Evaluation of Encephaloduroarteriosynangiosis Efficacy Using Probabilistic Independent Component Analysis Applied to Dynamic Susceptibility Contrast Perfusion MRI.

BACKGROUND AND PURPOSE: Indirect cerebral revascularization has been successfully used for treatment in Moyamoya disease and symptomatic intracranial atherosclerosis. While angiographic neovascularization has been demonstrated after surgery, measurements of local tissue perfusion are scarce and may not reflect the reported successful clinical outcomes. We investigated probabilistic independent component analysis and conventional perfusion parameters from DSC-MR imaging to measure postsurgical changes in tissue perfusion. MATERIALS AND METHODS: In this prospective study, 13 patients underwent unilateral indirect cerebral revascularization and DSC-MR imaging before and after surgery. Conventional perfusion parameters (relative cerebral blood volume, relative cerebral blood flow, and TTP) and probabilistic independent components that reflect the relative contributions of DSC signals consistent with arterial, capillary, and venous hemodynamics were calculated and examined for significant changes after surgery. Results were compared with postsurgical DSA studies to determine whether changes in tissue perfusion were due to postsurgical neovascularization. RESULTS: Before surgery, tissue within the affected hemisphere demonstrated a high probability for hemodynamics consistent with venous flow and a low probability for hemodynamics consistent with arterial flow, whereas the contralateral control hemisphere demonstrated the reverse. Consistent with symptomatic improvement, the probability for venous hemodynamics within the affected hemisphere decreased with time after surgery (P = .002). No other perfusion parameters demonstrated this association. Postsurgical DSA revealed an association between an increased preoperative venous probability in the symptomatic hemisphere and neovascularization after surgery. CONCLUSIONS: Probabilistic independent component analysis yielded sensitive measurements of changes in local tissue perfusion that may be associated with newly formed vasculature after indirect cerebral revascularization surgery.

Angiographic Structural Differentiation between Native Arteriogenesis and Therapeutic Synangiosis in Intracranial Arterial Steno-Occlusive Disease.

BACKGROUND AND PURPOSE: Encephaloduroarteriosynangiosis has been shown to generate collateral vessels from the extracranial-to-intracranial circulation in patients with Moyamoya disease and intracranial arterial steno-occlusive disease. The mechanisms involved are not well-understood. We hypothesized that angiogenesis is the leading mechanism forming collaterals after encephaloduroarteriosynangiosis because there are no pre-existing connections. Angiogenesis-generated collaterals should exhibit higher architectural complexity compared with innate collaterals. MATERIALS AND METHODS: Pre- and postoperative digital subtraction angiograms were analyzed in patients enrolled in a prospective trial of encephaloduroarteriosynangiosis surgery. Branching angioscore, tortuosity index, and local connected fractal dimension were compared between innate and postoperative collaterals. RESULTS: One hundred one angiograms (50 preoperative, 51 postoperative) were analyzed from 44 patients (22 with intracranial atherosclerosis and 22 with Moyamoya disease). There was a significantly higher median branching angioscore (13 versus 4, P < .001) and a lower median tortuosity index (1.08 versus 1.76, P < .001) in the encephaloduroarteriosynangiosis collaterals compared with innate collaterals. Higher mean local fractal dimension peaks (1.28 ± 0.1 versus 1.16 ± 0.11, P < .001) were observed in the encephaloduroarteriosynangiosis collaterals compared with innate collaterals for both intracranial atherosclerosis (P < .001) and Moyamoya disease (P < .001) groups. The observed increase in high connectivity was greater in the intracranial atherosclerosis group compared with patients with Moyamoya disease (P = .01). CONCLUSIONS: The higher median branching angioscore and local connected fractal dimension, along with the lower median tortuosity index of encephaloduroarteriosynangiosis collaterals, are consistent with the greater complexity observed in the process of sprouting and splitting associated with angiogenesis.

First Report of Fruit Rot Disease of Mango Caused by Botryosphaeria dothidea and Neofusicoccum mangiferae in Taiwan.

Mango (Mangifera indica L.) is an economically important fruit crop in the tropical and subtropical areas of the world. In southern Taiwan, mango is grown on 18,000 ha of hilly land mainly located in Tainan, Kaohsiung, and Pingtung. Tons (180,000) of mango with a value of NT$6.6 billion (US$206 million) are produced annually. In 2008, mango fruit rot disease was observed 1 week after harvest on 30 to 72% of stored mangoes collected from seven orchards in southern Taiwan. The initial symptom was a small, brown lesion and rot symptoms advanced progressively. Two predominant fungi were isolated from the margin of lesions on acidified potato dextrose agar (PDA with lactic acid, pH 3.8). Isolates of each fungal type were transferred to 2% water agar containing sterilized pine needles and exposed to near UV light to induce sporulation. For the first fungus, conidia obtained from pycnidia were ovate, one-celled, and hyaline, with an average length and width of 12.93 ± 0.93 × 6.98 ± 0.40 µm and an average length/width ratio of 1.85. To confirm the identity of the fungus, PCR amplification by universal primers, ITS1/ITS4, and DNA sequencing of the internal transcribed spacer (ITS1-5.8S-ITS2 rRNA gene cluster) were conducted. The internal transcribed spacer (ITS) sequence of ribosomal DNA of this fungus was analyzed and submitted to GenBank (Accession No. GQ421486). It showed a sequence identity of 100% with Neofusicoccum mangiferae (Syd. & P. Syd.) Crous, Slippers & A. J. L. Phillips) (GenBank Accession No. AY615185). For the second fungus, conidia obtained from pycnidia were fusiform, one-celled, and hyaline, with an average length and width of 22.87 ± 1.32 × 6.42 ± 0.46 µm and a length/width ratio of 3.53. The ITS sequence of ribosomal DNA of this fungus was analyzed and submitted to GenBank (Accession No. GQ421485). It showed a sequence identity of 100% with Botryosphaeria dothidea (Moug.: Fr.) Ces & De Not.) (GenBank Accession No. AY 786321). To test pathogenicity, four mango fruits were wounded with a sterile needle, inoculated with mycelium agar plugs (0.5 mm in diameter) excised from separate monoconidial cultures, and incubated in a plastic box with a 100% relative humidity for 2 days at room temperature. Brown lesions appeared on all wounded sites of each fungus 2 days postinoculation. In control experiments, sterile agar plugs were placed on the wounded mango fruits. These fruits remained completely free from symptoms throughout the experiment. The pathogen was reisolated from the lesions of inoculated fruits and identified as N. mangiferae and B. dothidea, thus fulfilling Koch's postulates. N. mangiferae and B. dothidea have been reported on mango trees in Australia and South Africa (1). To our knowledge, this is the first report of these fungi causing fruit rot of mango in Taiwan. References: (1) B. Slippers et al. Mycologia 97:99, 2005.

First Report of a Fruit Rot Disease of Avocado Caused by Neofusicoccum mangiferae.

Production of avocado (Persea americana) has increased significantly during the last 10 years in Taiwan and the area of cultivation is approximately 500 ha. The most important postharvest disease of avocado is anthracnose caused by Colletotrichum gloeosporioides (Penz.) in Taiwan (1). In 2008, a new disease was found to be infecting avocado fruit at some orchards in Tainan County of southern Taiwan. Infected avocados developed smooth, brown, circular spots first on the surface of harvested fruits. A fungus was always isolated from the margin of lesions and could also be found from symptomless fruit pedicles and stems. Fungal colonies cultured on acidified potato dextrose agar (PDA with lactic acid; pH 3.8) were initially colorless, turned dark gradually, and ultimately became gray to dark gray. After 4 days under fluorescent light at 25°C, pycnidia formed on PDA. Conidia obtained from fruiting bodies were ovate, one celled, and hyaline, with an average length and width of 12.9 (9.9 to 15.6) × 6.4 (5.2 to 7.2) µm. The internal transcribed spacer (ITS) sequence of ribosomal DNA of this fungus was analyzed and submitted to GenBank (No. EU847427). It showed a sequence identity of 99% with Neofusicoccum mangiferae ((Syd. & P. Syd.) Crous, Slippers & A.J.L. Phillips) (GenBank No. AY615185). Thus, both morphological and molecular results confirmed the isolated fungus as N. mangiferae. Five avocado fruits were used to test the pathogenicity with three different treatment inoculation sites on each fruit. Wounded and unwounded sites on fruit were inoculated with mycelia agar plugs (0.5 mm in diameter) excised from a monoconidial culture and the fruit was kept in a plastic box with high humidity for 2 days at room temperature. Brown lesions appeared on all wounded sites 2 days postinoculation (dpi) and on unwounded sites at 4 dpi. The pathogen was reisolated from the lesions of inoculated fruits and found to be N. mangiferae, thus fulfilling Koch's postulates. In control experiments, sterile agar plugs were placed on the wounded avocado fruits. These fruits remained completely free from symptoms throughout the experiment. Several species of Botryosphaeria have been reported on avocado, including N. parvum (anamorph of B. parva), Fusicoccum aesculi (anamorph of B. dothidea), and Dothiorella aromatica (= F. luteum). To our knowledge, this is the first report of N. mangiferae causing fruit rot of avocado in Taiwan. Previously, N. mangiferae has been reported on mango trees worldwide, especially in Australia and Thailand (2). The presence of N. mangiferae in the subtropical area presents a serious disease problem not only to avocado but also to mango. References: (1) Y. P. Tsai, ed. List of Plant Diseases in Taiwan. 4th ed. Taiwan Phytopathological Society, 2002. (2) B. Slippers et al. Mycologia 97:99, 2005.

Complete nucleotide sequence and genome organization of a Cactus virus X strain from Hylocereus undatus (Cactaceae).

The complete nucleotide sequence of a strain of Cactus virus X (CVX-Hu) isolated from Hylocereus undatus (Cactaceae) has been determined. Excluding the poly(A) tail, the sequence is 6614 nucleotides in length and contains seven open reading frames (ORFs). The genome organization of CVX is similar to that of other potexviruses. ORF1 encodes the putative viral replicase with conserved methyltransferase, helicase, and polymerase motifs. Within ORF1, two other ORFs were located separately in the +2 reading frame, we call these ORF6 and ORF7. ORF2, 3, and 4, which form the "triple gene block" characteristic of the potexviruses, encode proteins with molecular mass of 25, 12, and 7 KDa, respectively. ORF5 encodes the coat protein with an estimated molecular mass of 24 KDa. Sequence analysis indicated that proteins encoded by ORF1-5 display certain degree of homology to the corresponding proteins of other potexviruses. Putative product of ORF6, however, shows no significant similarity to those of other potexviruses. Phylogenetic analyses based on the replicase (the methyltransferase, helicase, and polymerase domains) and coat protein demonstrated a closer relationship of CVX with Bamboo mosaic virus, Cassava common mosaic virus, Foxtail mosaic virus, Papaya mosaic virus, and Plantago asiatica mosaic virus.

Molecular evidence that aphid-transmitted Alpinia mosaic virus is a tentative member of the genus Macluravirus.

Alpinia mosaic virus (AlpMV), once assigned to the genus Potyvirus, infects primarily plants of the ginger family. To seek molecular evidence for correct classification of this virus, a cDNA clone corresponding to the 3' portion of the AlpMV genome was obtained by reverse transcriptase-PCR and TA cloning. The authenticity of the cDNA clone was confirmed by expression of the coat protein (CP) in E. coli followed by immunoblot analysis. Sequence analysis indicated that, in contrast to its low identity with all the other genera of the family Potyviridae, the deduced amino acid sequence of AlpMV CP was 42.9 - 61.9% identical to members of the genus Macluravirus. Phylogenetic analysis also demonstrated that the AlpMV CP clustered with those of Cardamom mosaic virus and Chinese yam necrotic mosaic virus. These results indicate that AlpMV should be classified as a tentative species within the genus Macluravirus rather than Potyvirus as proposed previously.

Diagnosis of pulmonary alveolar proteinosis: usefulness of papanicolaou-stained smears of bronchoalveolar lavage fluid.

BACKGROUND: The globules (stained green, orange, or orange in the center coated with a green rim) seen in Papanicolaou-stained smears of bronchoalveolar lavage fluid are suggested to be characteristic of pulmonary alveolar proteinosis (PAP). OBJECTIVE: To evaluate the usefulness of Papanicolaou-stained smears of bronchoalveolar lavage fluid in aiding a diagnosis of PAP. METHODS: Papanicolaou-stained smears of bronchoalveolar lavage fluid obtained from 7 patients (5 idiopathic, 2 secondary) with PAP were evaluated. To serve as controls, the smears of 11 normal subjects and 128 patients with other pulmonary disorders were also examined. The findings on the presence and number of globules were recorded. To differentiate PAP from other pulmonary disorders, the highest globule value obtained from the control group was chosen as the cutoff point. RESULTS: The characteristic globules were not found in normal subjects and only found in 6 of 128 patients with other pulmonary disorders. Their clinical diagnoses were Sjögren syndrome in 2 cases; polymyositis, idiopathic pulmonary fibrosis, asbestosis, and hypersensitivity pneumonitis in 1 case each. The numbers of globules in these 6 patients were 1, 3, 17, 7, 3, and 2. In contrast, more than 100 globules were found in all patients with PAP. The number of globules was highly sensitive and specific in aiding a diagnosis of PAP when the cutoff value was set at 18. CONCLUSION: The globules seen in Papanicolaou-stained smears of bronchoalveolar lavage fluid may be valuable in aiding a diagnosis of PAP, especially when the number of globules is more than 18.

First Report of Cactus virus X on Hylocereus undatus (Cactaceae) in Taiwan.

Hylocereus undatus Britt. & Rose (Cactaceae), commonly known as pitaya, produces edible fruits with red thorny peel and sweet white pulp containing numerous small soft seeds. In recent years, this fruit crop has become increasingly important in Taiwan. During a survey of diseases of pitaya, some plants were found with systemic mild mottling on the stems. A virus was mechanically transmitted that caused necrotic local lesions on Chenopodium amaranticolor and chlorotic lesions on C. quinoa. This virus also caused necrotic lesions with chlorotic halos on Gomphrena globosa and small chlorotic spots followed by systemic infection in Celosia argentea. Back inoculation from C. quinoa by sap transmission caused mild mottling on pitaya similar to that observed on the naturally infected plants and thus confirmed that the agent was the cause of the mottle symptom. Electron microscopic examination of negatively stained extracts from diseased plants revealed a flexuous rod-shaped virus with a length of 480 to 520 nm. Purified viral particles contained a single major protein of approximately 26 KDa as estimated by SDS polyacrylamide gel electrophoresis. In immunodiffusion tests, this virus reacted with antiserum to Cactus X virus (CVX) (ATCC #PVAS245), but did not react with antisera to Bamboo mosaic or Papaya mosaic viruses. These results establish the identity of the virus causing mottle disease on H. undatus as a strain of CVX.

The effect of intravenous administration of a chimeric anti-IgE antibody on serum IgE levels in atopic subjects: efficacy, safety, and pharmacokinetics.

CGP 51901 is a non-anaphylactogenic mouse/human chimeric anti-human IgE antibody that binds to free IgE and surface IgE of IgE-expressing B cells but not to IgE bound to high affinity IgE receptors (Fc epsilonR1) on mast cells and basophils or low affinity IgE receptors (Fc epsilonR2) on other cells. A phase 1 double-blind, placebo-controlled, single dose study with doses of 3, 10, 30, and 100 mg of CGP 51901 was conducted in 33 pollen-sensitive subjects who had raised levels of serum IgE and received either intravenous CGP 51901 or placebo. The administration of CGP 51901 was well tolerated and resulted in a decrease of serum free IgE levels in a dose-dependent manner, with suppression after 100 mg of CGP 51901 reaching > 96%. Time of recovery to 50% of baseline IgE correlated with the dose of administered antibody and ranged from a mean of 1.3 d for the 3 mg to 39 d for the 100 mg dose. Total IgE, comprised of free and complexed IgE, increased as stored and newly synthesized IgE bound to CGP 51901. Complexed IgE was eliminated at a rate comparable with the terminal half-life of free CGP 51901 (11-13 d at all doses). Only one subject showed a weak antibody response against CGP 51901. We conclude that the use of anti-human IgE antibody is safe and effective in reducing serum IgE levels in atopic individuals and provides a potential therapeutic approach to the treatment of atopic diseases.

Efficacy, pharmacodynamics, and pharmacokinetics of CGP 51901, an anti-immunoglobulin E chimeric monoclonal antibody, in patients with seasonal allergic rhinitis.

The efficacy, pharmacodynamics, and pharmacokinetics of CGP 51901, a recombinant monoclonal mouse-human chimeric anti-human immunoglobulin E (IgE) antibody were evaluated for 153 patients with seasonal allergic rhinitis treated with placebo or with 15, 30, or 60 mg CGP 51901 in six biweekly doses. Seasonal allergic rhinitis was chosen to validate the concept of anti-IgE therapy because the causal and temporal relation between allergen confrontation and IgE-mediated evocation of symptoms is firmly established. A sustained 85% or greater reduction of serum free IgE levels was shown to be effective in improving clinical symptoms. The concentration of CGP 51901 needed to maintain 85% or greater reduction of IgE was estimated to be about 5000 ng/ml. Baseline IgE levels and body weights of the patients greatly influenced the pharmacokinetic and pharmacodynamic profiles of CGP 51901. A population model was developed and refined to take into account patient baseline IgE level and body weight. The model was able to help predict multiple-dose pharmacokinetic and pharmacodynamic profiles on the basis of single-dose pharmacokinetic and pharmacodynamic measurements in the therapeutically effective dose range.

Hypoxic-ischaemic injury of the spinal cord in systemic shock: MRI.

The imaging characteristics of concurrent spinal cord injury in children with hypoxic-ischaemic injury of the brain have not been described. We present the MRI findings of hypoxic-ischaemic injury of the brain and spinal cord following hypovolaemic shock in a 2-year-old girl.

Structural features of the extracellular portion of membrane-anchoring peptides on membrane-bound immunoglobulins.

Membrane-bound immunoglobulins, mIgs, are displayed as transmembrane proteins on the surface of B cells, where they serve as antigen receptors. The mIgs are anchored to the membrane through a carboxy-terminal extension of the immunoglobulin heavy chain. Three distinct structural regions of these membrane-anchor peptides, of mouse and human mIgs, have been delineated: (1) a central conserved stretch of 25 hydrophobic, unchanged amino acid residues, which spans the membrane lipid bilayer; (2) a C-terminal hydrophilic region of 3-28 amino acids, which is intracytoplasmic; and (3) an N-terminal extracellular hydrophilic region of 13-67 amino acids, which is isotype-specific. Here we report predicted secondary and tertiary structures of the third structural region of the membrane anchoring peptide along with corroborating experimental evidence. The predictions of secondary and tertiary structure indicate that most of these regions can assume an chi-helical conformation. Circular dichroism spectroscopy of corresponding synthetic peptide confirms this essential feature. The choice of solvent and pH have dramatic effects on peptide helicity; solvent conditions consistent with a membrane-proximal environment promote helicity. Additional studies suggest that the two adjacent extracellular peptides may be stabilized through coiled-coil interactions similar to those described for some other transmembrane proteins.

Complete nucleotide sequence of Beauveria bassiana 5.8s rRNA coding gene and flanking internal transcribed spacers.

The nucleotide sequence of two clones of Beauveria bassiana in 5.8s rRNA coding gene and ITS regions were completely sequenced. The overall sequence similarity of these two clones is 96%. The identities of internal transcribed spacer (ITS) regions are 91 % (ITSI) and 100% (ITSII), respectively. Both of 5.8s rRNA sequences have 98% homology.

A phase I/IIA clinical study with a chimeric mouse-human monoclonal antibody to the V3 loop of human immunodeficiency virus type 1 gp120.

A phase I/IIA clinical trial with the chimeric mouse-human monoclonal antibody CGP 47,439 to the principal neutralization determinant in the V3 region of human immunodeficiency virus type 1 (HIV-1) strain IIIB envelope protein gp120 is reported. The trial was an uncontrolled single-center, open-label, multidose tolerability, immunogenicity, and pharmacokinetic study in homosexual men with advanced HIV disease. Patient groups were formed on the basis of the reactivity of the antibody with the gp120 of their HIV-1 isolates. Intravenous infusions of 1, 10, and 25 mg of antibody were followed by seven escalated doses of 50, 100, and 200 mg, every 3 weeks. The antibody was well tolerated; no toxicity was observed. Some patients showed a transient but insignificant antibody response to the antibody with no apparent adverse reactions or accelerated elimination of it. Substantial serum levels of the antibody were maintained with a mean t1/2 beta of 8-16 days. A virus burden reduction was observed in some patients.

Construction and characterization of chimeric and humanized forms of a broadly neutralizing monoclonal antibody to HIV-1.

Murine monoclonal antibody (MAb) G3-519 has been shown to recognize a conserved neutralizing epitope in the fourth constant (C4) region of the external glycoprotein gp120 of HIV-1. Inasmuch as this antibody effectively neutralized the infectivity of diverse HIV-1 isolates, it has been selected to be developed for passive immunization against HIV-1 infection in humans. In order to minimize the problem of immunogenicity of murine antibodies and to confer additional accessory immune functions, we have constructed mouse/human chimeric and humanized forms of the antibody. The chimeric antibody was constructed by cloning the murine variable regions and replacing the mouse constant regions with those from human Ig gamma 1,kappa. The humanized antibody was constructed using the human KAS variable region framework sequences as template. Engineering was guided by a three dimensional model of the murine variable region. The murine, chimeric and humanized forms of the antibody exhibited similar reactivity with the peptidic antigen in ELISA, and comparably neutralized the infectivity of HIV-1 in vitro. Taken together, our results show that the chimeric and humanized forms of G3-519 essentially retain the binding activity of the mouse parental antibody. Clinical development is planned to assess the prophylactic and therapeutic usefulness of these reshaped antibodies in humans.

Conversion of a trans-spliced C. elegans gene into a conventional gene by introduction of a splice donor site.

In Caenorhabditis elegans, pre-mRNAs that are trans-spliced are distinguished by the presence of an 'outron', intron-like RNA at the 5' end followed by a splice acceptor. We report that trans-splicing of the rol-6 gene can be completely suppressed simply by introducing a donor site into its 173 nt outron, at a site 50 nt upstream of the trans-splice site, thereby converting rol-6 into a conventional gene with a spliced intron near its 5' end. When the consensus donor site was inserted at sites further upstream it was less effective in replacing transplicing with cis-splicing. Surprisingly, the length of the intron was not the important variable, since lengthening of the 50 nt intron to 250 nt did not restore trans-splicing. Apparently the context into which the splice site was introduced determined the efficiency of its use. These results support the conclusion that the sole signal for trans-splicing is the presence of an outron. Clearly, cis- and trans-splice acceptor sites are interchangeable, allowing the possibility of competition between the two types of splicing.

Snake venom cardiotoxin can rapidly induce actin polymerization in intact platelets.

The action of Taiwan cobra (Naja naja atra) venom cardiotoxin on rabbit platelets at 37 degrees C was characterized by observing cytoskeletal alterations and cell lysis. At a concentration of 21.4 microM the toxin produced no cell lysis within 30 s, and less than 5% of the total lactate dehydrogenase activity of intact cells was detected in the suspending medium after the interaction had proceeded for 3 min. The extent of cell lysis was proportional to toxin concentration and interaction time. Before cell lysis, the toxin caused rapid incorporation of actin monomers into cross-linked actin filaments. The actin incorporation could be inhibited by either the presence of cytochalasin B or increased CaCl2 concentration in the suspending medium. However, addition of indomethacin did not influence the toxin-induced cytoskeletal change.