Viroids proper can be distinguished from hammerhead viroids and satellite RNAs through their dinucleotide composition

Summary G + C-rich viroids proper exhibit a unique dinucleotide composition in that AU and UA are much less frequent than expected. Thus, evaluation of the dinucleotide pattern allows a quick discrimination between viroids proper and similar RNAs from plants, such as hammerhead-containing viroids, satellite RNAs, and defective interfering RNAs. In addition to sequence alignment, this method might be useful for the classification of a newly found small RNA replicon.     

Stem cells and experimental leukemia can be distinguished by lipid raft protein composition

The stable transfection of the canine CD34(-) multipotent cell line DO64 with retroviral constructs containing the cDNA for the canine major histocompatibility complex (MHC) class II DR genes led to the cell clone DO64#14, which is characterized by malignant transformation and tumor growth in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The additional expression of p27(kip-1) in the transformed cell clone partially reversed the malignant phenotype. Because several proteins associated with lipid rafts are involved in signal transduction and because changes of lipid raft composition are linked to the pathogenesis of leukemias, raft-associated proteins in DO64 cells and the deduced transformed cell clones were compared using a proteomic approach. Raft-associated proteins were separated by two-dimensional electrophoresis and identified by MALDI-TOF-MS. Here we show that the stem cell line DO64 and the deduced cell clones can clearly be distinguished by differences in the expression of a number of raft-associated proteins, namely caveolin-1, flotillin- 1, vimentin, galectin-3, and glyceraldehyde-3-phosphate dehydrogenase. All identified proteins play an important role in cellular functions and may therefore participate in raft-mediated leukemic transformation. Therefore, our study suggests that the analysis of lipid raft protein composition may be useful for the identification of molecular markers of the transformation process.     

Several groups among human herpesvirus 6 strains can be distinguished by Southern blotting and polymerase chain reaction.

Eight human herpesvirus 6 (HHV-6) strains were studied by Southern blot and polymerase chain reaction. DNA from infected cells was digested by a panel of restriction enzymes and hybridized with cloned BamHI fragments corresponding to about 30% of the HHV-6 strain SIE genome. In parallel, this DNA was amplified by polymerase chain reaction using pairs of primers derived from the strain SIE nucleotide sequence. Subsequently, amplification products were analyzed by hybridization, digestion with restriction endonucleases, and partial nucleotide sequencing. Overall results indicated that all strains were closely related to one another. However, concordant differences in restriction patterns allowed at least two groups to be distinguished, typified by strains SIE and HST, respectively. Differences between the two groups were found to reflect a limited number of punctual changes in nucleotide sequences. These results strengthen the idea of a unique HHV-6 species with genetic polymorphism. In addition, this study provides useful markers for the diagnosis and molecular epidemiology of HHV-6 infections.     

Follicular lymphoma can be distinguished from benign follicular hyperplasia by flow cytometry using simultaneous staining of cytoplasmic bcl-2 and cell surface CD20

The distinction between benign follicular hyperplasia (FH) and follicular lymphoma (FL) is sometimes problematic. We wanted to determine whether the expression of bcl-2 of FH was quantitatively different from that of FL, using surface CD20 expression as a discriminator of the various lymphoid compartments. Lymph node cell suspensions from 12 cases of FH and 17 cases of FL were analyzed by flow cytometry using a combined surface CD20 and intracellular bcl-2 staining. CD20- T cells in FH demonstrated the same bcl-2 expression as the CD20+ mantle cells, but the bright CD20+ germinal center cells showed near absence of bcl-2 expression. In contrast, the neoplastic cells of FL showed greater bcl-2 expression than the T cells of the same tumors and all cell populations of FH. This difference was particularly significant between the neoplastic B cells of FL and the germinal center cells of FH. The combined analysis of CD20 and bcl-2 should be useful for the differential diagnosis between FH and FL and particularly applicable to limited samples or when B-cell clonality is in question. Whether the quantitation of bcl-2 expression can be of further discriminatory value in malignant lymphomas remains to be determined.     

Lesional HPV types of cutaneous warts can be reliably identified by surface swabs

Background

Large numbers of HPV types infect the human skin and members from the HPV genera alpha, gamma and mu are associated with cutaneous warts.

Objectives

The aim of this study was to test if the HPV genotypes in swabs of the overlying skin are identical to the types present within these warts.

Study design

To this purpose, 25 persons being treated for persistent cutaneous warts were enrolled. Swabs of the overlying skin of the wart were collected from each participant. Additionally, scabs of the wart and deeper portions of the warts were surgically removed. HPV genotyping was performed on all samples using the novel HSL-PCR/MPG assay and the HPV genotyping results were compared.

Results

From the 25 wart biopsies one was HPV negative. 15 were positive for HPV27, 3 for HPV57, 2 for HPV2, 2 for HPV1, 1 for HPV3 and 1wart biopsy was positive for both HPV41 and HPV65. Scabs and swabs of the warts both showed identical typing results as the biopsies in 24 of the 25 cases (sensitivity: 96%).

Conclusions

There was an excellent agreement between HPV types in the swabs of the skin that overlies the warts and the biopsies of these warts validating the use of wart swabs for future studies of wart-associated HPV types. HPV27 was highly prevalent (70%) in the in adults of the investigated population of patients with persistent cutaneous warts.     

Mycobacterium avium subsp. paratuberculosis strains from cattle and sheep can be distinguished by a PCR test based on a novel DNA sequence difference

A DNA sequence differing between sheep and cattle types of Mycobacterium avium subsp. paratuberculosis was identified and used to develop a PCR test. The test unequivocally distinguished all sheep types from cattle types and was negative for a wide range of other strains from the Mycobacterium avium-Mycobacterium intracellulare complex. The test will be useful for epidemiological purposes, particularly in hosts such as deer that can be easily infected with either type.     

The motility of hepatic Ito cells can be acquired by their myofibroblastic transformation

We investigated the relationship between the motility of hepatic Ito cells and their myofibroblastic transformation in cultures. Ito cells were freshly isolated from rat liver and cultured in a 10% FBS-supplemented medium. On day 2 after beginning the culture, transmission electron microscopy and oil red O staining showed that Ito cells possessed numerous lipid droplets but not actin filaments in the cytoplasm. On day 8, actin filaments were abundantly found in the cytoplasm, whereas lipid droplets dramatically decreased in number. Western blot analysis also demonstrated that protein levels of alpha-smooth muscle actin in the cell markedly increased with time, but no obvious change was detected in those of desmin and tubulin beta. In Boyden's chamber assay, the migration of Ito cells, which was induced by platelet-derived growth factor-BB (PDGF-BB), was activated in a time-dependent manner. This migration of transformed Ito cells was independent of the degree of their adhesion to various substances of the extracellular matrix. Among these molecules, laminin showed the highest effect upon the migratory activity. The migration of transformed Ito cells on laminin was completely inhibited by cytochalasin D, colchicine, or taxol. Furthermore, their adhesion to the matrix was also decreased by cytochalasin D or colchicine, but not by taxol. These findings indicate that the motility of Ito cells is acquired in conjunction with their myofibroblastic transformation, which is accompanied by morphological changes with a new organization of actin filaments. The results also suggest that microtubules as well as the extracellular matrix are deeply associated with the motility of Ito cells.     

Cell micropatterning using photopolymerization with a liquid crystal device commercial projector

Photopolymerization has been widely used for surface micropatterning. The technique often requires photomasks and light sources with appropriate energies or filters. For rapid prototyping of surface photo-micropatterning, we have developed a novel device by modifying a commercially available liquid crystal device projector. In place of the image expansion unit of the projector, we attached an image reduction unit, an adjustable stage, and an optical monitoring unit. The device projected computer-generated images onto surfaces and subjected these patterns to photopolymerization. Micropatterned images can be easily prepared with various software run on personal computers. With the developed photopolymerization device, micropatterning of poly(ethylene glycol) (PEG) was achieved with PEG-diacrylate and a visible light photopolymerization initiator, camphorquinone. Selective cell adhesion control was also achieved on the micropatterned surfaces.     

Pharmacologically-distinct subsets of astroglia can be identified by their calcium response to neuroligands.

Type 1 astroglia have been reported to exhibit in excess of 20 different neuroligand receptors in vitro. The aim of this study was to determine if astroglia, like neurons, are heterogeneous with respect to their ability to respond to different neuroligand receptor agonists. Type 1 astroglia were loaded with the calcium indicator dye fura-2 and the influence of six different neuroligands on their intracellular calcium levels was examined using a video-based imaging system. Each of the six different neuroligands tested increased calcium levels in a subpopulation of glial fibrillary acidic protein immunopositive cells (astroglia). The percentage of cerebral cortical type 1 astroglia responding to a given neuroligand varied with the agonist and generally followed the order: 2-methylthio-ATP greater than phenylephrine greater than carbachol = serotonin greater than glutamate = histamine. The results also indicate that a single astroglial cell can respond to one agonist with a sustained rise in calcium levels and to an alternate agonist with oscillations in calcium levels. The pharmacological heterogeneity evident among astroglia does not appear to depend on cell proliferation or association with neurons. The results of our studies indicate that cultures of cerebral cortical type 1 astroglia are composed of distinct subsets of cells that can be distinguished by qualitative differences in their ability to respond to specific neuroligands with a rise in intracellular calcium.     

Hammondia heydorni-like oocysts shed by a naturally infected dog and Neospora caninum NC-1 cannot be distinguished

This study describes transmission experiments using Hammondia heydorni-like oocysts isolated in 1996 from a naturally infected dog. The isolate was designated as H. heydorni-Berlin-1996. Examination of sera from infected intermediate hosts showed immunoblot reactions that resembled patterns observed after Neospora caninum NC-1 infection. Furthermore, N. caninum DNA could be demonstrated in tissue samples (e.g. heart, brain) of experimentally infected intermediate hosts and in oocyst preparations from H. heydorni-Berlin-1996. The isolated oocysts did not induce any detectable disease in any of the inoculated adult intermediate hosts (goats, sheep, gerbils, guinea pigs, multimammate rats, BALB/c mice, SCID mice), even upon immunosuppression. Furthermore, neither histological lesions nor parasite stages could be identified in the tissues of all fetuses recovered from two multimammate rats that had been infected prior to pregnancy. An experiment with one dog fed a second time on infected intermediate host tissue indicated that immunity may prevent repeated oocyst shedding in N. caninum-infected dogs. In addition, the study clearly demonstrates that N. caninum can be readily transmitted by dogs that have ingested exclusively skeletal muscles of infected intermediate hosts. Therefore, the study has consequences for the recommendations for farmers to prevent postnatal transmission of N. caninum to cattle. It indicates that feeding of any tissues of potential intermediate hosts (including sheep, goats, rodents) to final hosts may induce the shedding of oocysts in these hosts and thus pose a risk for post-natal infection of cattle. With respect to oocyst morphology and the infectivity of muscle tissues for final hosts, no differences were seen in comparison with observations made in the past on Isospora bigemina/I. heydorni/H. heydorni. Therefore, earlier studies made on I. bigemina/I. heydorni/H. heydorni have to be re-evaluated critically to determine whether they may have included N. caninum or other protozoan parasites that use dogs as final hosts and have an oocyst morphology resembling that of I. bigemina/I. heydorni/H. heydorni.     

Myeloid malignancies with isolated 7q deletion can be further characterized by their accompanying molecular mutations

Deletions in the long arm of chromosome 7 (del(7q)) are recurrent cytogenetic aberrations in myeloid neoplasms. They occur either isolated or as part of a complex karyotype and are associated with unfavorable prognosis in certain disease entities. We performed detailed cytogenetic analysis, molecular analysis, and array comparative genomic hybridization in a cohort of 81 patients with a variety of myeloid malignancies and del(7q) as sole chromosomal alteration. In 70% (57/81) of patients, we identified a commonly deleted region (size: 18 Mb) involving the genomic region 101 912.442 (7q22.1)‐119 608.824 (7q31.31). Furthermore, in 80 patients, we analyzed 17 genes commonly mutated in myeloid neoplasms and identified high mutation frequencies in ASXL1 34% (27/80), TET2 33% (26/80), RUNX1 25% (20/80), DNMT3A 25% (20/80), while TP53 was rarely affected (5%, 4/80). ASXL1 and TET2 showed similar mutation frequencies across all analyzed entities while RUNX1, CBL, and JAK2 were specifically mutated in patients with acute myeloid leukemia (AML), chronic myelomonocytic leukemia, and myeloproliferative neoplasms, respectively. We detected a significantly higher frequency of RUNX1 (42% vs 13%, P = .0001) and ASXL1 (32% vs 14%, P = .008) mutations in AML patients with del(7q) compared to other AML patients in the Medical Research Council unfavorable risk group (n = 464), indicating a cooperative leukemogenic potential. Our data provide further insight into the pathomechanism of this cytogenetic subgroup.     

Adhesion and ingestion activities of fish phagocytes induced by bacterium Aeromonas salmonicida can be distinguished and directly measured from highly diluted whole blood of fish

The phagocytes of fish play an important role in innate host defense against bacterial infection, and participate in various immunoregulatory processes. Here, we investigated the effects of various opsonins in the ingestion and adhesion processes by examining respiratory burst (RB) activity in blood and head kidney (HK) fish phagocytes. RB activity was induced in rainbow trout phagocytes with the bacterium Aeromonas salmonicida (strain MT004) in the presence of various opsonins [purified antibodies (Ab), immune serum (IS), normal serum (NS) and heat-inactivated immune serum (HI-IS)], and measured in terms of luminol-amplified chemiluminescence (CL) emission at 20 degrees C for 210 min. The RB activity of blood phagocytes was measured directly from highly diluted whole blood and compared to that observed in isolated head kidney (HK) phagocytes measured under similar conditions. In addition, the extracellular RB activity of adhesion (extracellular degranulation) and the intracellular RB activity of ingestion were distinguished through their inhibition by gelatin and cytochalasin D. Our results showed that the first CL peak appeared within 50 min, and decreased or vanished when gelatin was added to the reaction or when the active complement was destroyed by heating. The second CL peak appeared after 50 min, depending on the utilized opsonin, and vanished when cytochalasin D was added to the reaction. Our results indicate that adhesion and ingestion compete for consumption of reactive oxygen intermediates. Specific IgM without an active complement was a relatively inefficient opsonin, whereas specific IgM with an active complement increased the magnitude of ingestion-mediated RB activity and accelerated the ingestion of target bacteria. Taken together, these results indicate that adhesion and ingestion responses competed for limited phagocyte resources and that the bacterial uptake by blood phagocytes can be measured directly from highly diluted blood.     

Localized superoxide release by neutrophils can be provoked by a cytosolic calcium 'cloud'.

We have used single-cell ratio imaging of Fura-2 loaded neutrophils to visualize release of cytosolic Ca2+ from an intracellular store in order to determine the location of this store and the relationship of release from it to oxidase activation. In the presence of extracellular Ca2+, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) produced an increase in free Ca2+ throughout the cytosol. In its absence, however, stimulation induced in 38% of neutrophils a highly localized increase in cytosolic free Ca2+, located between the nuclear lobes and the plasma membrane, at a region of cytosol which stained positively with 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)]. Calcium release from the store was transient, without oscillation and occurred after delays of up to 120 seconds. Addition of Ca2+ ionophore also released Ca2+ from this, and other stores, within the cell, up to three foci being detected in some cells. Localized oxidase activation occurred at the plasma membrane when the calcium concentration ([Ca2+]) of the 'cloud' exceeded 250 nM. Surprisingly, localized activation occurred at the plasma membrane at a site separate from but near to a region of high Ca2+. It was concluded that release of Ca2+ from a single receptor-releasable, Ca2+ store in neutrophils was insufficient to trigger oxidase activation throughout the cell, but could provide a localized activation of the oxidase.     

Benign hematogone-rich lymphoid proliferations can be distinguished from B-lineage acute lymphoblastic leukemia by integration of morphology, immunophenotype, adhesion molecule expression, and architectural features

Distinction of normal B-lymphoid proliferations including precursors known as hematogones from acute lymphoblastic leukemia (ALL) is critical for disease management. We present a multiparameter assessment of 27 bone marrow samples containing at least 25% hematogones (range, 25%-72%) by morphologic review. We used flow cytometry to evaluate B-cell differentiation antigen and adhesion molecule expression and immunohistochemistry on clot sections to evaluate architectural distribution. Flow cytometry revealed that intermediately differentiated cells (CD19+, CD10+) predominated, followed in frequency by CD20+, surface immunoglobulin-positive cells, with CD34+, terminal deoxynucleotidyl transferase (TdT)-positive cells as the smallest subset. Adhesion molecules (CD44, CD54) were expressed more heterogeneously compared with expression in acute lymphoblastic leukemia. Immunohistochemistry revealed that CD34+, TdT-positive cells were dispersed without significant clustering, while CD20+ cells exceeded CD34/TdT-positive cells in 24 of 25 cases. This multidisciplinary study demonstrates that hematogone-rich lymphoid proliferations exhibit a spectrum of B-lymphoid differentiation antigen expression with predominance of intermediate and mature B-lineage cells, heterogeneity of adhesion molecule expression, and nonclustered bone marrow architectural distribution.     

Typical dipole locations can be estimated using averaged somatosensory-evoked potentials and a standard brain model

It is reasonable to hypothesize that dipoles estimated from grand averaged event-related potentials based on summed-up data obtained from multiple subjects and standard head models could correspond to typical brain regions associated to a particular event. Six healthy subjects were enrolled in a study to test this hypothesis. We estimated dipoles from somatosensory-evoked potentials (SEP) elicited by electrical stimulation to the left median nerve. We also created individual three-layered (scalp, skull, and brain) head models from each subject’s magnetic resonance imaging scan, and dipoles were estimated from the individual averaged SEP with each individual head model. We then estimated dipoles using grand averaged SEP across all subjects on the standard head model created from the Montreal Neurological Institute (MNI) standard coordinate system brain template to compare the estimated dipoles located on our own head model and those on the MNI. The dipoles in the post-central gyrus were estimated from negative potentials at 20 ms from the grand averaged data incorporated with the MNI head model, corresponding to a typical location related to SEP stimulation. The results suggest the validity of estimating the dipole location from the grand averaged potential of all subjects with the MNI model if we focus on typical regions related to the task.     

Prediction of hip fracture can be significantly improved by a single biomedical indicator

Femoral neck fractures are a relevant clinical and social problem. The aim of this study was to improve the prediction of patients at-risk of femoral neck fracture with respect to the current densitometric-based methods. In particular, finite element models were used to assess the prediction accuracy obtained by combining together data from the bone density distribution, the proximal femur anatomy, and the fall-related loading conditions. Two-dimensional finite element models were developed based on dual energy x-ray absorptiometry data. A population of 93 elder Caucasian women (half of them reporting a femoral neck fracture) were retrospectively classified both using the standard clinical protocol and Bayes' linear classifiers. This study showed that the bone mineral density in the femoral neck region dominated the fracture event (65% accuracy). Adding the subject's height and the neck-shaft angle to the bone density increased the accuracy to 77%. The classification accuracy was further improved to 82% by including the peak principal tensile strain obtained from the finite element analyses. This research demonstrated that adding one single biomechanical indicator to the standard clinical measurements improves the identification of patients at-risk of femoral neck fracture. © 2002 Biomedical Engineering Society. PAC2002: 8759Ls, 8719Rr     

Blood pressure reactivity can be reduced by a cognitive behavioral stress management program

Although enhanced cardiovascular reactivity is extensively discussed as a relevant negative factor in the alteration of vascular structure, only a few controlled studies have been published presenting approaches to alter reactivity. Therefore, we examined whether enhanced reactivity could be reduced by stress management training (SMT). To control for expectation effects, progressive muscular relaxation (PMR) was the control condition. Forty-four patients with a blood pressure response greater than 15 mm Hg to a mental stress test participated in this study. Participants who took part in SMT showed a significantly stronger reduction of diastolic blood pressure reactivity to a mental stress test from pretest to posttest than the controls. Furthermore, patients who took part in SMT showed significantly smaller systolic blood pressure reactions to mental arithmetic and 2 social stress tests than the controls after the trainings. This study indicates that enhanced blood pressure reactivity can be reduced by SMT.