly affecting the incidence of intersucking, such as the rearing system, composition of the ration and the space allotted to each cow for feeding were not studied because of insufficient information or inadequate variation of the material.