The design and conduct of clinical trials to limit missing data.

This article summarizes recommendations on the design and conduct of clinical trials of a National Research Council study on missing data in clinical trials. Key findings of the study are that (a) substantial missing data is a serious problem that undermines the scientific credibility of causal conclusions from clinical trials; (b) the assumption that analysis methods can compensate for substantial missing data is not justified; hence (c) clinical trial design, including the choice of key causal estimands, the target population, and the length of the study, should include limiting missing data as one of its goals; (d) missing-data procedures should be discussed explicitly in the clinical trial protocol; (e) clinical trial conduct should take steps to limit the extent of missing data; (f) there is no universal method for handling missing data in the analysis of clinical trials - methods should be justified on the plausibility of the underlying scientific assumptions; and (g) when alternative assumptions are plausible, sensitivity analysis should be conducted to assess robustness of findings to these alternatives. This article focuses on the panel's recommendations on the design and conduct of clinical trials to limit missing data. A companion paper addresses the panel's findings on analysis methods.

Protracted emergence of overwintering Amyelois transitella (Lepidoptera: Pyralidae) from pistachios and almonds in California.

The navel orangeworm, Amyelois transitella (Walker), is the primary insect pest of pistachios and almonds in California. Four years of research (2002-2006) were conducted in Madera and Kern Counties to elucidate the pattern of adult emergence of the overwintering navel orangeworm population. Springtime emergence from unharvested (mummy) nuts was protracted (600 degree-days or more from 1 January of each year) and in 2004 and 2006 extended to mid-July. The population structure, sex ratio, and timing of emergence differed between pistachio and almond mummies. Pistachio populations had a significantly greater proportion of late stage individuals compared with almond mummies, 85.7 versus 34.1%. The sex ratio of adults emerging from pistachio mummies was significantly skewed with a ratio 57:43 male:female compared with 50:50 in almond mummies. Emergence from mummies held outdoors (variable temperature) began in early March and continued through early June in both pistachio mummies and almond mummies. The adult emergence pattern from pistachio mummies contained a single emergence peak, whereas emergence from almond mummies occurred in multiple peaks. These same patterns occurred when mummies were held at constant temperature, and the emergence peak from pistachio mummies occurred sooner. The impact of these findings on understanding navel orangeworm population dynamics and current control recommendations is discussed.

Recovery of commercially produced Bacillus thuringiensis var. israelensis and Bacillus sphaericus from tires and prevalence of bacilli in artificial and natural containers.

We conducted surveys to identify the species of spore-forming bacteria present in natural and artificial containers. Most of our samples came from Illinois. Identification was based on the cellular fatty acid composition of the bacterial cell wall. In addition, we utilized a custom database for commercially produced strains of Bacillus thuringiensis var. israelensis (Bti) and B. sphaericus, to differentiate between larvicidal isolates with commercial or native origin. Native Bti was present at low levels in almost all habitats but was not recovered from bromeliads and metal containers. In temporary woodland pools, 27.9% of the colonies recovered were native Bti. We did not recover larvicidal B. sphaericus in untreated habitats. VectoBac and VectoLex were applied to tires containing water and the tires were sampled 3 months and 9 months after treatment. Isolates of Bti and B. sphaericus with commercial origin were recovered as long as 9 months after application. We noticed numerous cadavers of Aedes triseriatus in several tires 9 months after treatment with VectoBac. We could not determine if this mortality resulted from recycling of Bti in these tires or whether insecticidal crystal proteins from the original treatment were resuspended. Bacillus thuringiensis var. israelensis isolates with commercial ancestry were recovered from untreated tires 9 months after application. Isolates of larvicidal B. sphaericus that differed from the bacteria in VectoLex were also recovered from untreated tires.

The mammalian safety of Bacillus thuringiensis-based insecticides.

The United States Environmental Protection Agency between the years 1961 and 1995 registered 177 products containing viable Bacillus thuringiensis (Bt). Numerous laboratory studies have demonstrated that Bt and Bt products are noninfectious and are toxic to mammals only at a dose > or =10(8) colony forming units (cfu) per mouse (a human equivalent based on the weight of >10(11) cfu). In contrast, as few as three vegetative cells of Bacillus anthracis can kill mice (a human equivalent of >10(3) cfu). There are only two literature reports of Bt infection in man between the year 1997 and the present, and all infected individuals had experienced either extensive burns or a blast injury, which predisposed them to infection. Two epidemiology studies conducted during large-scale aerial Bt serovar kurstaki spray campaigns reported no increased incidence of illness. Some recent papers have expressed concern about the production of Bacillus cereus enterotoxins by Bt isolates. Laboratory studies found no evidence of illness in rats and sheep fed Bt products, nor have epidemiology studies found increased incidence of diarrhea during Bt aerial spray campaigns. Increases in human antibody levels following exposure to Bt products have been reported but there was no increased incidence in asthma or other illness. Based on laboratory studies and field experience, Bt insecticides have an excellent safety record.

Impact of a North American isolate of the microsporidium Nosema carpocapsae on a laboratory population of the codling moth, Cydia pomonella.

Nosema carpocapsae is a microsporidian pathogen of the codling moth, Cydia pomonella. We report the occurrence of this pathogen in a colony originating from collections made in the United States. This is the first record of N. carpocapsae infecting North American codling moths. This North American isolate of N. carpocapsae was indistinguishable from isolates received from New Zealand and Bulgaria, based on small subunit ribosomal RNA sequencing, but was more virulent than the previously described New Zealand isolate. In the laboratory, infected larvae and pupae had increased mortality compared to their uninfected counterparts and developmental time increased by 1 week. There was no effect on female fecundity. Within a cohort of eggs laid by infected females, neonates that emerged first were more likely to be uninfected. We established an uninfected colony by interrupting horizontal transmission and only utilizing the larvae that emerged from the first-laid eggs.

Evaluation of CD4+ T cell function In vivo in HIV-infected patients as measured by bacteriophage phiX174 immunization.

Bacteriophage phiX174 immunization was used to measure CD4(+) T cell function in vivo in human immunodeficiency virus (HIV)-infected patients across all disease stages. Function was evaluated by measuring the ability of T cells to provide help to B cells in antibody production, amplification, and isotype switching. A total of 33 patients and 10 controls received 3 bacteriophage phiX174 immunizations 6 weeks apart. The patients' responses regarding bacteriophage-specific total antibody titers and IgG titers were quantitatively and qualitatively inferior to the controls' responses. Overall, 7 of 33 patients had normal T cell function. Baseline CD4 counts provided the strongest correlation with total antibody and IgG titers. HIV RNA had a weaker association with responses but had some predictive power among patients with a CD4 count >200 cells/microL. Bacteriophage phiX174 immunization seems to be a useful tool for measuring immune function in vivo, which suggests that most HIV-infected patients may have abnormal CD4(+) T cell function despite adequate antiretroviral treatment.

The effects of proportional representation and gender orientation of the task on emergent leadership behavior in mixed-gender work groups.

Much of the research that has examined the behavioral consequences of membership in mixed-gender work groups suggests that men are more participative and influential in task-related behavior. Drawing from elements of sociological, structural, and psychological perspectives, this study examined the effects of group gender composition and gender orientation of the group's task on patterns of emergent leadership behavior. Participants were assigned to male-dominated, female-dominated, or balanced-gender groups for the purpose of discussing and generating solutions for two business-related cases--each case emphasized either male-oriented or female-oriented expertise. The findings suggest that the proportional representation of men and women in a work group, along with the gender orientation of the group's task, can significantly influence the level of leadership behavior exhibited in group activity.

Duration of activity of the microbial larvicide VectoLex CG (Bacillus sphaericus) in Illinois catch basins and waste tires.

The duration of activity of a formulation of Bacillus sphaericus, VectoLex CG, for control of Culex species was evaluated in 338 catch basins in Urbana, IL, and compared to Altosid in 346 catch basins in Champaign, IL. The activity of VectoLex in car and truck waste tires was evaluated in a tire dump located in Pembroke Township, IL. In catch basins, 1 g of VectoLex per catch basin gave the same control as one Altosid briquet. Both larvicides were effective against Culex sp. in catch basins for 1 month, and the duration of control with VectoLex lasted 44 days in one catch basin. VectoLex was considerably cheaper to apply than Altosid briquets, at 0.64 cents per catch basin compared to 90.75 cents, respectively. However, the Altosid briquets were judged to be easier to apply from a vehicle than VectoLex granules. VectoLex (22.6 kg) was used to treat approximately 6,000 car and truck tires; some of the tires were in direct sunlight whereas others were shaded. Aedes triseriatus was the dominant species in these tires. Tires treated with VectoLex contained significantly fewer mosquitoes than control tires, and even 65 days after application, control tires were 16.7 times more likely to contain larvae. We conclude that VectoLex was effective when used in Illinois catch basins and tire dumps, and emphasize that it is more appropriate to base tire treatment rates on the total number of tires present than on a kilogram per hectare basis.

Production of IL-10 by human natural killer cells stimulated with IL-2 and/or IL-12.

Human NK cell activity can be augmented in vitro by stimulation with IL-2 or IL-12, both of which also induce the production of IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF by NK cells. For the first time, we demonstrate that freshly purified NK cells stimulated with IL-2 proliferated and produced IL-10 in a dose-dependent manner. IL-10 mRNA expression, as detected by semiquantitative reverse transcription-PCR, reached peak levels at 24 h. IL-10 protein was detectable on day 2 and further increased on days 3 and 6 as measured by ELISA. However, IL-12 alone induced neither substantial proliferation nor detectable IL-10 production by fresh NK cells, but it synergized with IL-2 in inducing IL-10 mRNA expression and protein synthesis. IL-10 production by activated NK cells was confirmed by intracytoplasmic cytokine staining by three-color immunofluorescence of CD16+ and/or CD56+ NK cells with anti-IL-10 antibody. IL-10 production by NK cells was further confirmed in the NK-like cell line, YT, which constitutively expressed IL-10 mRNA and protein. IL-12 alone did not induce NK proliferation, but it inhibited IL-2-induced proliferation. Neutralization of endogenously produced IL-10 with anti-IL-10 antibodies did not overcome the inhibition of IL-2-induced proliferation by IL-12. Together, these results demonstrate that IL-2 and IL-12 synergize to induce IL-10 production by human NK cells and that IL-12 inhibits IL-2 induced NK cell proliferation by an IL-10-independent mechanism.

Comparison of the cellular fatty acid composition of a bacterium isolated from a human and alleged to be Bacillus sphaericus with that of Bacillus sphaericus isolated from a mosquito larvicide.

The cellular fatty acid (CFA) composition of the cytoplasmic membrane of a bacillus isolated from a human lung and deposited in the National Collection of Type Cultures as Bacillus sphaericus NCTC 11025 was determined by gas-liquid chromatography. The CFA composition of B. sphaericus 2362, isolated from a microbial larvicide, and those of B. sphaericus reference strains obtained from public collections were also determined. Samples were grouped by hierarchical cluster analysis based on the unpaired-group method using arithmetic averages. Samples that linked at a Euclidean distance of < or = 2.0 U were considered to belong to the same strain. NCTC 11025 and the type strain of B. sphaericus, ATCC 14577, were mixed; all other isolates were monotypic. The predominant fatty acid in NCTC 11025 was 12-methyltetradecanoic acid, while the predominant fatty acid in the remaining isolates was 13-methyltetradecanoic acid. NCTC 11025 linked to the other isolates at a Euclidean distance of 83.8 U, and we concluded that it belongs to a different species that we could not identify. We could distinguish among six DNA homology groups of B. sphaericus by using fatty acids. Within DNA homology group IIA, strain 2362 could be distinguished from other strains belonging to serotype H5a, 5b. We concluded that CFA analysis is a useful technique to determine if future human isolates identified as B. sphaericus in fact belong to other species of bacteria or whether the isolates originated from commercial products.

Field trials of VectoLex CG, a Bacillus sphaericus larvicide, in Illinois waste tires and catch basins.

The susceptibilities of Aedes triseriatus (Say), Anopheles punctipennis (Say), Culex restuans (Theobald), and Culex pipiens (L.) larvae to VectoLex CG were determined. VectoLex, formulated on corncob granules (10-14 mesh) was applied to 2 tire dumps and numerous catch basins located in east-central Illinois. VectoLex, formulated as effervescent tablets, was also applied to catch basins. In a sunlit dump, there was a 99.6% reduction in Ae. triseriatus, Cx. restuans, and Cx. pipiens larvae as long as 32 days after treatment, and no pupae were recovered during this interval. There was still a 71.6% larval reduction 74 days after treatment. There was an overall reduction of 22% for An. punctipennis larvae. In a shaded dump, no larval Ae. triseriatus, Cx. restuans, or Cx. pipiens were recovered for 25 days after treatment, and no pupae were recovered 25-67 days after treatment. There was still a 68.7% larval reduction 74 days after treatment. An. punctipennis was unaffected. In one catch basin study, VectoLex was comparable to Altosid (average of 46 days vs. 50 days until larvae were recovered from catch basins). As the summer progressed, the duration of VectoLex control was reduced to 30 days. VectoLex effervescent tablets (evaluated mid-August through September) gave 18-day control. VectoLex was effective against Ae. triseriatus, Cx. restuans, and Cx. pipiens, but was not effective against An. punctipennis in waste tires. VectoLex was effective against Cx. restuans and Cx. pipiens in catch basins.

Dispersal of 10-14-mesh corncob granules in stacked tires.

Dispersal of 10-14-mesh corncob granules was evaluated in 2 random-stacked tire piles, one shingle-stacked tire pile, and one column-stacked tire pile located in a used-tire storage facility in Chicago, IL. Ninety percent and 98%, respectively, of the tires in the 2 random-stacked piles contained granules. In the shingle-stacked tire pile 87% of the tires sampled contained granules, and the number of granules per tire was dependent on depth. The 2 bottom rows of tires were 73.9% less likely to contain granules than the 5 rows above them. In the column-stacked tire pile 91.2% of the tires contained granules and the relationship between granule recovery and tire depth was logarithmic. Overall, the dispersal of 10-14-mesh corncob granules was comparable to that of 8-mesh corncob granules evaluated in a previous study at this site.

Evaluation of a reverse transcriptase-polymerase chain reaction assay for detecting St. Louis encephalitis virus using field-collected mosquitoes (Diptera: Culicidae).

A reverse transcriptase-polymerase chain reaction assay (RT-PCR) was compared with a tissue culture assay (TC) and an enzyme immunoassay (EIA) to detect St. Louis encephalitis virus (SLEV) in mosquito pools. Overall, 1,725 mosquito pools with a low viral prevalence (3.3%-5.0%) were tested. The comparison of the EIA to TC showed that the EIA was 0.947 sensitive, 0.988 specific, and 0.987 accurate. Comparison of RT-PCR to TC showed that the RT-PCR was 0.947 sensitive, 0.980 specific, and 0.979 accurate. Comparison of the RT-PCR to EIA showed that the RT-PCR was 0.878 sensitive, 0.987 specific, and 0.982 accurate. Because of speed, accuracy, and cost, either the RT-PCR or the EIA is recommended as the primary screen. RT-PCR has an advantage over EIA, because the amplified product contains sequence information which can confirm its identity. TC, EIA, or both can be applied as a 2nd assay for the confirmation of samples suspected as positives by the RT-PCR.

Cytolytic effector mechanisms of human CD4+ cytotoxic T lymphocytes.

To elucidate mechanisms by which human CD4+ cells mediated cytolytic activity, we studied the expression of cytolytic proteins and the effects of inhibitors and mAbs on T-cell clones. Of seven cytolytic CD4+ clones, three were specific for the HLA-DR17, while four recognized DR18. Anti-HLA-DR mAb and anti-CD4 mAb blocked lysis. In addition, N alpha-p-tosyl-L-lysine chloromethylketone (TLCK), a serine esterase inhibitor, as well as cytochalasin B and monensin, antagonists of secretory pathways, inhibited CD4+ CTLs, whereas the absence of extracellular Ca+2 or the presence of Ca+2 channel blockers partially inhibited cytotoxicity. CD4+ CTLs induced apoptosis of target cell nuclei and membrane damage simultaneously. The CD4+ clones synthesized perforin and granzyme B and expressed the granule-associated protein TIA-1. Our studies indicate that two distinct mechanisms may contribute to cytolysis by CD4+ clones: (1) a Ca+2-dependent mechanism associated with the cytotoxic granules and (2) a Ca+2-insensitive mechanism.

Cellular fatty acid analysis of Bacillus thuringiensis ssp. israelensis (ONR-60A).

The cellular fatty acid composition of Bacillus thuringiensis ssp. israelensis (B.t.i.) ONR-60A was determined by gas-liquid chromatography (GLC). Isolates of ONR-60A were obtained from the Bacillus Genetic Stock Center, Pasteur Institute, and USDA culture collections. These isolates were compared to one another and the culture collections differed in the strains that they contained. Eleven separate strains of ONR-60A were identified, based on fatty acid composition of the cell envelope. The current Pasteur Institute international standard for B.t.i., IPS 82, was also analyzed and it consisted of 3 strains using these same criteria. Profiles were created for each strain found in ONR-60A and IPS 82, and validated against profiles of commercially produced B.t.i. from 2 time periods, 1983-84, and 1990-94. Approximately 84% of the commercially produced B.t.i. (134 out of 160 agar plates) was identified as belonging to the strains found in ONR-60A and in IPS 82. The most prevalent strain found in IPS 82 is also the most prevalent strain in the recently produced commercial material, 40.8% of the commercial plates analyzed, but commercially produced B.t.i. also retained a strong ONR-60A ancestry (35% of the plates analyzed).