Nematode transmission of tobacco rattle virus serves as a bottleneck to clear the virus population from defective interfering RNAs.

DI7 is a defective interfering RNA derived from RNA 2 of tobacco rattle tobravirus (TRV) isolate PpK20. Tobacco was transformed with DI7 cDNA fused to the CaMV 35S promoter. Upon infection of the transgenic plants with TRV isolate PpK20 or the serologically unrelated isolate PaY4, the transgenic DI7 RNA started to accumulate at high levels and strongly interfered with accumulation of wild-type (wt) RNA 2. When DI7 transgenic plants infected with isolate PpK20 were used as source plants in nematode-transmission experiments, the vector Paratrichodorus pachydermus efficiently transmitted virus to healthy bait plants. However, the nematodes transmitted only the wt virus present in the transgenic source plants, whereas virus particles containing the abundant, accumulated DI7 RNA were excluded from transmission. Evidence is presented that wt RNA 2 and DI7 RNA are encapsidated in cis by their encoded CPs, which are known to be functional and nonfunctional in transmission, respectively. This mechanism would result in defective interfering RNAs, which rapidly arise after mechanical transmission of the virus in the laboratory, being eliminated from tobraviruses under natural field conditions. Also this mechanism which acts with nematode transmitted virus isolates contrasts with that of vector-transmission of defective potyviruses and luteoviruses by wt helper viruses.     

Engineering of Alfalfa mosaic virus RNA 3 into an expression vector

Summary.  RNA 3 of alfalfa mosaic virus (AMV) encodes the 5′-proximal movement protein (MP) gene and the 3′-proximal coat protein (CP) gene which is expressed from a subgenomic RNA. Several strategies were explored to use this RNA as a vector for expression of the green fluorescent protein (GFP) in Nicotiana tabaccum plants expressing the viral polymerase proteins P1 and P2 (P12 plants). Insertion of a subgenomic promoter (sgp)-GFP cassette between the CP gene and the 3′-untranslated region (UTR) interfered with RNA accumulation in protoplasts, indicating that cis-acting sequences required for replication were disrupted. When GFP was fused to the N-terminus of MP or CP, the chimeric RNAs accumulated in protoplasts but cell-to-cell movement in plants was blocked. Insertion of a GFP-sgp cassette immediately upstream of the CP gene caused a hypersensitive host response. However, insertion of a GFP-sgp cassette upstream of the MP gene did not affect symptom formation and yielded a vector that expressed GFP in inoculated but not in the systemic leaves of both P12 tobacco and non-transgenic N. benthamina plants. When the size of the GFP gene was reduced from 700 to 300 nucleotides, virus infection was observed in the non-inoculated leaves. Analysis of the progeny of some chimera revealed novel data on replication, encapsidation and recombination of AMV RNA 3. Received August 7, 2000 Accepted December 18, 2000     

Assessment of rigid multi-modality image registration consistency using the multiple sub-volume registration (MSR) method

Registration of different imaging modalities such as CT, MRI, functional MRI (fMRI), positron (PET) and single photon (SPECT) emission tomography is used in many clinical applications. Determining the quality of any automatic registration procedure has been a challenging part because no gold standard is available to evaluate the registration. In this note we present a method, called the 'multiple sub-volume registration' (MSR) method, for assessing the consistency of a rigid registration. This is done by registering sub-images of one data set on the other data set, performing a crude non-rigid registration. By analysing the deviations (local deformations) of the sub-volume registrations from the full registration we get a measure of the consistency of the rigid registration. Registration of 15 data sets which include CT, MR and PET images for brain, head and neck, cervix, prostate and lung was performed utilizing a rigid body registration with normalized mutual information as the similarity measure. The resulting registrations were classified as good or bad by visual inspection. The resulting registrations were also classified using our MSR method. The results of our MSR method agree with the classification obtained from visual inspection for all cases (p < 0.02 based on ANOVA of the good and bad groups). The proposed method is independent of the registration algorithm and similarity measure. It can be used for multi-modality image data sets and different anatomic sites of the patient.     

Improvement of myocardial blood flow to ischemic regions by angiotensin-converting enzyme inhibition with quinaprilat IV: a study using [15O] water dobutamine stress positron emission tomography.

OBJECTIVES: This study was designed to analyze the effects of acute angiotensin-converting enzyme (ACE) inhibition on myocardial blood flow (MBF) in control and ischemic regions. BACKGROUND: Although animal studies indicate an improvement of MBF to ischemic regions after ACE inhibition, this effect has not been conclusively demonstrated in patients with coronary artery disease. METHODS: Myocardial blood flow was analyzed in ischemic and nonischemic regions of 10 symptomatic patients with coronary artery disease using repetitive [15O] water positron emission tomography at rest and during maximal dobutamine stress before and after ACE inhibition with quinaprilat 10 mg i.v. To exclude the possibility that repetitive ischemia may cause an increase in MBF, eight patients underwent the same protocol without quinaprilat (placebo patients). RESULTS: Rate pressure product in control and quinaprilat patients was comparable. In placebo patients, repetitive dobutamine stress did not change MBF to ischemic regions (1.41 +/- 0.17 during the first stress vs. 1.39 +/- 0.19 ml/min/g during the second stress, p = 0.93). In contrast, MBF in ischemic regions increased significantly after acute ACE inhibition with quinaprilat during repetitive dobutamine stress (1.10 +/- 0.13 vs. 1.69 +/- 0.17 ml/min/g, p < 0.015). Dobutamine coronary reserve in ischemic regions remained unchanged in placebo patients (1.07 +/- 0.11 vs. 1.10 +/- 0.16, p = 0.92), but increased significantly after quinaprilat (0.97 +/- 0.10 vs. 1.44 +/- 0.14, p < 0.002). Total coronary resistance decreased after ACE inhibition (123 +/- 19 vs. 71 +/- 10 mm Hg x min x g/ml, p < 0.02). CONCLUSIONS: Angiotensin-converting enzyme inhibition by quinaprilat significantly improves MBF to ischemic regions in patients with coronary artery disease.     

High-affinity growth hormone (GH)-binding protein (GHBP), body fat mass, and insulin-like growth factor-binding protein-3 predict the GHBP response to GH therapy in adult GH deficiency syndrome

The study objective was to investigate which baseline factors can accurately predict plasma high-affinity growth hormone (GH)-binding protein (GHBP) levels after GH replacement therapy in patients with GH deficiency (GHD). The study group consisted of 36 GHD patients (22 men and 14 women; mean age, 43.1 years; (range, 21 to 60) known to have adult-onset GHD for many years (range, 4 to 22). They were randomly divided into a GH-treated group (n = 19) and a placebo group (n = 17). Body composition (assessed by bioelectrical impendance analysis [BIA]), plasma GHBP (fast protein liquid chromatography [FPLC] size-exclusion gel chromatography), insulin-like growth factor-I (IGF-I), and IGF-binding protein-3 ([IGFBP-3] radioimmunoassays) were measured before and after 6 months. A stepwise multiple linear regression analysis with the plasma GHBP level after 6 months as the dependent variable was used to unravel significant explanatory (or predictor) variables. In contrast to placebo therapy, GH replacement therapy increased the mean plasma levels of IGF-I and IGFBP-3 to the normal range, whereas a small but statistically significant increase in plasma GHBP was observed. The combination of baseline plasma GHBP, body fat mass, and IGFBP-3 predicts posttreatment GHBP levels accurately (adjusted R2 = .97), indicating that baseline variables such as age, gender, fat-free mass, and IGF-I have no contribution. Furthermore, reliability analysis showed that the observed and predicted values for GHBP fit a strict parallel model. These findings indicate that the variations in body fat mass and IGFBP-3 among adult GHD subjects explain the reported variable response of GHBP to GH replacement therapy.     

Anti-leishmanial activity of a new formulation of amphotericin B

The effectiveness of albumin microspheres loaded with amphotericin B was tested in an in vivo model of visceral leishmaniasis using the golden hamster. Free and encapsulated amphotericin B was tested at the dose of 1 mg/kg given by the intracardiac route on days 25, 26 and 27 post-infection (p.i.) to treat animals previously infected with 10(7) stationary promastigotes by the intracardiac route. Encapsulated amphotericin was highly effective against infection causing a reduction of 88.8% and 87.2% in the early stage of infection (day 32 p.i.) and of 66.7% and 54% in a later stage of infection (day 135 p.i.) in liver and spleen parasite load respectively, compared with untreated animals, whereas free amphotericin was inactive. Lymphocyte proliferation was restored together with an increase in CD4(+) subsets in animals treated with encapsulated amphotericin B, but not in those treated with the non-encapsulated compound. Antibody responses did not increase after treatment with encapsulated amphotericin B with antibody levels remaining at base levels for most animals in contrast to those of untreated or treated with free amphotericin, where in most animals the antibody levels sharply increased. This new formulation could be a more economical alternative to liposomes for the treatment of visceral leishmaniasis with amphotericin B.     

Corticosteroid therapy increases membrane-tethered while decreases secreted mucin expression in nasal polyps

Background: Mucus hypersecretion is a hallmark of nasal polyposis (NP). Corticosteroids (CS) are first‐line treatment for NP, decreasing their size and inflammatory component. However, their effect on mucin production is not well‐understood. The aim of this (pilot) study was to investigate CS effect on mucin expression in NP. Methods: Patients were randomized in control (n = 9) and treatment (oral prednisone for 2 weeks and intranasal budesonide for 12 weeks; n = 23) groups. Nasal polyposis from nonasthmatic (NP; n = 13), aspirin‐tolerant (NP‐ATA; n = 11) and aspirin‐intolerant (NP‐AIA; n = 8) asthmatics were studied. Nasal polyposis biopsies were obtained before (w0) and after 2 (w2) and 12 (w12) weeks of CS treatment. Secreted (MUC5AC, MUC5B and MUC8) and membrane‐tethered (MUC1, MUC4) mucins (immunohistochemistry) and goblet cells (Alcian blue‐periodic acid Schiff) were quantified in both epithelium and glands. Rhinorrea and nasal obstruction were also assessed. Results: At w2, steroids increased MUC1 (from 70 to 97.5) and MUC4 (from 80 to 100) in NP‐ATA patients’ epithelium compared with baseline (w0). At w12, steroids decreased MUC5AC (from 40 to 5) and MUC5B (from 45 to 2.5) in NP‐ATA patients’ epithelium and glands, respectively, compared with baseline. No mucin presented significant changes in NP‐AIA patients. MUC5AC and MUC5B expression correlated with goblet and mucous cell numbers, respectively, and MUC5AC also with rhinorrea score. Conclusions: These results suggest: (i) CS up‐regulate membrane (MUC1, MUC4) while down‐regulate secreted (MUC5AC, MUC5B) mucins; (ii) there exists a link between secreted mucin expression and goblet cell hyperplasia; and (iii) NP from AIA may develop resistance to CS treatment.