Inhibition of miR-9 de-represses HuR and DICER1 and impairs Hodgkin lymphoma tumour outgrowth in vivo.

MicroRNAs are important regulators of gene expression in normal development and disease. miR-9 is overexpressed in several cancer forms, including brain tumours, hepatocellular carcinomas, breast cancer and Hodgkin lymphoma (HL). Here we demonstrated a relevance for miR-9 in HL pathogenesis and identified two new targets Dicer1 and HuR. HL is characterized by a massive infiltration of immune cells and fibroblasts in the tumour, whereas malignant cells represent only 1% of the tumour mass. These infiltrates provide important survival and growth signals to the tumour cells, and several lines of evidence indicate that they are essential for the persistence of HL. We show that inhibition of miR-9 leads to derepression of DICER and HuR, which in turn results in a decrease in cytokine production by HL cells followed by an impaired ability to attract normal inflammatory cells. Finally, inhibition of miR-9 by a systemically delivered antimiR-9 in a xenograft model of HL increases the protein levels of HuR and DICER1 and results in decreased tumour outgrowth, confirming that miR-9 actively participates in HL pathogenesis and points to miR-9 as a potential therapeutic target.

The linked units of 5S rDNA and U1 snDNA of razor shells (Mollusca: Bivalvia: Pharidae).

The linkage between 5S ribosomal DNA and other multigene families has been detected in many eukaryote lineages, but whether it provides any selective advantage remains unclear. In this work, we report the occurrence of linked units of 5S ribosomal DNA (5S rDNA) and U1 small nuclear DNA (U1 snDNA) in 10 razor shell species (Mollusca: Bivalvia: Pharidae) from four different genera. We obtained several clones containing partial or complete repeats of both multigene families in which both types of genes displayed the same orientation. We provide a comprehensive collection of razor shell 5S rDNA clones, both with linked and nonlinked organisation, and the first bivalve U1 snDNA sequences. We predicted the secondary structures and characterised the upstream and downstream conserved elements, including a region at -25 nucleotides from both 5S rDNA and U1 snDNA transcription start sites. The analysis of 5S rDNA showed that some nontranscribed spacers (NTSs) are more closely related to NTSs from other species (and genera) than to NTSs from the species they were retrieved from, suggesting birth-and-death evolution and ancestral polymorphism. Nucleotide conservation within the functional regions suggests the involvement of purifying selection, unequal crossing-overs and gene conversions. Taking into account this and other studies, we discuss the possible mechanisms by which both multigene families could have become linked in the Pharidae lineage. The reason why 5S rDNA is often found linked to other multigene families seems to be the result of stochastic processes within genomes in which its high copy number is determinant.

Ambient fauna impairs parasite transmission in a marine parasite-host system.

To understand possible factors controlling transmission of trematode larvae between first and second intermediate hosts we examined the impact of ambient fauna on parasite transmission in a marine intertidal parasite-host association. Cockle hosts (Cerastoderma edule) kept together with selected co-occurring macrozoobenthic species in mesocosms acquired a lower parasite load compared to cockles kept alone, when targeted by cercariae of the trematode Himasthla elongata. The reduction of parasite load in the cockles differed between the 7 macrozoobenthic species tested and was between 35 and 91%. Three different types of reduction could be distinguished: (1) predators (Carcinus maenas, Crangon crangon) actively preying upon cercariae, (2) non-host filter feeders (Crepidula fornicata, Mya arenaria, Crassostrea gigas) filtering cercariae but not becoming infected and (3) alternative hosts (Mytilus edulis, Macoma balthica) becoming infected by the cercariae and thus distracting cercariae from the target hosts. In addition, interference competition may occur in the form of disturbance of cockles by ambient organisms resulting in lower filtration rates and subsequently lower parasite loads. Our results suggest that the species composition and relative abundance of the ambient fauna of parasite-host systems play an important role in controlling trematode transmission rates in benthic marine systems.

The role of biotic factors in the transmission of free-living endohelminth stages.

The transmission success of free-living larval stages of endohelminths is generally modulated by a variety of abiotic and biotic environmental factors. Whereas the role of abiotic factors (including anthropogenic pollutants) has been in focus in numerous studies and summarized in reviews, the role of biotic factors has received much less attention. Here, we review the existing body of literature from the fields of parasitology and ecology and recognize 6 different types of biotic factors with the potential to alter larval transmission processes. We found that experimental studies generally indicate strong effects of biotic factors, and the latter emerge as potentially important, underestimated determinants in the transmission ecology of free-living endohelminth stages. This implies that biodiversity, in general, should have significant effects on parasite transmission and population dynamics. These effects are likely to interact with natural abiotic factors and anthropogenic pollutants. Investigating the interplay of abiotic and biotic factors will not only be crucial for a thorough understanding of parasite transmission processes, but will also be a prerequisite to anticipate the effects of climate and other global changes on helminth parasites and their host communities.

Neurohormonal activation late after cavopulmonary connection.

OBJECTIVE: To determine whether patients with cavopulmonary connection have higher levels of vasoactive/water-salt regulating hormones and if so, whether hormone levels are related to postoperative haemodynamics and postoperative follow up. DESIGN: Cross sectional study. SETTING: University hospital. PATIENTS: 20 patients (New York Heart Association functional class I-II), mean age 11 years (range 4 to 22), were studied at a mean of 2 years (0.5 to 6) after a total cavopulmonary connection (TCPC, n = 12) or a bidirectional Glenn anastomosis (BDG, n = 8). INTERVENTIONS: Cardiac catheterisation was performed and blood samples were drawn. Control blood samples were drawn from 33 healthy children, mean age 12 years (6 to 16). MAIN OUTCOME MEASURES: Plasma levels of angiotensin II, renin, aldosterone, arginine, vasopressin, atrial natriuretic factor (ANF), brain natriuretic peptide (BNP). RESULTS: All neurohormones were significantly increased in both TCPC and BDG patients (p < 0. 05), with a fourfold increase in angiotensin II, renin, and aldosterone, and a twofold increase in vasopressin, ANF, and BNP (compared with healthy controls). There was no correlation between haemodynamic variables and hormone levels. Angiotensin II and renin were inversely correlated with time to follow up. All subjects over 15 years (n = 5) had normal neurohormonal levels. CONCLUSIONS: Neurohormones were raised for years after successful cavopulmonary operations but lower levels were observed with time on follow up. This supports the hypothesis that neurohormonal activation is primarily related to altered postoperative physiology and that adaptation takes place over time.

Replication of Aleutian mink disease parvovirus in mink lymph node histocultures.

Aleutian mink disease parvovirus (ADV), causes an immune disorder with a persistent infection of lymphoid organs in adult mink. We studied replication of ADV in gel-supported histocultures prepared from adult mink mesenteric lymph node (MLN). Evidence of virus replication in the histocultures was first observed by indirect immunofluorescence 72 h after incubation with virus. Cells resembling lymphocytes and macrophages contained both ADV capsid (VP2) and nonstructural (NS1 and NS2) proteins, and were present in a distribution suggestive of infected cells within germinal centres. ADV replicative form and encapsidated virion DNA were also detected in infected histocultures at time-points after 72 h. In addition, we were able to passage ADV-Utah to a new round of histocultures. These results suggested that the infected cells were actual target cells for ADV replication and that productive ADV-Utah replication, complete with the generation of virus, was occurring in the histocultures. The mink MLN histocultures provide a system to study the replication and molecular pathogenesis of ADV in target tissues.

Pulsatile secretion of atrial natriuretic peptide and brain natriuretic peptide in healthy humans.

Both atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are involved in sodium and water homoeostasis in healthy humans. The plasma concentrations of the natriuretic peptides can be used to differentiate between dyspnoea of cardiac and pulmonary origin, and the degree of elevation of the peptide levels in the plasma in heart failure is a measure of the severity of the disease. However, the patterns of secretion of ANP and BNP are not clear either in healthy humans or in patients. The purpose of the present study was to test the hypotheses that both ANP and BNP are secreted in pulses in healthy humans, and that this phenomenon can be revealed by determination of ANP and BNP in peripheral venous blood samples. In 12 healthy subjects, blood samples were drawn every 2 min through an intravenously inserted plastic needle over a period of 2 h. Plasma concentrations of ANP and BNP were determined by RIAs, and the results were analysed for pulsatile behaviour by Fourier transformation. Pulsatile secretion of ANP was seen in 10 out of 12 subjects [nu=0.028 min(-1) (median; range 0.013-0.047 min(-1)), i.e. a pulse of ANP with an interval of 36 min (range 21-77 min)]. Pulsatile secretion of BNP was seen in nine out of 12 patients [nu=0. 021 min(-1) (range 0.013-0.042 min(-1)), i.e. a pulse of BNP with an interval of 48 min (range 24-77 min)]. The main conclusion is that the secretion patterns of both ANP and BNP are pulsatile in most healthy humans. Consequently, it is important to study whether pulsatile secretion also occurs in heart failure in order to obtain the most informative predictive values both in the differential diagnosis of dyspnoea and in the evaluation of the severity of the disease.

Epitope mapping of Aleutian mink disease parvovirus virion protein VP1 and 2.

Six overlapping fragments of the Aleutian Mink Disease parvoVirus (AMDV) virion protein VP1 and 2 (VP1/2) gene were inserted into the expression vector pMAL-c2. Four of the clones carried large overlapping fragments covering the entire VP1/2 gene. The remaining two clones covered specifically chosen regions within the VP1/2 gene. Using a Western blotting detection system, sera from AMDV-infected mink were tested against the recombinant polypeptides. These studies showed reactions primarily directed against the two AMDV polypeptides ranging from amino acids 297 to 518. Weaker reactions against other regions of the VP1/2 were also observed. The small fusion protein designed to cover the presumed AMDV VP1/2 loop 4 was purified by affinity chromatography and used to develop solid-phase immunoassays. Twelve small synthetic peptides were constructed and used as inhibitors. A peptide covering amino acids S428 to T448 was shown to block the reactivity of a pool of positive mink sera, indicating the presence of one dominant linear epitope.

Renal effects of brain natriuretic peptide in patients with congestive heart failure.

The effect of a continuous infusion of human brain natriuretic peptide, 2 pmol.min-1.kg-1, during 60 min was studied in nine patients with congestive heart failure and in 10 healthy control subjects. Brain natriuretic peptide increased from 1.6 to 101 pmol/l in control subjects and from 25 to 173 pmol/l in congestive heart failure during infusion. Urinary sodium excretion increased significantly in both congestive heart failure (60%) and control subjects (71%), but the absolute increase was significantly lower in congestive heart failure (27 micromol/min) than in control subjects (190 micromol/min). Urinary flow rate did not change. The lithium clearance technique was used to evaluate the segmental tubular function; the distal fractional reabsorption of sodium decreased significantly less in congestive heart failure (DFRNa: -0.8%) than in control subjects (DFRNa: -3.7%). Baseline values for glomerular filtration rate and renal plasma flow were reduced in congestive heart failure, but brain natriuretic peptide induced no significant changes between congestive heart failure and control subjects. Brain natriuretic peptide induced the same absolute increase in secondary messenger cGMP in plasma and urine in both patients and healthy subjects. It is concluded that the natriuretic response to brain natriuretic peptide infusion was impaired in patients with congestive heart failure compared with healthy subjects, and it is likely that the impaired natriuretic response was caused by a reduced responsiveness in the distal part of the nephron.

Renal effects of a urodilatin infusion in patients with liver cirrhosis, with and without ascites.

This study reports the effects of a short-term (60 min) low-dose (20 ng x kg(-1) x min(-1)) infusion of synthetic urodilatin (URO) in patients with liver cirrhosis. URO is a natriuretic peptide. A total of 15 cirrhotic patients with ascites and nine without ascites participated in a randomized, double-blind, placebo-controlled study in a crossover design. Renal hemodynamics were estimated by a clearance technique using radioactive tracers, and tubular handling of sodium was evaluated by the lithium clearance method. The renal effects of URO were characterized by a significant increase in urine sodium excretion rate (UNa) and urine flow rate (V) in the cirrhotic patients without ascites (UNa: 173%; V: 94%) and with ascites (UNa: 219%, P < 0.01; V: 42%, P < 0.01) when compared with placebo infusions. Fractional excretion of sodium increased significantly, indicating a tubular effect of URO on sodium handling. Filtration fraction, lithium clearance (a marker of end-proximal fluid delivery), and fractional excretion of lithium increased, fractional proximal tubular sodium reabsorption decreased, and absolute proximal tubular sodium reabsorption remained unchanged, suggesting increased delivery of isotonic fluid from the proximal tubule during URO infusion. In addition, a significant decrease in fractional distal tubular sodium reabsorption contributed to the natriuresis. In conclusion, URO improved sodium and urine output in cirrhotic patients with and without ascites by enhancing fluid delivery from the proximal tubules in addition to inhibiting fractional sodium reabsorption in the distal nephron.

Metabolism and action of urodilatin infusion in healthy volunteers.

OBJECTIVES: The objective of this investigation was to study both the pharmacokinetics and renal pharmacodynamic properties of intravenously infused urodilatin in human beings. METHODS: Twelve healthy subjects received a short-term infusion (90 minutes) of urodilatin and placebo with a graded infusion rate (from 7.5 to 15 to 30 ng.kg body weight-1.min-1) in a randomized, double-blind, crossover study design. The renal parameters were evaluated by clearance technique with the use of 51Cr-ethylenediaminetetraacetic acid, 125I-hippuran, and lithium. Urodilatin concentrations were determined by a radioimmunoassay with a urodilatin-specific antibody. RESULTS: Kinetics were characterized by a high apparent volume of distribution (43.7 +/- 11.2 L), a high total body clearance (5383 +/- 581 ml/min), and a short plasma half-life (5.57 +/- 0.8 minutes). The maximal plasma urodilatin level was 177.2 +/- 25.8 pmol/L. Less than 1% of total infused urodilatin was recovered in urine. Urodilatin significantly increased glomerular filtration rate (urodilatin, 7.0%, versus placebo, -1.9%; p < 0.05), reduced effective renal plasma flow (urodilatin, -17%, versus placebo, -3%; p < 0.01), increased fractional excretion of sodium (urodilatin, 137%, versus placebo, 27%; p < 0.05), and increased urine flow rate (urodilatin, 46%, versus placebo, -15%; p < 0.01). Fractional excretion of lithium did not change. Mean blood pressure decreased and vasoactive hormone levels remained unchanged or increased. CONCLUSION: The natriuretic and diuretic effects of urodilatin closely followed the profile of urodilatin concentration in plasma. A major part of the synthetic urodilatin was removed from circulation by a route other than filtration through the glomeruli.

Evaluation of various cytokines (IL-6, IFN-alpha, IFN-gamma, TNF-alpha) as markers for acute bacterial infection in swine--a possible role for serum interleukin-6.

A total of 64 specific pathogen free pigs were divided into eight experimental groups. Pigs in Group I served as non-infected controls while the other 56 pigs were infected intranasally with approximately 7 x 10(8) CFU of Actinobacillus pleuropneumoniae serotype 2 (strain 700/89) in 1 ml saline. When more than 25% of the infected animals showed clinical signs of disease, i.e. 20 h post infection, 48 of the infected pigs were treated with different antibiotics (8 pigs per group), leaving 8 infected animals untreated. Serum samples collected 0, 10, 20, 28 and 44 h, and 3, 4, 7, 13 and 17 days post infection were analysed for their content of interferon (IFN)-alpha, IFN-gamma, tumor necrosis factor (TNF)-alpha by immunoassays and interleukin-6 (IL-6) by a bioassay. In addition, the development of specific antibodies was determined in sera. Among the cytokines analysed, the experimental infection only induced detectable serum levels of IL-6. The appearance of IL-6 positive animals coincided with the onset of clinical signs of disease and increased body temperatures. Varying levels of IL-6 (range, 1-220 U ml-1) were detected in serum from a majority of the infected pigs (80%). In general, the highest levels of IL-6 were detected in serum collected for 10 or 20 h after infection. Among the animals not treated with antibiotics, the number of pigs displaying IL-6 in serum continued to increase until 28 h post infection and then declined. The duration of the IL-6 response varied between individuals and lasted from eight hours to three days. Treatment with antibiotics that ceased the infection also terminated the IL-6 production in most of the pigs. In a pilot field survey, IL-6 was detected in an approximately 30% of serum samples collected from conventional reared pigs before allocation to finishing units. Thus, serum IL-6 seems to be a potential marker for ongoing bacterial infections in swine.

Analysis of the immunological cross reactivities of 213 well characterized monoclonal antibodies with specificities against various leucocyte surface antigens of human and 11 animal species.

213 Monoclonal antibodies (mAbs) raised against leucocyte surface antigens from human and 11 animal species were analyzed for reactivities against leucocytes from human and 15 different animal species. We found 77 mAbs (36%) to cross-react. Altogether, 217 cross reactions were registered out of 3195 possible combinations (7%). Most of the cross reacting mAbs had integrin or MHC class II specificities. This study defined cross reactions on the following markers: CD1a, 1c, 2, 4, 5, 8, 9, 11a, 11b, 14, 18, 20, 21, 23, 29, 31, 41, 43, 44, 45, 45R, 46, 49, 61, 62L, TCR gamma/delta, BCR, Thy-1, MHC class I and MHC class II, Swine-WC7 and Cattle-WC1. In order to characterize the molecular weight (MW) of the corresponding cross reacting antigens, selected mAbs were used to immunoprecipitate the antigens. The MW's of the analyzed precipitated antigens were in good agreement with the MWs of the homologous antigens. The followed strategy was found to be efficient and economical in defining new leucocyte antigen reactive mAbs.

Dietary sodium affects systemic and renal hemodynamic response to NO inhibition in healthy humans.

Animal studies have indicated that increased nitric oxide (NO) synthesis plays a significant role in the renal adaptation to increased sodium intake. To investigate the role of NO during increased sodium intake in humans, we studied the effect of acute, systemic injection of NG-monomethyl-L-arginine (L-NMMA) on renal hemodynamics [glomerular filtration rate and renal plasma flow (GFR and RPF, respectively)], urinary sodium excretion (FENa), systemic hemodynamics [mean arterial blood pressure and heart rate (MAP and HR)], and plasma levels of several vasoactive hormones in 12 healthy subjects during high (250 mmol/day) and low (77 mmol/day) sodium intake in a crossover design. The sodium diets were administered for 5 days before the L-NMMA treatments, in randomized order, with a washout period of 9 days between each diet and L-NMMA treatment. GFR and RPF were measured using the renal clearance of 51Cr-labeled EDTA and 125I-labeled hippuran by the constant infusion technique in clearance periods of 30-min duration. Two baseline periods were obtained, after which L-NMMA was given (3 mg/kg over 10 min), and the effect of treatment was followed over the next five clearance periods. During high sodium intake, L-NMMA induced a more pronounced relative decrease in RPF (P = 0.0417, ANOVA), a more pronounced relative decrease in FENa (P = 0.0032, ANOVA), and a more pronounced relative increase in MAP (P = 0.0231, ANOVA). During low sodium intake, the effect of L-NMMA on FENa was abolished. During low sodium intake, L-NMMA induced a sustained drop in plasma renin (31 +/- 5 vs. 25 +/- 5 microU/ml, P < 0.001), which was not seen during high sodium intake. The data indicate that increased production of NO is an important part of the adaptation to increased dietary sodium intake in healthy humans, with respect to renal hemodynamics, sodium excretion, and the secretion of renin.

Summary of the first round analyses of the Second International Swine CD Workshop.

The reactivity of 176 monoclonal antibodies (mAb) submitted to the Second International Swine CD Workshop, together with 19 internal standards, was analyzed by flow cytometry on 16 different cell types as a means of establishing the proper cell subset for later detailed clustering analyses. The exact CD subset reactivity of the 19 internal standard mAb had been characterized in the First International Swine CD Workshop. The flow cytometric analyses resulted in 40 data sets which were then subjected to statistical clustering using the Leukocyte Typing Database IV (LTDB4) software. As result of this work, 22 clusters were defined. After review of these results, panels of mAb from the defined first round clusters were assigned to cell subsets. The respective mAb in those first round clusters were then distributed to subset group researchers for further examination during the second round of the workshop.

Effect of food intake on the pharmacokinetics and antidiuretic activity of oral desmopressin (DDAVP) in hydrated normal subjects.

OBJECTIVE: The effect of food ingestion on the gastrointestinal absorption and antidiuretic action of oral desmopressin. An oral preparation of desmopressin, a synthetic analogue of vasopressin, has recently become available for clinical use. DESIGN: A randomized, single-blind, crossover study with four treatment arms. Day A, no meal + placebo; B, no meal + 400 micrograms oral desmopressin; C, standard meal + 400 micrograms oral desmopressin; D, standard meal + 400 micrograms oral desmopressin after 1.5 hours. Plasma desmopressin was measured every 15-30 minutes for 6 hours after drug intake. An intravenous hydration regimen was employed on each study day. SUBJECTS: Sixteen healthy, non-smoking, mean aged 20-35 years (mean 27.8 years). MEASUREMENTS: Plasma desmopressin concentrations were measured throughout each study day to calculate the area under the desmopressin plasma-concentration-time curve to infinity (AUCinf), the maximum plasma desmopressin concentration (Cmax), the time at which Cmax was reached (Tmax) and the time at which plasma desmopressin was first detected (Tlag). Urine volume, urine osmolality and plasma sodium concentrations were also measured at specified times on each study day. RESULTS: The total absorption of oral desmopressin, reflected by the AUCinf, was significantly higher when taken during the fasting state (day B) compared with its administration with or 1.5 hours after a standard meal (days C and D). In addition, Cmax was higher and both Tmax and Tlag were shorter on day B compared with days C and D. No effect of food ingestion was observed on the pharmacodynamics of oral desmopressin: urine volume was decreased and urine osmolality was increased to similar extents on all active treatment days (B, C and D). No significant reductions in plasma sodium concentrations (a safety parameter) was observed during the trial. CONCLUSIONS: The gastrointestinal absorption of desmopressin is reduced and delayed if administered with or 1.5 hours after a meal. This decreased absorption of desmopressin did not have an impact on the antidiuretic action of the drug since all treatment regimens elicted a maximal response. It is possible that administration of desmopressin in the fasting state may prolong its duration of action.

Effect of BNP on renal hemodynamics, tubular function and vasoactive hormones in humans.

The effect of a continuous infusion of human brain natriuretic peptide (BNP) was studied in 48 healthy men. The study was randomized, placebo controlled, and single blind. BNP was given in doses of 1, 2, or 4 pmol.kg-1.min-1 for 60 min, and peak values of BNP in plasma were 38, 85, and 199 pmol/l, giving increments in plasma as seen in heart or renal failure. BNP infusion increased the urinary flow rate and the excretion of sodium in a dose-dependent way. The maximal effects were +65 and +156%, respectively. GFR increased and RPF decreased, the latter in a dose-dependent manner. Blood pressure, heart rate, angiotensin II, and aldosterone were all unaffected by infusion of BNP, whereas a direct inhibition of renin secretion was seen. With the use of the lithium clearance technique, we concluded that the tubular site of action is in both the proximal and distal segments, and the major effect on sodium handling is in the distal parts of the nephron.

A new, fast and reliable radioimmunoassay of brain natriuretic peptide in human plasma. Reference values in healthy subjects and in patients with different diseases.

A new, fast and reliable radioimmunoassay for measurement of brain natriuretic peptide (BNP) in human plasma has been developed and its application is reported in healthy subjects and in patients with congestive heart failure, chronic renal failure, liver cirrhosis and essential hypertension. The antibody was raised in rabbits, the tracer was made by the iodogen method and polyethylene glycol was used for separation of free and bound tracer. BNP was extracted from plasma using Sep-Pak C18 cartridges. The recovery of unlabelled BNP added to plasma was 77.5 +/- 6.2% (mean +/- SD). The detection limit in plasma was 0.55 pmol l-1. No cross-reactivity existed with the natriuretic peptides ANP, CNP or urodilatin. In 124 healthy subjects the mean BNP was 1.8 +/- 1.0 pmol l-1 (SD), range 0.6-5.5. BNP increased slightly with age, was higher in women than men and had no circadian rhythm. In eight patients with congestive heart failure the median BNP level was 30.5 pmol l-1, range 3.9-65.3. In 14 patients with chronic renal failure the median BNP level was 50.5 pmol l-1, range 10.9-219.8 before dialysis, and 38.0 pmol l-1, range 9.4-180.0 immediately following dialysis. In 25 patients with liver cirrhosis the median BNP value was 7.8 pmol l-1, range 1.2-43.1. There was no difference between patients with or without ascites. In 18 medically treated patients with essential hypertension the median BNP level was 5.0 pmol l-1, range 1.2-45.5 pmol l-1.