Paracrine effects of uterine leucocytes on gene expression of human uterine stromal fibroblasts

The endometrium contains a distinct population of immune cells that undergo cyclic changes during the menstrual cycle and implantation. The majority of these leucocytes are uterine NK (uNK) cells, however how these cells interact with uterine stromal fibroblasts remains unclear. We therefore investigated the paracrine effect of medium conditioned by uterine decidual leucocytes (which are enriched for uNK cells) on the gene expression profile of endometrial stromal fibroblasts in vitro using a cDNA microarray. Our results, verified by real-time PCR, ELISA and FACS analysis, reveal that soluble factors from uterine leucocytes substantially alter endometrial stromal fibroblast gene expression. The largest group of up-regulated genes found was chemokines and cytokines. These include IL-8, CCL8 and CXCL1, which have also been shown to be stimulated by contact of stromal fibroblasts with trophoblast, suggesting that uNK cells work synergistically to support trophoblast migration during implantation. The decidual leucocytes also up-regulated IL-15 and IL-15Ralpha in stromal fibroblasts which could produce a niche for uNK cells allowing proliferation within and recruitment into the uterus, as seen in bone marrow. Overall this study demonstrates, for the first time, the paracrine communication between uterine leucocytes and uterine stromal fibroblasts, and adds to the understanding of how the uterine immune system contributes to the changes seen within the cycling endometrium.

The Akt/PKB pathway is constitutively activated in Theileria-transformed leucocytes, but does not directly control constitutive NF-kappaB activation

The intracellular protozoan parasites Theileria parva and Theileria annulata transform leucocytes by interfering with host cell signal transduction pathways. They differ from tumour cells, however, in that the transformation process can be entirely reversed by elimination of the parasite from the host cell cytoplasm using a specific parasiticidal drug. We investigated the state of activation of Akt/PKB, a downstream target of PI3-K-generated phosphoinositides, in Theileria-transformed leucocytes. Akt/PKB is constitutively activated in a PI3-K- and parasite-dependent manner, as judged by the specific phosphorylation of key residues, in vitro kinase assays and its cellular distribution. In previous work, we demonstrated that the parasite induces constitutive activation of the transcription factor NF-kappaB, providing protection against spontaneous apoptosis that accompanies transformation. In a number of other systems, a link has been established between the PI3-K-Akt/PKB pathway and NF-kappaB activation, resulting in protection against apoptosis. In Theileria-transformed leucocytes, activation of the NF-kappaB and the PI3-K-Akt/PKB pathways are not directly linked. The PI3-K-Akt/PKB pathway does not contribute to the persistent induction of IkappaBalpha phosphorylation, NF-kappaB DNA-binding or transcriptional activity. We show that the two pathways are downregulated with different kinetics when the parasite is eliminated from the host cell cytoplasm and that NF-kappaB-dependent protection against apoptosis is not dependent on a functional PI3-K-Akt/PKB pathway. We also demonstrate that Akt/PKB contributes, at least in part, to the proliferation of Theileria-transformed T cells.

WASP plays a novel role in regulating platelet responses dependent on alphaIIbbeta3 integrin outside-in signalling

The most consistent feature of Wiskott Aldrich syndrome (WAS) is profound thrombocytopenia with small platelets. The responsible gene encodes WAS protein (WASP), which functions in leucocytes as an actin filament nucleating agent -yet- actin filament nucleation proceeds normally in patient platelets regarding shape change, filopodia and lamellipodia generation. Because WASP localizes in the platelet membrane skeleton and is mobilized by alphaIIbbeta3 integrin outside-in signalling, we questioned whether its function might be linked to integrin. Agonist-induced alphaIIbbeta3 activation (PAC-1 binding) was normal for patient platelets, indicating normal integrin inside-out signalling. Inside-out signalling (fibrinogen, JON/A binding) was also normal for wasp-deficient murine platelets. However, adherence/spreading on immobilized fibrinogen was decreased for patient platelets and wasp-deficient murine platelets, indicating decreased integrin outside-in responses. Another integrin outside-in dependent response, fibrin clot retraction, involving contraction of the post-aggregation actin cytoskeleton, was also decreased for patient platelets and wasp-deficient murine platelets. Rebleeding from tail cuts was more frequent for wasp-deficient mice, suggesting decreased stabilisation of the primary platelet plug. In contrast, phosphatidylserine exposure, a pro-coagulant response, was enhanced for WASP-deficient patient and murine platelets. The collective results reveal a novel function for WASP in regulating pro-aggregatory and pro-coagulant responses downstream of integrin outside-in signalling.

Cellular actors, Toll-like receptors, and local cytokine profile in acute coronary syndromes

Inflammation plays a key role in acute coronary syndromes (ACS). Toll-like receptors (TLR) on leucocytes mediate inflammation and immune responses. We characterized leucocytes and TLR expression within coronary thrombi and compared cytokine levels from the site of coronary occlusion with aortic blood (AB) in ACS patients.

Eosinophilic vasculitis: an inhabitual and resistant manifestation of a vasculitis

A 55-year-old woman was referred because of diffuse pruritic erythematous lesions and an ischemic process of the third finger of her right hand. She was known to have anaemia secondary to hypermenorrhea. She presented six months before admission with a cutaneous infiltration on the left cubital cavity after a paravenous leakage of intravenous iron substitution. She then reported a progressive pruritic erythematous swelling of her left arm and lower extremities and trunk. Skin biopsy of a lesion on the right leg revealed a fibrillar, small-vessel vasculitis containing many eosinophils.Two months later she reported Raynaud symptoms in both hands, with a persistent violaceous coloration of the skin and cold sensation of her third digit of the right hand. A round 1.5 cm well-delimited swelling on the medial site of the left elbow was noted. The third digit of her right hand was cold and of violet colour. Eosinophilia (19 % of total leucocytes) was present. Doppler-duplex arterial examination of the upper extremities showed an occlusion of the cubital artery down to the palmar arcade on the right arm. Selective angiography of the right subclavian and brachial arteries showed diffuse alteration of the blood flow in the cubital artery and hand, with fine collateral circulation in the carpal region. Neither secondary causes of hypereosinophilia nor a myeloproliferative process was found. Considering the skin biopsy results and having excluded other causes of eosinophilia, we assumed the diagnosis of an eosinophilic vasculitis. Treatment with tacrolimus and high dose steroids was started, the latter tapered within 12 months and then stopped, but a dramatic flare-up of the vasculitis with Raynaud phenomenon occurred. A new immunosuppressive approach with steroids and methotrexate was then introduced. This case of aggressive eosinophilic vasculitis is difficult to classify into the usual forms of vasculitis and constitutes a therapeutic challenge given the resistance to current immunosuppressive regimens.

[SPECT/CT diagnostics for skeletal infections]

CLINICAL/METHODICAL ISSUE: Skeletal infections are often a diagnostic and clinical challenge. STANDARD RADIOLOGICAL METHODS: Nuclear imaging modalities used in the diagnostic workup of acute and chronic skeletal infections include three-phase bone scintigraphy and scintigraphy with labelled leucocytes. METHODICAL INNOVATIONS: The introduction of hybrid technologies, such as single photon emission computed tomography/computed tomography (SPECT/CT) has dramatically changed nuclear medical imaging of infections. PERFORMANCE: In general SPECT/CT leads to a considerably more accurate diagnosis than planar or SPECT imaging. ACHIEVEMENTS: Given the integrated acquisition of metabolic, functional and morphological information, SPECT/CT has increased in particular the specificity of three-phase skeletal scanning and scintigraphy with labeled leucocytes.

Clinical, laboratory and pathological findings in dogs experimentally infected with Angiostrongylus vasorum

The aim of this comparative study was to investigate the development of clinical signs and accompanying haematological, coproscopic and pathological findings as a basis for the monitoring of health condition of Angiostrongylus vasorum infected dogs. Six beagles were orally inoculated with 50 (n=3) or 500 (n=3) A. vasorum third stage larvae (L3) obtained from experimentally infected Biomphalaria glabrata snails. Two dogs were treated with moxidectin/imidacloprid spot-on solution and two further dogs with an oral experimental compound 92 days post infection (dpi), and were necropsied 166 dpi. Two untreated control dogs were necropsied 97 dpi. Prepatency was 47-49 days. Dogs inoculated with 500 L3 exhibited earlier (from 42 dpi) and more severe respiratory signs. Clinical signs resolved 12 days after treatment and larval excretion stopped within 20 days in all four treated dogs. Upon necropsy, 10 and 170 adult worms were recovered from the untreated dogs inoculated with 50 and 500 L3, respectively. Adult worms were also found in two treated dogs, in the absence of L1 or eggs. Despite heavy A. vasorum infection load and severe pulmonary changes including vascular thrombosis, only mild haematological changes were observed. Eosinophilia was absent but the presence of plasma cells was observed. Neutrophilic leucocytes showed a transient increase but only after treatment. Signs for coagulopathies were slight; nevertheless coagulation parameters were inoculation dose dependent. Ten weeks after treatment pulmonary fibrosis was still present. Infections starting from 50 L3 of A. vasorum had a massive impact on lung tissues and therefore on the health of affected dogs, particularly after prepatency, although only mild haematological abnormalities were evident.

Inflammation and atrial remodeling after a mountain marathon

Endurance athletes have an increased risk of atrial fibrillation. We performed a longitudinal study on elite runners of the 2010 Jungfrau Marathon, a Swiss mountain marathon, to determine acute effects of long-distance running on the atrial myocardium. Ten healthy male athletes were included and examined 9 to 1 week prior to the race, immediately after, and 1, 5, and 8 days after the race. Mean age was 34.9 ± 4.2 years, and maximum oxygen consumption was 66.8 ± 5.8 mL/kg*min. Mean race time was 243.9 ± 17.7 min. Electrocardiographic-determined signal-averaged P-wave duration (SAPWD) increased significantly after the race and returned to baseline levels during follow-up (128.7 ± 10.9 vs. 137.6 ± 9.8 vs. 131.5 ± 8.6 ms; P < 0.001). Left and right atrial volumes showed no significant differences over time, and there were no correlations of atrial volumes and SAPWD. Prolongation of the SAPWD was accompanied by a transient increase in levels of high-sensitivity C-reactive protein, proinflammatory cytokines, total leucocytes, neutrophil granulocytes, pro atrial natriuretic peptide and high-sensitivity troponin. In conclusion, marathon running was associated with a transient conduction delay in the atria, acute inflammation and increased atrial wall tension. This may reflect exercise-induced atrial myocardial edema and may contribute to atrial remodeling over time, generating a substrate for atrial arrhythmias.

Effects of tributyltin on the immune system of Japanese flounder (Paralichthys olivaceus)

Effects of tributyltin (TBT) which has been used for antifouling paint of ship's hulls and fishing nets on the immune system in Japanese flounder (Paralichthys olivaceus) were investigated. After short-term exposure to a high level of TBT, leucocytes in the head kidney from 1-year-old flounder were examined for the proportion of neutrophils in total leucocytes. Also examined were their respiratory burst activities using flow cytometry, the reduction of nitroblue tetrazolium (NBT) and lysozyme activities. Furthermore, long-term exposures to a relatively low level of TBT using young flounder were also carried out. The proportion of neutrophils in total leucocytes prepared from head kidney in each fish exposed to TBT at 20 microg/L for 5 days and the reduction of NBT by leucocytes prepared from the same experimental conditions increase compared to the control group. The contents were 42.0+/-6.8 and 52.5+/-6.3%, respectively. Significant differences of the NBT reduction were observed between 0 and 20 microg/L TBT exposure groups. On the other hand, the respiratory burst activity of cells in the exposure group clearly showed a tendency to decrease compared to the control group. Furthermore, high level of TBT also inhibited lysozyme activity which plays an important role for the bacteriocidal procedures. However, similar results were not obtained in the exposure group with a relatively low level of TBT. To determine the immunotoxic effects of TBT, infection experiments using pathogens which are naturally occurring should be further investigated.

Significantly shorter telomeres in T-cells of patients with ZAP-70+/CD38+ chronic lymphocytic leukaemia

In contrast to other B-cell neoplasias, chronic lymphocytic leukaemia (CLL) is characterized by increased non-leukaemic T-cells. In order to assess their replicative history, telomere length was analyzed in subsets of leucocytes from CLL patients. Naive and memory T-cells from ZAP-70(+)/CD38(+) patients exhibited significantly shorter average telomere lengths than ZAP-70(-)/CD38(-) patients. Compared to the age-related percentiles of telomere length values from healthy individuals practically all values of the naive and memory T-cells from the ZAP-70(+)/CD38(+) CLL patients fell below the 50th percentile. This indicated an extensive expansion and a role for T-cells in ZAP-70(+)/CD38(+) CLL patients.

CD39 is incorporated into plasma microparticles where it maintains functional properties and impacts endothelial activation

Plasma microparticles (MPs, <1.5 mum) originate from platelet and cell membrane lipid rafts and possibly regulate inflammatory responses and thrombogenesis. These actions are mediated through their phospholipid-rich surfaces and associated cell-derived surface molecules. The ectonucleotidase CD39/ecto-nucleoside triphosphate diphosphohydrolase1 (E-NTPDase1) modulates purinergic signalling through pericellular ATP and ADP phosphohydrolysis and is localized within lipid rafts in the membranes of endothelial- and immune cells. This study aimed to determine whether CD39 associates with circulating MPs and might further impact phenotype and function. Plasma MPs were found to express CD39 and exhibited classic E-NTPDase ecto-enzymatic activity. Entpd1 (Cd39) deletion in mice produced a pro-inflammatory phenotype associated with quantitative and qualitative differences in the MP populations, as determined by two dimensional-gel electrophoresis, western blot and flow cytometry. Entpd1-null MPs were also more abundant, had significantly higher proportions of platelet- and endothelial-derived elements and decreased levels of interleukin-10, tumour necrosis factor receptor 1 and matrix metalloproteinase 2. Consequently, Cd39-null MP augment endothelial activation, as determined by inflammatory cytokine release and upregulation of adhesion molecules in vitro. In conclusion, CD39 associates with circulating MP and may directly or indirectly confer functional properties. Our data also suggest a modulatory role for CD39 within MP in the exchange of regulatory signals between leucocytes and vascular cells.

Clinical parameters associated with collateral development in patients with chronic total coronary occlusion

OBJECTIVE Well-developed collaterals provide survival benefit in patients with obstructive coronary artery disease (CAD). Therefore, in this study we sought to determine which clinical variables are associated with arteriogenesis. DESIGN Clinical and laboratory variables were collected before percutaneous coronary intervention. Multivariate analysis was performed to determine which variables are associated with the collateral flow index (CFI). PATIENTS Data from 295 chronic total occlusion (CTO) patients (Bern, Switzerland, Amsterdam, the Netherlands and Jena, Germany) were pooled. In earlier studies, patients had varying degrees of stenosis. Therefore, different stages of development of the collaterals were used. In our study, a unique group of patients with CTO was analysed. INTERVENTIONS Instead of angiography used earlier, we used a more accurate method to determine CFI using intracoronary pressure measurements. CFI was calculated from the occlusive pressure distal of the coronary lesion, the aortic pressure and central venous pressure. RESULTS The mean CFI was 0.39 ± 0.14. After multivariate analysis, β blockers, hypertension and angina pectoris duration were positively associated with CFI (B: correlation coefficient β=0.07, SE=0.03, p=0.02, B=0.040, SE=0.02, p=0.042 and B=0.001, SE=0.000, p=0.02). Furthermore also after multivariate analysis, high serum leucocytes, prior myocardial infarction and high diastolic blood pressure were negatively associated with CFI (B=-0.01, SE=0.005, p=0.03, B=-0.04, SE=0.02, p=0.03 and B=-0.002, SE=0.001, p=0.011). CONCLUSIONS In this unique cohort, high serum leucocytes and high diastolic blood pressure are associated with poorly developed collaterals. Interestingly, the use of β blockers is associated with well-developed collaterals, shedding new light on the potential action mode of this drug in patients with CAD.

Clinical parameters, intestinal function, and IGF1 concentrations in colostrum-deprived and colostrum-fed newborn pony foals.

Colostrum (COL) contains cytokines and growth factors that may enhance intestinal development in neonates. The hypothesis of this study was that besides providing immunoglobulins, COL is important for intestinal function and meconium release in foals. Newborn foals were either fed COL (n = 5) or an equal amount of milk replacer (MR, n = 7) during the first 24 hours of life. To ensure passive immunity, all foals received 1 L plasma. Postnatal development, meconium release, intestinal motility, white blood cell count, insulin-like growth factor 1, and intestinal absorptive function (xylose absorption test) were evaluated. Clinical findings and meconium release were not affected by feeding of COL or MR. Ultrasonography revealed a slightly larger jejunum and stomach in group COL versus MR (P < 0.05). The percentage of polymorphonuclear leucocytes was higher in foals of group MR versus group COL (P < 0.05) and the percentage of lymphocytes was lower in MR compared with COL foals (P < 0.05). Plasma insulin-like growth factor 1 concentration increased during the first 14 days after birth in both groups. A xylose absorption test on Day 5 revealed similar increases in plasma xylose concentrations after oral intake. In conclusion, feeding of COL versus MR was without effect on meconium release and intestinal absorptive function. Differences between foals fed COL and MR with regard to intestinal function are apparently without clinical relevance. In foals that have not received maternal COL, there is no major risk of intestinal problems if they are fed MR and provided with immunoglobulins by transfusion of plasma.

IL-9 and its receptor are predominantly involved in the pathogenesis of UC.

OBJECTIVE Several pathogenic roles attributed over the past two decades to either T helper (Th)1 or Th2 cells are increasingly becoming associated with interleukin (IL)-17 and most recently IL-9 signalling. However, the implication of IL-9 in IBD has not been addressed so far. DESIGN We investigated the expression of IL-9 and IL-9R by using peripheral blood, biopsies and surgical samples. We addressed the functional role of IL-9 signalling by analysis of downstream effector proteins. Using Caco-2 cell monolayers we followed the effect of IL-9 on wound healing. RESULTS IL-9 mRNA expression was significantly increased in inflamed samples from patients with UC as compared with controls. CD3(+) T cells were major IL-9-expressing cells and some polymorphonuclear leucocytes (PMN) also expressed IL-9. IL-9 was co-localised with the key Th9 transcription factors interferon regulatory factor 4 and PU.1. Systemically, IL-9 was abundantly produced by activated peripheral blood lymphocytes, whereas its receptor was overexpressed on gut resident and circulating PMN. IL-9 stimulation of the latter induced IL-8 production in a dose-dependent manner and rendered PMN resistant to apoptosis suggesting a functional role for IL-9R signalling in the propagation of gut inflammation. Furthermore, IL-9R was overexpressed on gut epithelial cells and IL-9 induced STAT5 activation in these cells. Moreover, IL-9 inhibited the growth of Caco-2 epithelial cell monolayers in wound healing experiments. CONCLUSIONS Our results provide evidence that IL-9 is predominantly involved in the pathogenesis of UC suggesting that targeting IL-9 might become a therapeutic option for patients with UC.

A quantitative reverse-transcriptase PCR assay for the assessment of drug activities against intracellular Theileria annulata schizonts.

Intracellular schizonts of the apicomplexans Theileria annulata and Theileria parva immortalize bovine leucocytes thereby causing fatal immunoproliferative diseases. Buparvaquone, a hydroxynaphthoquinone related to parvaquone, is the only drug available against Theileria. The drug is only effective at the onset of infection and emerging resistance underlines the need for identifying alternative compounds. Current drug assays employ monitoring of proliferation of infected cells, with apoptosis of the infected host cell as a read-out, but it is often unclear whether active compounds directly impair the viability of the parasite or primarily induce host cell death. We here report on the development of a quantitative reverse transcriptase real time PCR method based on two Theileria genes, tasp and tap104, which are both expressed in schizonts. Upon in vitro treatment of T. annulata infected bovine monocytes with buparvaquone, TaSP and Tap104 mRNA expression levels significantly decreased in relation to host cell actin already within 4 h of drug exposure, while significant differences in host cell proliferation were detectable only after 48-72 h. TEM revealed marked alterations of the schizont ultrastructure already after 2 h of buparvaquone treatment, while the host cell remained unaffected. Expression of TaSP and Tap104 proteins showed a marked decrease only after 24 h. Therefore, the analysis of expression levels of mRNA coding for TaSP and Tap104 allows to directly measuring impairment of parasite viability. We subsequently applied this method using a series of compounds affecting different targets in other apicomplexan parasites, and show that monitoring of TaSP- and Tap104 mRNA levels constitutes a suitable tool for anti-theilerial drug development.