Comparing the efficacy of three techniques to reduce isoagglutinin titers in AB0 incompatible kidney transplant recipients.

ABO incompatible (ABOi) organ transplantation requires pre-transplant reduction of the recipient's IgG and IgM isoagglutinin titer against the donor to prevent hyperacute rejection. Over the past four years we primarily used unspecific IgG immunoadsorption (IA) for this purpose and combined this selectively with membrane filtration (IAc) to reduce IgM isoagglutinines. In patients with an initial IgG titer against donor below 1:64, plasma exchange (PE) was initiated. In this retrospective analysis covering January 2012 to August 2015 we compared how efficiently IgG and IgM isoagglutinines in a total of 22 ABOi kidney transplant recipients were reduced by either IA (n = 75 sessions), IAc (n = 14 sessions) or PE (n = 40 sessions). Median pre-treatment IgG isoagglutinin titers were 32 (4-4096) while IgM titers were 16 (1-256) respectively. Mean IgG reduction by either treatment modality was 1.3 ± 0.9 (IA), 1.8 ± 1.0 (IAc) and 2.6 ± 1.3 (PE) titer steps per session (p < 0.001 IA vs. PE; p < 0.04 PE vs. IAc). Mean IgM reduction was 0.6 ± 0.6 (IA), 1.8 ± 0.8 (IAc) and 2.4 ± 1.9 (PE) titer steps (p < 0.001 for both IA vs. PE and IA vs. IAc). Our data indicate that PE efficiently removed IgG- and IgM isoagglutinines. By processing only half the plasma volume per treatment PE was twice as effective as IA in terms of IgG-type isoagglutinin removal in our patient group. This is best explained by the presence of soluble AB0 antigens in the FFP used as plasma replacement. These advantages in efficacy have to be weighed against the potential hazards of PE. Combination of IA and plasma filtration effectively removes IgM-type and even enhances net IgG-type isoagglutinin elimination compared to IA alone. When trying to avoid PE, combined application of IA and IAc is a possible and effective way to reduce isoagglutinin titers before ABOi transplantation.

Methodological and clinical aspects of alloimmunization after granulocyte transfusion in patients undergoing allogeneic stem cell transplantation.

In allogeneic hematopoietic stem cell transplantation (HSCT), granulocyte transfusions (GT) may be required in immunocompromised, neutropenic patients. In this context, alloimmunization against alloantigens may occur and affect HSCT outcome. Anti-human leukocyte antigen (HLA) and -MHC class I chain related antigens A (MICA) antibody response after the administration of GT in 29 patients undergoing allogeneic HSCT (n = 27) encompassing 109 sera was investigated by multianalyte microbead assay before and up to 6 month after HSCT. Anti-HLA class I and II antibodies emerged de novo in 11 (38%) and 4 (14%) patients, respectively. Similarly, preformed antibodies were observed in four cases (14%) for anti-HLA class I and also four patients for anti-HLA class II antibodies. Anti-MICA antibodies were observed in eight granulocyte recipients of which three patients developed anti-MICA antibodies after GT, whereas preformed antibodies were seen in five patients. The conversion to positivity for any of the investigated antibodies did not significantly affect overall survival or the incidence of GVHD. GT-associated alloantibody conversion observed did not significantly correlate with outcome. Thus, surveillance of anti-HLA antibodies in the course of GT in the context of HSCT may not be required routinely. The role of MICA antibodies in HSCT and GT, however, requires further study.

First Report of Recombinant Potato virus Y Strains Infecting Potato in Jordan.

Potato (Solanum tuberosum L.) is an important vegetable crop in Jordan, occupying second position after olives. In 2012, potatoes were planted on about 6,000 ha with a production of about 141,000 t (2). Potato virus Y (PVY) is a serious problem for potato production worldwide. Recombinant strains of the virus were reported to cause tuber necrotic ringspot disease (PTNRD) in many potato-growing regions of the world. In the last few years, a new recombinant PVYNTN-NW that belongs to PVYZ (3) has been reported in the neighboring Syria. It included three recombination patterns, SYR-I, SYR-II, and SYR-III, and caused severe PTNRD (1). Since PVY is easily transmitted from one region to another by aphid vectors and infected potato seeds, this study was initiated to investigate the possible occurrence of PVY strains in Jordan. In October 2013, 33 leaf samples were collected from symptomatic potato plants cv. Spunta from Wadi Rum, Jordan (GPS coordinates 29°31'37.76″ N, 35°42'48.75″ E), the largest potato-producing area in Jordan. Sampled plants displayed leaf mottling and yellowing, symptoms similar to those caused by PVY. All samples were tested for PVY by DAS-ELISA using the ELISA kit (monoclonal cocktail) developed by BIOREBA (Reinach, Switzerland) to detect all PVY isolates. Twenty-nine samples were found positive for PVY by ELISA. To confirm virus infection, total RNA was extracted from all ELISA-positive samples and used as template in uniplex RT-PCR using strain-specific primers (1). The band pattern of PCR amplicons showed that 12 samples were infected with PVYNTN-NW genotype SYR-III and produced bands of 1,085, 441, and 278 bp. One sample was infected with PVYNTN (A) and produced bands of 1,307, 633, and 441 bp, and one other sample was infected with PVYNTN-NW genotype SYR-II and produced bands of 1,085 and 441 bp. Mixed infection with PVYNTN-NW genotype SYR-III and PVYNTN (B) was also detected in one sample producing bands of 278, 441, 1,085, and 1,307 bp. To confirm infection with the recombinant strains, PCR fragments of 278 bp amplified from three samples and 1,085 bp obtained from another three samples were directly sequenced and sequences were deposited in GenBank under accession numbers KJ159968, KJ159969, and KJ159970 for the 278-bp fragment and KJ159974, KJ159975, and KJ159976 for the 1,085-bp fragment. Sequence comparison with other PVY strains available in the NCBI database showed that the 278-bp fragment had the highest nucleotide sequence identity (100%) with PVY isolates SYR-III-A26 (AB461467) and SYR-III-2-4 (AB461457) from Syria. BLAST searches also showed that the 1,085-bp fragment shared 99% nucleotide identities with PVY isolates SYR-II-L3 (AB461482) and SYR-II-Be4 (AB461474) from Aleppo, Syria. To our knowledge, this is the first report of PVY recombinants in Jordan, and the first report of PVYNTN-NW recombinants infecting potato crop outside Syria. Since Europe is the main supplier of potato seeds for farmers in Jordan and Syria, the introduction of PVYNTN-NW to the region could have happened through infected potato seeds. Results of this study create new challenges for potato growers in Jordan as well as other countries in the region. References: (1) M. Chikh Ali et al. J. Virol. Methods 165:15, 2010. (2) FAO. http://faostat.fao.org/ (3) A. V. Karasev and S. M. Gray. Ann. Rev. Phytopathol. 51:571, 2013.

A New Pathotype of Pepper mild mottle virus (PMMoV) Overcomes the L4 Resistance Genotype of Pepper Cultivars.

The biological, serological, and molecular characteristics of a newly isolated L4 resistance-breaking isolate of Pepper mild mottle virus (PMMoV) were studied. The new pathotype of PMMoV is closely related to the Israeli pathotypes P1,2 and P1,2,3 of the virus; however, the mosaic symptoms caused by this new pathotype on pepper plants with an L4 genotype were more severe than the mild mosaic symptoms caused by other common pathotypes of the virus in susceptible plants. The predicted amino acid sequence of the putative coat protein (CP) of the newly described pathotype has two amino acid mismatches when compared with the CP of pathotype P1,2, leucine to glutamine at position 47, and alanine to glycine at position 87. The CP of the new pathotype has one amino acid mismatch when compared with P1,2,3, having alanine instead of glycine at position 87. Based on its biological characteristics, the new pathotype was designated P1,2,3,4 of PMMoV-Is. A method is described for the differentiation among the three PMMoV pathotypes using restriction cleavage analysis of reverse-transcription polymerase chain reaction products made from virus-infected plants. An additional unique MnlI site in the CP gene of the newly isolated P1,2,3,4 allows its distinction from the other two isolates, while BglI cleaved only products of the P1,2 pathotype.

The DDX3 subfamily of the DEAD box helicases: divergent roles as unveiled by studying different organisms and in vitro assays.

DDX3 (or Ded1p), the highly conserved subfamily of the DEAD-box RNA helicase family (40 members in humans), plays important roles in RNA metabolism. DDX3X and DDX3Y, the two human paralogous genes of this subfamily of proteins, have orthologous candidates in a diverse range of eukaryotes, from yeast and plants to animals. While DDX3Y, which is essential for normal spermatogenesis, is translated only in the testes, DDX3X protein is ubiquitously expressed, involved in RNA transcription, RNA splicing, mRNA transport, translation initiation and cell cycle regulation. Studies of recent years have revealed that DDX3X participates in HIV and hepatitis C viral infections, and in hepatocellular carcinoma, a complication of hepatitis B and hepatitis C infections. In the urochordates (i.e., Botryllus schlosseri) and in diverse invertebrate phyla (represented by model organisms such as: Drosophila, Hydra, Planaria), DDX3 proteins (termed also PL10) are involved in developmental pathways, highly expressed in adult undifferentiated soma and germ cells and in some adult and embryo's differentiating tissues. As the mechanistic and functional knowledge of DDX3 proteins is limited, we suggest assembling the available data on DDX3 proteins, from all studied organisms and in vitro assays, depicting a unified mechanistic scheme for DDX3 proteins' functions. Understanding the diverse functions of DDX3 in multicellular organisms may be particularly important for effective strategies of drug design.

AML1 deletion in adult mice causes splenomegaly and lymphomas.

AML1 (RUNX1) encodes a DNA-binding subunit of the CBF transcription factor family and is required for the establishment of definitive hematopoiesis. AML1 is one of the most frequently mutated genes associated with human acute leukemia, suggesting that genetic alterations of the gene contribute to leukemogenesis. Here, we report the analysis of mice carrying conditional AML1 knockout alleles that were inactivated using the Cre/loxP system. AML1 was deleted in adult mice by inducing Cre activity to replicate AML1 deletions found in human MDS, familial platelet disorder and rare de novo human AML. At a latency of 2 months after induction, the thymus was reduced in size and frequently populated by immature double negative thymocytes, indicating defective T-lymphocyte maturation, resulting in lymphatic diseases with 50% penetrance, including atypical hyperplasia and thymic lymphoma. Metastatic lymphomas to the liver and the meninges were observed. Mice also developed splenomegaly with an expansion of the myeloid compartment. Increased Howell-Jolly body counts indicated splenic hypofunction. Thrombocytopenia occurred due to immaturity of mini-megakaryocytes in the bone marrow. Together with mild lymphocytopenia in the peripheral blood and increased fractions of immature cells in the bone marrow, AML1 deficient mice display features of a myelodysplastic syndrome, suggesting a preleukemic state.

DNA markers for identifying biotypes B and Q of Bemisia tabaci (Hemiptera: Aleyrodidae) and studying population dynamics.

The two most widespread biotypes of Bemisia tabaci (Gennadius) in southern Europe and the Middle East are referred to as the B and Q-type, which are morphologically indistinguishable. In this study various DNA markers have been developed, applied and compared for studying genetic diversity and distribution of the two biotypes. For developing sequence characterized amplified regions (SCAR) and cleaved amplified polymorphic sequences (CAPS) techniques, single random amplified polymorphic DNA (RAPD) fragments of B and Q biotypes, respectively, were used. The CAPS were investigated on the basis of nuclear sodium channel and the mitochondrial cytochrome oxidase I genes (mtCOI) sequences. In general, complete agreement was found between the different markers used. Analysis of field samples collected in Israel for several years, using these markers, indicated that the percentage of the Q biotype tends to increase in field populations as time progresses. This may be attributed to the resistance of the Q biotype to neonicotinoids and pyriproxyfen and the susceptibility of the B biotype to these insecticides.

Development and initial validation of a new preference-based disease-specific health-related quality of life instrument for erectile function.

UNLABELLED: Health-related quality of life instruments may be generic or specific. In general, only generic instruments use preference-based scoring. We report on a novel approach to combine in one instrument the strengths of the specific approach, greater disease relevance and responsiveness, with those of preference-based scoring, generalizability through utilities. OBJECTIVES: The primary objective was to develop a self-administered, preference-based instrument capable of measuring utilities in the disease-specific context of erectile dysfunction (ED). METHODS: Content derivation/validation began with a literature review. Eight attributes (domains) were selected to provide clinical experts structure for focus group discussion. Four levels describing a continuum of dysfunction-function were defined for each domain. Each domain, including functional levels, was reviewed and modified until consensus was achieved regarding content. This content was then integrated into a preference based scoring instrument using two visual analogue scales (VAS) with which patients rated three 'marker' health states (representing mild, moderate and severe ED), their self-state and a previously validated external marker state. The instrument was pilot tested, and implemented in a clinical trial. Initial validation analyses have been performed. RESULTS: A self-administered, preference-based, VAS instrument was developed for use in the ED population, and the instrument was feasible to complete, was reliable beyond the threshold of acceptability established a priori and demonstrated good validity. Evidence of these properties accumulates over time and this study begins that process with this instrument. Responsiveness is being assessed in the context of a clinical trial.

Characterization of a distinct carlavirus isolated from Verbena.

Avirus was isolated from Verbena plants that bore virus-like symptoms. The virus, for which the name Verbena latent virus (VeLV) is proposed, was consistently isolated from these plants, both with and without disease symptoms. Electron microscopy studies of ultrathin sections of infected Verbena tissues revealed the presence of elongated flexuous virus particles, ca. 650 nm in length. Its experimental host range was limited to Verbena spp. and Nicotiana clevelandii. No inclusion bodies or specific cytopathological effects, were observed. Electrophoresis of dissociated purified virus preparation in sodium dodecyl sulfate-polyacrylamide gel revealed a major protein component with a molecular mass of 38.9 kDa. Polyclonal antibodies which could specifically bind to virus particles were produced. A portion of the viral RNA was cloned and sequenced; it comprised 2503 nucleotides and contained part of three open reading frames (ORFs) which from the 5' to the 3'-ends, potentially encode for 489 amino acids (ORF1), a 25.8-kDa protein (ORF2) and a 12-kDa protein (ORF3). Comparison of the predicted amino acid sequence with those of other plant viruses revealed 40-60% identity with several carlaviruses. In the light of particle morphology, absence of specific cytopathological effects in ultrathin sections, and genomic and serological properties, it is suggested that this virus belongs to the genus Carlavirus.

Quality of life and costs associated with micronized progesterone and medroxyprogesterone acetate in hormone replacement therapy for nonhysterectomized, postmenopausal women.

BACKGROUND: Because natural progesterone is poorly absorbed and rapidly metabolized, synthetic derivatives of progesterone, such as medroxyprogesterone acetate (MPA), are used in combination with estrogen in hormone replacement therapy. A micronized form of natural progesterone is available that is readily absorbed and reaches peak serum concentrations from 1 to 4 hours after administration. OBJECTIVE: The purpose of this study was to compare the quality of life (QOL), menopausal symptoms, and costs associated with a natural micronized progesterone (MP) formulation versus MPA as add-on therapy to estrogen in hormone replacement for post-menopausal women. METHODS: This prospective, multicenter, randomized, fixed-dose, open-label, parallel-group study enrolled postmenopausal, otherwise healthy, nonhysterectomized women 45 to 65 years of age who had been amenorrheic for > or =6 months and exhibited symptoms of estrogen deficiency. All women received 0.625 mg conjugated equine estrogens on days 1 to 25 of a 30-day cycle; on days 12 to 25, women were randomized to receive either MP 200 mg or MPA 5 mg; patients were followed for 9 months. QOL, the primary end point, was measured at baseline and months 3, 6, and 9 using the 36-Item Short-Form Health Survey (SF-36), the Nottingham Health Profile (NHP), and the condition-specific Women's Health Questionnaire (WHQ). Bleeding pattern, compliance, menopausal symptoms, and cost were evaluated as secondary end points. Costs (in 1997 Canadian dollars) were assessed from the societal perspective and included costs of study medication, hormone therapy monitoring, concomitant medication, outpatient resources, out-of-pocket expenses, and patient and caregiver time loss. RESULTS: A total of 182 women were enrolled; 89 received MP and 93 received MPA. Improvements in climacteric symptoms were observed from baseline to month 9 for both treatments. Mean scores on all domains of the SF-36 at month 9 were greater than scores at baseline in both treatment groups but the increases were not statistically significant. All domains within the NHP and WHQ improved significantly over this period for both groups (P < or = 0.008). Only patients receiving MP showed specific improvements in the menstrual problems and cognitive domains of the WHQ. The difference in average 9-month cost per patient was not statistically significant, at Can 367 dollars +/- 120 dollars and Can 360 dollars +/- 369 dollars for patients receiving MP and MPA, respectively. CONCLUSIONS: MP is a clinically effective, well-tolerated, and cost-comparable alternative to MPA.

Differentiating PVY(NTN) from PVY(N) by annealing to reference RNA transcripts.

A method for the differentiation of virus strains based on the shift in electrophoretic mobility of partially annealed RNA transcripts is described. Oppositely oriented RNA transcripts of the NTN- and N-strains of PVY, complementary at their 3'-end variable (strain-specific) region, were annealed to form a partial duplex which moved more slowly in gel than heterologous (NTN+N) unpaired transcripts. Thus, the two virus strains could be identified by annealing to a known reference transcript. The rate of duplex migration was correlated with transcript lengths and could be tightly controlled thereby. Thus, a higher degree of resolution was obtained than with transcript conformation polymorphism, which is empirical and unpredictable in nature.

Economic evaluation of a new acellular vaccine for pertussis in Canada.

OBJECTIVE: Pertussis is a highly contagious infection affecting mainly children. Acellular pertussis vaccines were recently introduced in Canada based on evidence of improved safety and efficacy over whole cell vaccines, the current standard of care. The following study reports the economic impact of replacing the whole cell vaccine (wP) by a new acellular vaccine (aP) in the Ontario pertussis immunisation programme. DESIGN: For a hypothetical cohort of 100,000 children from birth to the age of 8 years, the costs and consequences of pertussis vaccination with either aP or wP were compared. A decision analytical model was constructed for vaccine delivery, treatment of pertussis cases and vaccine adverse events, with analyses from the viewpoints of the Ontario Ministry of Health and society. MAIN OUTCOME MEASURES AND RESULTS: The main outcomes were expected number of pertussis cases, hospitalisations, and workdays lost by parents. Data on vaccine effectiveness, pertussis incidence, and other parameters used in the model were from published literature. Costs were discounted at 5%, and extensive sensitivity analyses were undertaken. Over 8 years, in a cohort of 100,000 children, the introduction of aP would prevent 10,500 cases of pertussis, avoiding 504 hospital admissions and 73,500 days of work absence. For Ontario, healthcare cost savings over the same period would amount to 275,585 Canadian dollars ($Can), and societal savings to $Can9,752,864

A case study of misrepresentation of the scientific literature: recent reviews of chiropractic.

Accurate use of published data and references is a cornerstone of the peer-review process. Statements, inferences, and conclusions based upon these references should logically ensue from the data they contain. When journal articles and textbook chapters summarizing the safety and efficacy of particular therapies or interventions use references inaccurately or with apparent intent to mislead, the integrity of scientific reporting is fundamentally compromised. Ernst et al.'s publication on chiropractic include repeated misuse of references, misleading statements, highly selective use of certain published papers, failure to refer to relevant literature, inaccurate reporting of the contents of published work, and errors in citation. Meticulous analysis of some influential negative reviews has been carried out to determine the objectivity of the data reported. The misrepresentation that became evident deserves full debate and raises serious questions about the integrity of the peer-review process and the nature of academic misconduct.

Improved electrophoretic resolution of RNA transcripts by specific homologous oligonucleotide gel retardation.

An improved procedure for the resolution of RNA transcripts by electrophoretic gel retardation, mediated by annealing to specific homologous oligonucleotiedes is described. The N and NTN strains of PVY served as a model system. Non-polymorphic but sequence-diverse RNA transcripts were copied from PCR products of the two virus strains. The transcripts were resolved by gel electrophoresis, because of the differential retardation effect caused by the binding of strain-specific homologous oligonucleotides. The two PVY strains were thus differentiated. Applicability of this method to virus strain differentiation in general is discussed.

Okra (Hibiscus esculentus)-A New Host of Turnip mosaic virus in Israel.

In July 1999, Hibiscus esculentus plants, grown in garden plots in Galilee, Israel, exhibited chlorosis, vein clearing accompanied by necrosis, and growth reduction. All samples (n = 10) tested positive for Turnip mosaic virus (TuMV) in enzyme-linked immunosorbent assay (ELISA), using a polyclonal antibody produced in our laboratory against purified virus. Virus in crude sap extracted from symptomatic tissue was mechanically transmitted to Chenopodium quinoa, C. amaranticolor, Nicotiana tabacum Xanthi nc and White Burley, N. clevelandii, N. benthamiana, N. sylvestris, and N. rustica, all of which developed symptoms characteristic of TuMV infection (1). ELISA testing of leaf sap extracted from mechanically inoculated indicator plants gave a strong positive reaction to TuMV. Leaf dip preparations of H. esculentus were analyzed by transmission electron microscopy. Filamentous virus particles typical of a potyvirus were observed in samples from symptomatic leaves. General primer pairs, which cover the complete 3'-end of the potyvirus genome were used in a reverse transcription-polymerase chain reaction assay (RT-PCR), gave an expected amplification product of approximately 300 bp. The nucleotide sequence of the PCR product was 97% identical to the CP sequence of other TuMV, thus verifying TuMV infection of H. esculentus. This is the first report of H. esculentus infection by TuMV. Reference: (1) A. Gera et al. J. Phytopathol. 145:289-293, 1997.

Cost-Effectiveness of tolterodine for patients with urge incontinence who discontinue initial therapy with oxybutynin: a Canadian perspective.

BACKGROUND: Tolterodine is a novel muscarinic receptor antagonist for the treatment of overactive bladder. OBJECTIVE: The purpose of this study was to examine the cost-effectiveness of tolterodine for patients with urge incontinence (UI) who discontinue initial therapy with oxybutynin in a Canadian setting. METHODS: We compared 2 treatment strategies for the management of adult patients with UI: (1) generic oxybutynin with no further treatment for patients who discontinue and (2) generic oxybutynin with switch to tolterodine (2 mg BID) for patients who discontinue. We developed a 1-year Markov model (4-week cycle length) with transitions between disease states of normal, mild, moderate, and severe. Transition probabilities over 12 weeks were obtained from randomized trial data, and drug discontinuation rates were obtained from Quebec prescription claims data. Outcome measures were time in "normal" health state and quality-adjusted life-years (QALYs) using EuroQol-5D utility scores from a survey of Swedish patients with overactive bladder. Costs to the health care payer and patient out-of-pocket costs (in Canadian dollars) were included. RESULTS: For patients who discontinue oxybutynin, the use of tolterodine is associated with approximately 6 months per year in a normal health or mild disease state, compared with approximately 3 months for those who do not receive further drug therapy after discontinuation. Tolterodine use resulted in an annual additional cost per patient of Can $163. The incremental cost per QALY was Can $9,982 and appeared to be robust to alternative model parameter assumptions. CONCLUSION: Use of tolterodine in patients with UI who discontinue initial therapy with generic oxybutynin lies within currently accepted benchmarks for cost-effectiveness.