Large genomic deletion linked to field-evolved resistance to Cry1F corn in fall armyworm (Spodoptera frugiperda) from Florida.

The fall armyworm (Spodoptera frugiperda) is a highly polyphagous lepidopteran pest of relevant food and fiber staple crops. In the Americas, transgenic corn and cotton producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have controlled and reduced the damage caused by S. frugiperda. However, cases of field-evolved S. frugiperda resistance to Bt corn producing the Cry1F insecticidal protein have been documented in North and South America. When characterized, field resistance to Cry1F is linked to insertions and mutations resulting in a modified or truncated ABC transporter subfamily C2 (SfABCC2) protein that serves as Cry1F receptor in susceptible S. frugiperda. In this work, we present detection of a large genomic deletion (~ 8 kb) affecting the SfABCC2 and an ABC transporter gene subfamily 3 -like gene (SfABCC3) as linked to resistance to Cry1F corn in a S. frugiperda strain from Florida (FL39). Monitoring for this genomic deletion using a discriminatory PCR reaction in field-collected S. frugiperda moths detected individuals carrying this allele in Florida, but not in surrounding states. This is the first report of a large genomic deletion being involved in resistance to a Bt insecticidal protein.

Rearing goat kids away from their dams 2. Understanding farmers' views on changing management practices.

Improving animal welfare is an important aim of livestock industries and is dependent on human management. Understanding attitudes to change and perceived barriers is therefore a key consideration for welfare scientists. A survey that aimed to investigate farmers' attitudes towards changing goat kid-rearing practices was distributed. Likert scales examined willingness to change and the importance of factors in decision-making alongside open-text responses for further explanation. A total of 242 farmers (United States of America (USA) 72; United Kingdom (UK) 71; Australia 33; Canada 23; New Zealand 20; European Union 14; Other 9) rearing goat kids away from their dams responded. All respondents rated from one (highly unwilling) to seven (highly willing), how willing they would be to supply three enrichment types. Willingness to provide enrichments differed (χ2(2) = 190.114,P < 0.001), with farmers most likely to provide climbing or loose items rather than swinging items. The most common reasons cited for unwillingness to provide enrichment were related to safety (101 responses/76.5%). Those currently abruptly weaning were asked how willing they would be to use gradual weaning methods. Those abruptly weaning from ad libitum milk systems (n = 47) showed no difference in willingness to change to different gradual weaning methods; the median (Interquartile Range (IQR)) for the willingness to change to removing teats was 2 (1-4), reducing milk temperature 3 (1-5) and diluting milk 2 (1-5), with most concerns relating to feasibility. Those abruptly weaning from bottle feeding (n = 18) also showed no difference in willingness to change to gradual weaning methods. Median (IQR) score for willingness to change to reduced number of bottle feeds was 4 (1-7), reducing milk quantity 3 (1-6.25), and diluting milk 1 (1-5), respectively. Health concerns were the most common reason for not being willing to change. All 242 respondents were asked to rate how important different factors are when deciding to implement a new management practice. There was a significant difference in importance between factors (χ2(2) = 34.779, P < 0.001). Median (IQR) importance of the factors was labour/time 5 (4-7), cost 5 (4-7), evidence beneficial to welfare 6 (5-7), evidence beneficial to health 6 (5-7), and evidence beneficial to growth 6 (4-7). To our knowledge, this is the first study to examine goat farmers' attitudes towards changing management practices and could help ensure that future research addresses farmer concerns and therefore has the best opportunity to be implemented on-farm.

Rearing goat kids away from their dams 1. A survey to understand rearing methods.

Despite an estimated global goat population of over one billion, little is known about methods being used to feed milk to artificially reared kids (reared away from their dams) and how kids are weaned from these systems. Quantifying and characterising current methods utilised on farms will enable future targeted research to investigate best practice methods for milk feeding and weaning of artificially rearedkids.A recall-based survey that investigated on-farmkid-rearingpractices (focusing on the milk feeding and weaning stages) was distributed via social media, and regional goat organisations across multiple countries. A total of 242 responses from 16 countries were collected and geographically grouped. Responses that could not be grouped sufficiently were removed (nine responses from eight countries). A total of 233 responses from eight countries (United States of America (USA) 72; United Kingdom (UK) 71; Australia 33; Canada 23; New Zealand 20; European Union (EU) 14), were analysed. Most farms (217; 93%) bred their own kids. The most common milk feeding method was bottle feeding, used on 135 farms (57.9%), followed by ad libitum feeding used by 72 (30.9%). A relationship between number of kids reared and feeding system was identified, χ2(3, N = 233) = 89.605,P < 0.001, with farms rearing > 100 kids more likely to feed milk ad libitum. A total of 170 farms (72.9%) were weaned based on a target age and 85 (36.4%) on a target weight, 53 (22.7%) used both and 45 (19.3%) neither. Target weaning ages and weights varied across countries; the median age was 84 days (interquartile range (IQR) 56-84), and the median weight was 16 kg (IQR 15-18). A difference was found between milk feeding systems for weaning method (Χ2(2, N = 232) = 63.797,P≤0.001), with kids most likely to be abruptly weaned from ad libitum systems (or gradually weaned from bottle feeding). Abrupt weaning was used by 67 farms (28.8%), and gradual weaning was used by 165 (71.1%). Gradual weaning strategies included reducing milk quantity (150 farms; 93% of farms providing detail) and diluting milk (six farms; 4%). A total of 169 (72.5%) supplied enrichment that met the survey's definition; items to climb on/hide in were most common, provided by 157 farms (92.8%). Findings suggest differing practices in smaller-scalebottle-fedversuslarger-scalead libitummilksystems, likely reflecting differing system needs.This highlights a requirement for welfare-focused research in kids reared artificiallyin order to identify and communicatebest practices to ensure on-farm welfare is optimised within each system.

Dissociation of systemic and mucosal autoimmunity in cystic fibrosis.

BACKGROUND: Pseudomonas aeruginosa accounts for ~80% of cystic fibrosis (CF) airway infection. It shows a remarkable correlation with presence of autoantibody to bactericidal/permeability-increasing protein (BPI), which is not understood. In this study, we sought to better understand the characteristics of systemic and mucosal autoimmunity and their relation to humoral immunity to P. aeruginosa. METHODS: Antibody titers and isotypes to BPI and P. aeruginosa were characterized in sera and bronchoalveolar lavage (BAL) of adult and pediatric CF patients (n = 131), by ELISA and/or immunoblot. RESULTS: Serum BPI autoantibodies were common (~43%) in adult while rare (≪5%) in pediatric (≤18 yrs) CF patients. Serum BPI IgG autoantibodies were of high avidity and strongly correlated with anti-P. aeruginosa IgG responses. A parallel relationship was observed with IgA, but not IgG, responses in adult and pediatric CF patient in the BAL. Thus, BAL IgA anti-BPI antibodies were independent of age and correlated with the presence of BPI cleavage in BAL. CONCLUSIONS: IgG and IgA autoreactivity to BPI in CF patients was demonstrated in serum and BAL, respectively, and correlated with the isotype of the antibody response to P. aeruginosa. The co-occurrence of anti-BPI and anti-P. aeruginosa IgA in the BAL, but not serum, of pediatric CF patients suggests that BPI tolerance is broken in the P. aeruginosa-infected airway and that serologic IgG autoantibodies are later induced, potentially through a separate pathway. The relationship between P. aeruginosa, BPI cleavage, and IgA autoantibodies in the BAL suggests a role for cryptic epitope generation in the breaking of tolerance.

Mobile oral heath technologies based on saliva.

In this review we have highlighted a few innovative microfluidic analytical technologies with mobile phone image processing tools for various bio-chemical tests performed using salivary biomarkers. Saliva-based assays for mobile monitoring with a smartphone sensor provide an excellent analytical technique which can be simple to perform. We describe several examples from the literature, utilizing different modalities of analysis, applied to several different applications of mobile health monitoring: cortisol monitoring, infectious disease testing, and drugs of abuse.

Delimiting Strategic Zones for the Development of Fall Armyworm (Lepidoptera: Noctuidae) on Corn in the State of Florida.

The fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), cannot survive prolonged periods of freezing temperatures, thereby limiting where it can overwinter in North America. Climate change is anticipated to reduce the frequency of freeze days in Florida over the decades, with the potential consequence of a significant expansion of the overwintering range, whose northern limit in North America was assessed between 27 and 28°N in the last century. To assess this possibility, the development of the fall armyworm on corn leaves, one of the main host plants in the United States, was determined at five constant temperatures ranging from 14 to 30°C. Based on the development time, the thermal constant and the lower threshold temperature were used to estimate the number of generations of fall armyworm at 42 locations in the state of Florida, from 2006 to 2016. Maps were constructed to provide a visual description of the interpolated data, using GIS (Geographic Information System). The highest number of generations was observed in the counties farther south, an area that showed the highest temperatures during the years and plays a strategic role in maintaining fall armyworm populations in corn fields. Additionally, we conclude that in the absence of freeze periods, the northern limit for fall armyworm overwintering should be between 28 and 29°N.

Long-term prognosis for 1-year relapse-free survivors of CD34+ cell-selected allogeneic hematopoietic stem cell transplantation: a landmark analysis.

CD34+ cell selection significantly improves GvHD-free survival in allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, specific information regarding long-term prognosis and risk factors for late mortality after CD34+ cell-selected allo-HSCT is lacking. We conducted a single-center landmark analysis in 276 patients alive without relapse 1 year after CD34+ cell-selected allo-HSCT for AML (n=164), ALL (n=33) or myelodysplastic syndrome (n=79). At 5 years' follow-up after the 1-year landmark (range 0.03-13 years), estimated relapse-free survival (RFS) was 73% and overall survival (OS) 76%. The 5-year cumulative incidence of relapse and non-relapse mortality (NRM) were 11% and 16%, respectively. In multivariate analysis, Hematopoietic Cell Transplantation Comorbidity Index score⩾3 correlated with marginally worse RFS (hazard ratio (HR) 1.78, 95% confidence interval (CI) 0.97-3.28, P=0.06) and significantly worse OS (HR 2.53, 95% CI 1.26-5.08, P=0.004). Despite only 24% of patients with acute GvHD within 1 year, this also significantly correlated with worse RFS and OS, with increasing grades of acute GvHD associating with increasingly poorer survival on multivariate analysis (P<0.0001). Of 63 deaths after the landmark, GvHD accounted for 27% of deaths and was the most common cause of late mortality, followed by relapse and infection. Although prognosis is excellent for patients alive without relapse 1 year after CD34+ cell-selected allo-HSCT, risks of late relapse and NRM persist, particularly due to GvHD.

A rapidly-prototyped microfluidic device for size-based nucleic acid fractionation using isotachophoresis.

We present a novel microfluidic device for size-based nucleic acid (NA) fractionation using isotachophoresis (ITP) and an ionic spacer. Our rapid-prototyped laser-cut plastic device has easily modifiable channel dimensions, can process up to 10 µL of sample, and contains an in-line extraction reservoir for minimally-disruptive manual collection of size-fractionated NAs. We designed custom buffering reservoirs using 1 mL pipette tips to provide high buffering capacity and prevent bubbles from entering the microfluidic channels. We demonstrated the utility of the device by implementing a proof-of-concept assay in which NAs were preconcentrated (via ITP) and then segregated by size (using the ionic spacer and sieving matrix) to generate two separate fractions, the first comprised of small (<50 nt) NA, and the second comprised of NAs of all sizes. Through this approach, we demonstrated size-based fractionation of both DNA and RNA samples (a mixture of synthetic ssDNA molecules, and a commercially-available RNA molecular weight standard, respectively). Our results indicate that this simple, rapid (≤10 min), and label-free approach is a promising and cost-effective alternative to the commercially-available size-selection kits currently on the market. We discuss the design and features of the device, as well as challenges which must be met in the future in order to further improve its performance and utility.

A simple check valve for microfluidic point of care diagnostics.

Check valves are often essential components in microfluidic devices, enabling automated sample processing for diagnostics at the point of care. However, there is an unmet need for a check valve design that is compatible with rigid thermoplastic devices during all stages of development-from initial prototyping with a laser cutter to final production with injection molding. Here, we present simple designs for a passive, normally closed check valve that is manufactured from commonly available materials with a CO2 laser and readily integrated into prototype and production thermoplastic devices. The check valve consists of a thermoplastic planar spring and a soft elastomeric pad that act together to seal against fluid backflow. The valve's cracking pressure can be tuned by modifying the spring's planar geometry and thickness. Seal integrity is improved with the addition of a raised annular boss beneath the elastomeric pad. To demonstrate the valve's usefulness, we employ these valves to create a finger-operated on-chip reagent reservoir and a finger-actuated pneumatic pump. We also apply this check valve to passively seal a device to enable portable detection of RNA from West Nile virus in a laser-cut device.

Fungal dimorphism in the entomopathogenic fungus Metarhizium rileyi: Detection of an in vivo quorum-sensing system.

This investigation documents the expression of the in vivo dimorphic program exhibited by the insect mycopathogen Metarhizium rileyi. This insect mycopathogen represents the key mortality factor regulating various caterpillar populations in legumes, including subtropical and tropical soybeans. Using two hosts and M. rileyi isolates, we have measured M. rileyi growth rates under in vivo and in vitro conditions and have assessed the pathogen's impact on host fitness. Significantly, the hyphal bodies-to-mycelia transition that occurs at the late infection stage is regulated by a quorum-sensing molecule(s) (QSM) that triggers hyphal bodies (Hb) to synchronously switch to the tissue-invasive mycelia. Within hours of this transition, the host insect succumbs to mycosis. The production of the QS chemical(s) occurs when a quorum of Hb is produced in the hemolymph (late-stage infection). Furthermore, the QS activity detected in late-stage infected sera is unique and is unrelated to any known fungal QSM. The lack of similar QS activity from conditioned media of M. rileyi suggests that the chemical signal(s) that mediates the dimorphic switch is produced by host tissues in response to a quorum of hyphal bodies produced in the host hemolymph. The serum-based QS activity is retained after lyophilization, mild heat treatment, and proteinase digestion. However, attempts to extract/identify the QSM have been unsuccessful. Results suggest that the observed hyphal body-to-mycelia transition is a multi-step process involving more than one chemical signal.

Using intron sequence comparisons in the triose-phosphate isomerase gene to study the divergence of the fall armyworm host strains.

The noctuid moth Spodoptera frugiperda (the fall armyworm) is endemic to the Western Hemisphere and appears to be undergoing sympatric speciation to produce two subpopulations that differ in their choice of host plants. The 'rice strain' and 'corn strain' are morphologically indistinguishable, requiring the use of genetic markers for identification. Because fall armyworm is a major pest of corn and several other agricultural crops, characterizing the strains has important economic consequences. In this study, comparisons were made of the intron sequences from the triose-phosphate isomerase (Tpi) gene isolated from 85 fall armyworm specimens collected from two host plants. Sixteen new strain-specific haplotypes based on intron polymorphisms are described that can facilitate the characterization of fall armyworm populations associated with different host plants. Comparisons of genetic diversity within and between the strains provides evidence that the corn strain is undergoing active selection and supports the proposal of directional interstrain mating occurring in the wild. Comparisons of the polymorphisms indicate that each intron undergoes different patterns of mutation that in some cases corresponds to host plant preferences. The results confirm that intron sequence comparisons are an effective approach to study fall armyworm population genetics.

Modeling seasonal migration of fall armyworm moths.

Fall armyworm, Spodoptera frugiperda (J.E. Smith), is a highly mobile insect pest of a wide range of host crops. However, this pest of tropical origin cannot survive extended periods of freezing temperature but must migrate northward each spring if it is to re-infest cropping areas in temperate regions. The northward limit of the winter-breeding region for North America extends to southern regions of Texas and Florida, but infestations are regularly reported as far north as Québec and Ontario provinces in Canada by the end of summer. Recent genetic analyses have characterized migratory pathways from these winter-breeding regions, but knowledge is lacking on the atmosphere's role in influencing the timing, distance, and direction of migratory flights. The Hybrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT) model was used to simulate migratory flight of fall armyworm moths from distinct winter-breeding source areas. Model simulations identified regions of dominant immigration from the Florida and Texas source areas and overlapping immigrant populations in the Alabama-Georgia and Pennsylvania-Mid-Atlantic regions. This simulated migratory pattern corroborates a previous migratory map based on the distribution of fall armyworm haplotype profiles. We found a significant regression between the simulated first week of moth immigration and first week of moth capture (for locations which captured ≥ 10 moths), which on average indicated that the model simulated first immigration 2 weeks before first captures in pheromone traps. The results contribute to knowledge of fall armyworm population ecology on a continental scale and will aid in the prediction and interpretation of inter-annual variability of insect migration patterns including those in response to climatic change and adoption rates of transgenic cultivars.

Guideline summary review: An evidence-based clinical guideline for the diagnosis and treatment of degenerative lumbar spondylolisthesis.

BACKGROUND CONTEXT: The North American Spine Society's (NASS) Evidence-Based Clinical Guideline for the Diagnosis and Treatment of Degenerative Lumbar Spondylolisthesis features evidence-based recommendations for diagnosing and treating degenerative lumbar spondylolisthesis. The guideline updates the 2008 guideline on this topic and is intended to reflect contemporary treatment concepts for symptomatic degenerative lumbar spondylolisthesis as reflected in the highest quality clinical literature available on this subject as of May 2013. The NASS guideline on this topic is the only guideline on degenerative lumbar spondylolisthesis included in the Agency for Healthcare Research and Quality's National Guideline Clearinghouse (NGC). PURPOSE: The purpose of this guideline is to provide an evidence-based educational tool to assist spine specialists when making clinical decisions for patients with degenerative lumbar spondylolisthesis. This article provides a brief summary of the evidence-based guideline recommendations for diagnosing and treating patients with this condition. STUDY DESIGN: A systematic review of clinical studies relevant to degenerative spondylolisthesis was carried out. METHODS: This NASS spondyolisthesis guideline is the product of the Degenerative Lumbar Spondylolisthesis Work Group of NASS' Evidence-Based Guideline Development Committee. The methods used to develop this guideline are detailed in the complete guideline and technical report available on the NASS website. In brief, a multidisciplinary work group of spine care specialists convened to identify clinical questions to address in the guideline. The literature search strategy was developed in consultation with medical librarians. Upon completion of the systematic literature search, evidence relevant to the clinical questions posed in the guideline was reviewed. Work group members used the NASS evidentiary table templates to summarize study conclusions, identify study strengths and weaknesses, and assign levels of evidence. Work group members participated in webcasts and in-person recommendation meetings to update and formulate evidence-based recommendations and incorporate expert opinion when necessary. The draft guidelines were submitted to an internal peer review process and ultimately approved by the NASS Board of Directors. Upon publication, the Degenerative Lumbar Spondylolisthesis guideline was accepted into the NGC and will be updated approximately every 5 years. RESULTS: Twenty-seven clinical questions were addressed in this guideline update, including 15 clinical questions from the original guideline and 12 new clinical questions. The respective recommendations were graded by strength of the supporting literature, which was stratified by levels of evidence. Twenty-one new or updated recommendations or consensus statements were issued and 13 recommendations or consensus statements were maintained from the original guideline. CONCLUSIONS: The clinical guideline was created using the techniques of evidence-based medicine and best available evidence to aid practitioners in the care of patients with degenerative lumbar spondylolisthesis. The entire guideline document, including the evidentiary tables, literature search parameters, literature attrition flow chart, suggestions for future research, and all of the references, is available electronically on the NASS website at https://www.spine.org/Pages/ResearchClinicalCare/QualityImprovement/ClinicalGuidelines.aspx and will remain updated on a timely schedule.

Early pair housing increases solid feed intake and weight gains in dairy calves.

Dairy calves have traditionally been kept in individual pens throughout the milk-feeding period. Social rearing is associated with increased solid feed intake and, hence, higher weight gains before and after weaning. Little is known about the effect of the age at which social housing begins. The aim of this study was to assess the effects of early versus late pairing on feeding behavior and weight gain before and after weaning. Holstein bull calves were reared individually (n=8 calves) or paired with another calf at 6±3 d (n=8 pairs) or 43±3 d of age (n=8 pairs). All calves were fed 8 L of milk/d for 4 wk, 6 L/d from 4 to 7 wk, and then milk was reduced by 20%/d until calves were completely weaned at 8 wk of age. Calves were provided ad libitumaccess to calf starter and a total mixed ration (TMR). Body weight and feed intake were measured weekly from 3 to 10 wk of age.Intake of calf starter was significantly higher for the early-paired calves than for individually reared and late-paired calves throughout the experimental period. At 10 wk of age, starter dry matter intake averaged 2.20±0.22, 1.09±0.25, and 1.26±0.33kg/d for early-paired, late-paired, and individually housed calves, respectively. Intake of TMR did not differ among treatments, TMR dry matter intake averaged 3.27±0.72, 3.08±0.46, and 2.89±0.54kg/d for the same 3 treatments. Calves in the early paired treatment also showed significantly higher average daily gain over the experimental period (0.89±0.04 vs. 0.76±0.04 and 0.73±0.04kg/d for the early-paired, individual, and late-paired calves, respectively). These results indicate that social housing soon after birth can increase weight gains and intake of solid feed.

Inheritance of Cry1F resistance, cross-resistance and frequency of resistant alleles in Spodoptera frugiperda (Lepidoptera: Noctuidae).

Transgenic maize, Zea maize L., expressing the Cry1F protein from Bacillus thuringiensis has been registered for Spodoptera frugiperda (J. E. Smith) control since 2003. Unexpected damage to Cry1F maize was reported in 2006 in Puerto Rico and Cry1F resistance in S. frugiperda was documented. The inheritance of Cry1F resistance was characterized in a S. frugiperda resistant strain originating from Puerto Rico, which displayed >289-fold resistance to purified Cry1F. Concentration-response bioassays of reciprocal crosses of resistant and susceptible parental populations indicated that resistance is recessive and autosomal. Bioassays of the backcross of the F1 generation crossed with the resistant parental strain suggest that a single locus is responsible for resistance. In addition, cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry2Aa and Vip3Aa was assessed in the Cry1F-resistant strain. There was no significant cross-resistance to Cry1Aa, Cry1Ba and Cry2Aa, although only limited effects were observed in the susceptible strain. Vip3Aa was highly effective against susceptible and resistant insects indicating no cross-resistance with Cry1F. In contrast, low levels of cross-resistance were observed for both Cry1Ab and Cry1Ac. Because the resistance is recessive and conferred by a single locus, an F1 screening assay was used to measure the frequency of Cry1F-resistant alleles from populations of Florida and Texas in 2010 and 2011. A total frequency of resistant alleles of 0.13 and 0.02 was found for Florida and Texas populations, respectively, indicating resistant alleles could be found in US populations, although there have been no reports of reduced efficacy of Cry1F-expressing plants.

Development and feeding of fall armyworm on Miscanthus x giganteus and switchgrass.

Observations of fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), larvae infesting plots of Miscanthus x giganteus Greef and Deuter ex Hodkinson and Renvoize prompted laboratory-based tests of survival, development, and feeding preferences on leaf tissue from M. x giganteus and switchgrass, Panicum virgatum L. Survival from hatch to pupation was >70 and 50% for fall armyworms reared on switchgrass and M. x giganteus, respectively, although survival of the S. frugiperda rice strain was significantly greater than the corn strain on both crops. Developmental times from hatch to pupation or adult emergence showed effects of crop and S. frugiperda host strain, but analysis of an interaction revealed developmental times for the rice strain were similar on both crops, whereas corn strain larvae showed delayed development on M. x giganteus relative to switchgrass. Analysis of larval (10 d) and pupal masses showed a similar pattern, with effects of crop and an interaction (at 10 d), but only the mass of corn strain larvae feeding on M. x giganteus was reduced relative to the other crop and strain combinations. In choice tests, neonates of both corn and rice strains showed a strong preference for feeding on young tissues rather than mature leaves of M. x giganteus or switchgrass, but they also clearly favored corn, Zea mays L., leaves over either of the perennial grasses. Results indicate both plants are potential hosts for S. frugiperda, but additional information is needed to understand under which scenarios and to what degree fall armyworms may damage perennial grasses grown for biofuel production.

Actual or ideal body weight to calculate CD34+ cell dose in patients undergoing autologous hematopoietic SCT for myeloma?

CD34+ cell dose calculations are usually based on actual body weight (ABW). We have shown that ideal body weight (IBW) may provide a better basis for this in a small population of patients with hematologic malignancies. This was studied further in 514 myeloma autografts. The CD34+ cell doses (10(6)/kg) by IBW and ABW were 1.37-39.36 (median 6.03) and 1.15-29.67 (median 4.84), respectively. IBW-based cell doses correlated slightly better with engraftment than ABW-based doses (higher r(2)): 0.5 x 10(9)/l neutrophils 0.83 versus 0.82, 1.0 x 10(9)/l neutrophils 0.78 versus 0.77, 20 x 10(9)/l platelets 0.54 versus 0.53 and 50 x 10(9)/l platelets 0.57 versus 0.55. When outliers (hematologic recovery in <8 or >16 days) were excluded, the findings were similar: 0.5 x 10(9)/l neutrophils 0.85 versus 0.84, 1.0 x 10(9)/l neutrophils 0.85 versus 0.84, 20 x 10(9)/l platelets 0.86 versus 0.85 and 50 x 10(9)/l platelets 0.85 versus 0.84. CD34+ cell doses based on IBW as well as ABW significantly affected engraftment when analyzed separately as continuous variables. However, when analyzed together, only the dose based on IBW retained significance. We conclude that calculation of CD34+ cell numbers for autotransplantation should be based on IBW.

Infusion of the allogeneic cell line NK-92 in patients with advanced renal cell cancer or melanoma: a phase I trial.

BACKGROUND: Renal cell cancer and malignant melanoma are two types of cancer that are responsive to immunotherapy. In this phase I dose-escalation study, the feasibility of large-scale expansion and safety of administering ex vivo-expanded NK-92 cells as allogeneic cellular immunotherapy in patients with refractory renal cell cancer and melanoma were determined. METHODS: Twelve patients (aged 31-74 years) were enrolled, three per cohort at cell dose levels of 1x10(8)/m(2), 3x10(8)/m(2), 1x10(9)/m(2) and 3x10(9)/m(2). One treatment course consisted of three infusions. Eleven patients had refractory metastatic renal cell cancer; one patient had refractory metastatic melanoma. RESULTS: The NK-92 cells were expanded in X-Vivo 10 serum-free media supplemented with 500 U/mL Proleukin recombinant human interleukin-2 (rhIL-2), amino acids and 2.5% human AB plasma. Final yields of approximately 1x10(9) cells/culture bag (218-250xexpansion) over 15-17 days were achievable with >or=80% viability. Infusional toxicities of NK-92 were generally mild, with only one grade 3 fever and one grade 4 hypoglycemic episode. All toxicities were transient, resolved and did not require discontinuation of treatment. One patient was alive with disease at 4 years post-NK-92 infusion. The one metastatic melanoma patient had a minor response during the study period. One other patient exhibited a mixed response. DISCUSSION: This study establishes the feasibility of large-scale expansion and safety of administering NK-92 cells as allogeneic cellular immunotherapy in advanced cancer patients and serves as a platform for future study of this novel natural killer (NK)-cell based therapy.

Electric and hydrodynamic stretching of DNA-polymer conjugates in free-solution electrophoresis.

The conjugation of an uncharged polymer to DNA fragments makes it possible to separate DNA by free-solution electrophoresis. This end-labeled free-solution electrophoresis method has been shown to successfully separate ssDNA with single monomer resolution up to about 110 bases. It is the aim of this paper to investigate in more detail the coupled hydrodynamic and electrophoretic deformation of the ssDNA-label conjugate at fields below 400 V/cm. Our model is an extension of the theoretical approach originally developed by Stigter and Bustamante [Biophys. J. 75, 1197 (1998)] to investigate the problems of a tethered chain stretching in a hydrodynamic flow and of the electrophoretic stretch of a tethered polyelectrolyte. These two separate models are now used together since the charged DNA is "tethered" to the uncharged polymer (and vice versa), and the resulting self-consistent model is used to predict the deformation and the electrophoretic velocity for the hybrid molecule. Our theoretical and experimental results are in good qualitative agreement.

Does younger donor age affect the outcome of reduced-intensity allogeneic hematopoietic stem cell transplantation for hematologic malignancies beneficially?

Sixty three patients aged 27-66 years (median 52) were allografted from HLA-matched sibling (n=47), 10 of 10 allele-matched unrelated (n=19), or one-antigen/allele-mismatched (n=7) donors aged 24-69 years (median 46) after a conditioning regimen comprising 100 mg/m(2) melphalan. Cyclophosphamide (50 mg/kg) was also administered to patients who had not been autografted previously. Cyclosporine or tacrolimus, and mycophenolate mofetil were administered to prevent graft-versus-host disease (GVHD). The 2-year cumulative incidences of relapse and TRM were 55 and 24% respectively, and 2-year probabilities of overall survival (OS) and disease-free survival (DFS) were 36 and 21%, respectively. Poor performance status, donor age >45 years and elevated lactate dehydrogenase (LDH) increased the risk of treatment-related mortality (TRM), refractory disease and donor age >45 years increased the risk of relapse, and OS and DFS were adversely influenced by refractory disease, poor performance status, increased LDH, and donor age >45 years. Our data suggest that younger donor age is associated with better outcome after sub-myeloablative allogeneic hematopoietic stem cell transplantation (HSCT) for hematologic malignancies due to lower TRM and relapse. This finding raises the question of whether a young 10-allele-matched unrelated donor is superior to an older matched sibling donor in patients where the clinical situation permits a choice between such donors.