Killing Agent Research Articles

Responses of <I>Rhyzopertha dominica</I> (Coleoptera: Bostrichidae) to Its Aggregation Pheromones as Influenced by Trap Design, Trap Height, and Habitat

Lindgren multiple funnel traps and Japanese beetle traps captured more lesser grain borers, Rhyzopertha dominica F. (Coleoptera: Bostrichidae), than did Pherocon II sticky traps or bucket traps when all were baited with the same aggregation pheromones. Bucket traps captured six-fold fewer beetles than Lindgren four-unit traps. Retentions of captured R. dominica were not significantly different in traps with soapy water or dry insecticide as killing agents for either trap design but were significantly higher than those retained in traps lacking a killing agent. Lindgren eight-unit funnel traps captured a similar number of R. dominica compared with the four-unit funnel traps. More beetles were captured near grain storage facilities than in forests or in open fields. Trap height (1, 2, or 4 m above the ground) had no detectable effect across habitat but significantly interacted with habitat. Traps placed 1 or 2 m high near grain elevators and in open fields captured similar numbers of beetles and yielded higher catches of R. dominica than traps placed 4 m high in these habitats. The reverse was true in forest habitats. Captured R. dominica were similarly female-biased in all trap designs, and the proportion of females to males did not differ among trap heights or habitats in which they were trapped.

Bactrocera zonata

<i>Bactrocera zonata</i>

A MATHEMATICAL MODEL FOR TUMOR CORDS INCORPORATING THE FLOW OF INTERSTITIAL FLUID

This work extends a previous model that described the evolution of a tumor cord (a cylindrical arrangement of tumor cells, generally surrounded by necrosis, growing around a blood vessel of the tumor) under the activity of cell killing agents. In the present model we include the most relevant aspects of the dynamics of extracellular fluid, by computing the longitudinal average of the radial fluid velocity and of the pressure field. We still assume that the volume fraction occupied by the cells always keeps the same constant value everywhere in the cord. The necrotic region is treated as a "fluid reservoir". To improve the modelling of therapeutic treatment, we have subdivided the viable cell population into a proliferating and a quiescent subpopulation. The transitions between the two states are both permitted, and are regulated by rates depending on the local oxygen concentration. For simplicity, the rates of death induced by treatment are assumed to be known functions of the radial distance and time. Existence and uniqueness of the stationary state in the absence of treatment has been shown, as well as the existence and uniqueness of the evolution that arises following a cell killing treatment.

Modulating apoptosis as a target for effective therapy

Alterations in cell proliferation and cell death are essential determinants in the pathogenesis and progression of several diseases such as cancer, neurodegenerative disorders or autoimmune diseases among others. Complex networks of regulatory factors determine whether cells proliferate or die. Recent progress in understanding the molecular changes offer the possibility of specifically targeting molecules and pathways to achieve more effective and rational therapies. Drugs that target molecules involved in apoptosis are used as treatment against several diseases. Candidates such as TNF death receptor family, caspase inhibitors, antagonists of the p53–MDM2 interaction, NF-κB and PI3K pathways and Bcl-2 family members have been targeted as cancer cell killing agents. Moreover, apoptosis of tumor cells can also be achieved by targeting the inhibitor of apoptosis proteins, IAPs, in addition to the classical antiproliferative approach. Disruption of STAT activation and interferon beta therapy have been used as a treatment to prevent the progression of some autoimmune diseases. In models of Parkinson's, Alzheimer's and amyotrophic lateral sclerosis, blocking of Par-4 expression or function, as well as caspase activation, prevents neuronal cell death. Finally, it has been shown that gene therapy may be an encouraging approach for treatment of neurodegenerative disorders.

Deferoxamine as a chelator for 67Ga in the preparation of antibody conjugates

67Ga antibodies (Abs) have been shown to be effective agents for single-cell killing due to the Auger electrons emitted, but their specific activities have not been as high as desired. We therefore evaluated deferoxamine (DFO) as a chelator, as opposed to the cyclic chelator NOTA, which was used previously. Use of DFO for Ab conjugation to 67Ga was reported previously by several laboratories. DFO was conjugated to Abs by two methods, one using Ablysine conjugation and another using mild reduction of Abs to generate thiols in the hinge region. Labeling with 67Ga was efficient, and the specific activities obtained under nonoptimized conditions were twice as high as those achieved previously. However, analysis of these conjugates revealed two problems that appear to prevent their further development. First, the stability was inadequate for the 3-day half-life of the nuclide. Second, the labels were poorly retained within cells after Ab internalization and catabolism. Also, it was found that stability was significantly affected by the incubation buffer used: buffers lacking physiological concentrations of divalent cations Ca and Mg resulted in much lower stability than buffers including them. In conclusion, DFO does not seem to be a suitable chelator for 67Ga conjugation for our purposes.

1038. Lentiviral Gene Transfer into HSC Is Enhanced by Early-Acting Cytokines without Impairing Stem Cell Properties and Involves Cellular Responses Distinct from Cell Cycle Control

Hematopoietic stem cells (HSC) are attractive targets for gene therapy because they can be transduced ex vivo and infused in the donor to generate gene-corrected progeny potentially for a lifespan. To broaden the scope of HSC-based gene therapy, transduction of the majority of HSC in a transplant sample would be required, without affecting stem cell features. HIV-derived lentiviral vectors (LV) are good candidates for this purpose, because they integrate in non-proliferating cells, thus bypassing the requirement of retroviral vectors for ex vivo cell cycle induction. We and others have demonstrated that LV transduce efficiently cord blood-derived SCID/NOD mouse repopulating cells (SRC), assumed to be closely related to HSC, by a short ex vivo incubation. Contrary to the expectations, however, hematopoietic progenitors treatment with the early-acting cytokines IL6, SCF, TPO and Flt3L, strongly enhanced SRC transduction. To determine if cytokine treatment affected SRC repopulating ability, we performed a competitive repopulation assay, injecting half CD34+ cells transduced with and half transduced without cytokines into the same NOD/SCID mouse. We observed that SRC transduced with and without stimulation contributed equally to host repopulation, indicating that cytokine stimulation did not impair engraftment and repopulating ability of HSC in this stringent model. We then asked if cytokines enhanced transduction triggering the cells through the S-phase of the cell cycle, a condition that could lead to the loss of some SRC features. To address this issue, we performed an S-phase suicide assay, transducing CD34+ cells with or without cytokines and exposing them or not to an agent that kills all the cells that pass through the S-phase, before injecting them into NOD/SCID mice. We found higher transduction efficiency in stimulated SRC, and this higher level was also observed for cells exposed to the killing agent, indicating that cytokine enhancement of SRC transduction was not due to progression into S-phase. We then aimed to determine at what stage of transduction cytokines enhanced LV gene transfer. We evaluated viral entry, transducing CD34+ cells with LV vectors pseudotyped with different envelopes, such as A-MLV and RD114/TR. In all cases, we observed higher transduction efficiency in stimulated cells, indicating that the cytokine effect was not linked to a specific entry pathway. Performing transduction with different vector doses, we showed a similar one slope-trend in the dose-response curve both for stimulated and not stimulated CD34+ cells, suggesting that the different transduction efficiency was not due to the expression of saturable restrictive factor by not stimulated cells. Preliminary results rather indicate that, in hematopoietic progenitors, susceptibility to LV transduction is differentially regulated by the activity of a specific enzymatic complex in the cytosol, and that interference with this system strongly up-regulates transduction efficiency. Overall, our results will help to establish experimental conditions for safe and efficient HSC gene therapy, and shed new light on the mechanism of HSC transduction by LV.

A Free Boundary Problem with Unilateral Constraints Describing the Evolution of a Tumor Cord Under the Influence of Cell Killing Agents

A system of tumor cords is schematized by an array of identical cords, each one having approximately a rotational symmetry around its central blood vessel. A mathematical model for the evolution of the cord is presented, taking into account the influence of a limiting nutrient on the proliferation and death of the cells, the volume reduction of the necrotic material due to fluid loss from the cord, and the influence of chemotherapy or radiation treatment. Both the steady state and the evolution problem are considered, showing existence and uniqueness of the solution. A peculiar feature of the evolution model is that the boundary conditions for nutrient concentration on the interface between viable cord and the necrotic region may change during the response to treatment, depending on whether or not new cells enter the necrotic region.

Redox Buffering of Hypochlorous Acid by Thiocyanate in Physiologic Fluids

The major antimicrobial products of neutrophilic myeloperoxidase (MPO) in physiologic fluids are hypochlorous acid (HOCl) and hypothiocyanite (OSCN-), and the former is generally believed to be the killing agent. However, we have determined that HOCl oxidizes SCN- in a facile nonenzymic reaction. The observed kinetics and computational models substantiate the hypothesis that SCN- serves to moderate the potential autotoxicity of HOCl by restricting its lifetime in physiologic fluids. Furthermore, the oxidizing equivalents of HOCl are preserved in OSCN-, a more discriminate biocide that is not lethal to mammalian cells.

Macrophages in spleen and liver direct the migration pattern of rat neutrophils during inflammation.

The exact fate of polymorphonuclear neutrophilic granulocytes (PMN; neutrophils) after their mobilization from the bone marrow is not known. It is believed that they, after a relatively short lifespan (1-3 d), become apoptotic and phagocytosed by macrophages. We have recently shown that transfused neutrophils sequestrate not only in lungs, liver and spleen, but also to a large extent in the bone marrow, possibly because of uptake by macrophages. Hence, we studied if inactivation of macrophages would alter the pattern of neutrophil migration. We used transfused congenic or syngeneic neutrophils in rats with or without sterile peritonitis, induced by a casein preparation (Bacto-Tryptone). To perturb macrophage function, we either killed them with liposome-encapsulated clodronate or overloaded them with inert phagocytosable particles. Transfused neutrophils were tracked with flow cytometric or radiometric methods. Not more than a small portion of the neutrophils migrated to the inflamed peritoneal cavity under any circumstance. Their ecotaxis to liver and spleen was reduced in rats with liver and spleen macrophages either congested with polystyrene particles or depleted by clodronate. The bone marrow uptake and blood retention of transfused neutrophils were increased in macrophage-depleted rats 18 h after transfusion. In rats depleted of liver macrophages only, the sequestration in the liver was reduced, without detectably changed uptake in bone marrow and spleen. Macrophages are instrumental to the neutrophil migration stream in the organism, and their function in this regard is robust and not easily decreased by inert phagocytosable particles or a killing agent.

Low temperature cure of a male killing agent in Drosophila melanogaster

Environmental factors can affect transmission or phenotype expression of selfish cytoplasmic endosymbionts such as embryonic male killers. Temperature is one factor that usually affects the transmission rate of selfish cytoplasmic endosymbionts. Heat cures have been described for several host-parasite systems, cold cures, however, are rare. We report a temperature cure of the Drosophila melanogaster male-killing agent, which occurs when flies are raised at 16.5 °C. Flies grown at 20, 24, and 28 °C maintained an extremely female biased sexual proportion.

Attractancy and Toxicity of an Attracticide for Indianmeal Moth, &lt;I&gt;Plodia interpunctella&lt;/I&gt; (Lepidoptera: Pyralidae)

Plodia interpunctella (Hübner) is a serious and widespread postharvest pest on cereal products, dried fruits, candy, and pet food. Due to the strong positive anemotactic flight response of P. interpunctella males to the main component of the female-produced pheromone [(Z,E)-9,12-tetradecadienyl acetate, herein referred to as ZETA], we evaluated the potential of an attracticide for this pest, in which ZETA as attractant was combined with permethrin as the killing agent. Two concentrations of ZETA [0.16 and 0.32% (wt:wt)] and five concentrations of permethrin [0, 3, 6, 12, and 18% (wt:wt)] were incorporated into Last Call gel (10 different permethrin:ZETA combinations). All attracticide gels were evaluated in a toxicity test, in which either the tip of a leg or an antenna of a virgin P. interpunctella male was touched < 3 s into a dot of attracticide gel. These males were subsequently transferred to jars with virgin females. The toxicity test showed that a brief and gentle contact of P. interpunctella males with attracticide gel containing 3-18% permethrin caused a significant reduction in mating and killed males moths within 24 h. A wind tunnel test was conducted to evaluate the flight responses of P. interpunctella males to the same 10 attracticide gels. Male moths displayed significantly higher levels of positive anemotactic flight and more males made contact with the attracticide gel when the ZETA concentration was 0.16% compared with 0.32%. P. interpunctella males showed no signs of repellency to permethrin concentrations within a range of 0-18% in the attracticide gel. Three densities of P. interpunctella pairs were released into small warehouse rooms, and we found that the attracticide gel suppressed oviposition when the moth density was at a low level, but it was ineffective when the moth density exceeded one male-female pair per 11.3 m3.

Activity of three disinfectants and acidified nitrite against Clostridium difficile spores.

To identify environmentally safe, rapidly acting agents for killing spores of Clostridium difficile in the hospital environment. Three classic disinfectants (2% glutaraldehyde, 1.6% peracetyl ions, and 70% isopropanol) and acidified nitrite were compared for activity against C. difficile spores. Four strains of C. difficile belonging to different serogroups were tested using a dilution-neutralization method according to preliminary European Standard prEN 14347. For peracetyl ions and acidified nitrite, the subjective cleaning effect and the sporicidal activity was also tested in the presence of organic load. Peracetyl ions were highly sporicidal and yielded a minimum 4 log10 reduction of germinating spores already at short exposure times, independent of organic load conditions. Isopropanol 70% showed low or no inactivation at all exposure times, whereas glutaraldehyde and acidified nitrite each resulted in an increasing inactivation factor (IF) over time, from an IF greater than 1.4 at 5 minutes of exposure time to greater than 4.1 at 30 minutes. Soiling conditions did not influence the effect of acidified nitrite. There was no difference in the IF among the 4 strains tested for any of the investigated agents. Acidified nitrite demonstrated a good subjective cleaning effect and peracetyl ions demonstrated a satisfactory effect. Cidal activity was shown against C. difficile spores by glutaraldehyde, peracetyl ions, and acidified nitrite. As acidified nitrite and peracetyl ions are considered to be environmentally safe chemicals, these agents seem well suited for the disinfection of C. difficile spores in the hospital environment.

Influence of extracellular bactericidal agents on bacteria within macrophages.

We employed gentamicin-sensitive and -resistant derivatives of Escherichia coli in a macrophage phagocytosis assay that compared lambda bacteriophage and gentamicin as extracellular bactericidal agents. Colony counts and direct microscopic examination of phagocytized E. coli supported the conclusion that gentamicin entered macrophages, even at low concentrations, and contributed to their bactericidal activity. Also, two E. coli strains differing in the ability to express the adhesin of type 1 pili (FimH) were distinguishably different in intracellular survival when lambda was used as the extracellular killing agent but were indistinguishable when gentamicin was employed.

ANALYSIS OF A CELL-CYCLE SPECIFIC MODEL FOR CANCER CHEMOTHERAPY

A class of mathematical models for cancer chemotherapy which has been described in the literature takes the form of an optimal control problem with dynamics given by a bilinear system. In this paper we analyze a three-dimensional model in which the cell-cycle is broken into three compartments. The cytostatic agent used as control to kill the cancer cells is active in a compartment which combines the second growth phase and mitosis where cell-division occurs. A blocking agent is used as a second control to slow down the transit of cells during synthesis, but does not kill cells. The cumulative effect of the killing agent is used to model the negative effect of the treatment on healthy cells. It is shown that singular controls are not optimal. This eliminates treatments where during some time only a portion of the full drug dose is administered. Consequently only treatments which alternate between a full and no dose, i.e., so-called bang-bang controls, canbe optimal for this model. Both necessary and sufficient conditions for optimality of treatment schedules of this type are given.

Endotoxin activity of Moraxella osloensis against the grey garden slug, Deroceras reticulatum.

Moraxella osloensis is a gram-negative bacterium associated with Phasmarhabditis hermaphrodita, a slug-parasitic nematode that has prospects for biological control of mollusk pests, especially the grey garden slug, Deroceras reticulatum. This bacterium-feeding nematode acts as a vector that transports M. osloensis into the shell cavity of the slug, and the bacterium is the killing agent in the nematode-bacterium complex. We discovered that M. osloensis produces an endotoxin(s), which is tolerant to heat and protease treatments and kills the slug after injection into the shell cavity. Washed or broken cells treated with penicillin and streptomycin from 3-day M. osloensis cultures were more pathogenic than similar cells from 2-day M. osloensis cultures. However, heat and protease treatments and 2 days of storage at 22 degrees C increased the endotoxin activity of the young broken cells but not the endotoxin activity of the young washed cells treated with the antibiotics. This suggests that there may be a proteinaceous substance(s) that is structurally associated with the endotoxin(s) and masks its toxicity in the young bacterial cells. Moreover, 2 days of storage of the young washed bacterial cells at 22 degrees C enhanced their endotoxin activity if they were not treated with the antibiotics. Furthermore, purified lipopolysaccharide (LPS) from the 3-day M. osloensis cultures was toxic to slugs, with an estimated 50% lethal dose of 48 microg per slug, thus demonstrating that the LPS of M. osloensis is an endotoxin that is active against D. reticulatum. This appears to be the first report of a biological toxin that is active against mollusks.

POST-DISPERSAL CHANGES IN THE SPATIAL DISTRIBUTION OF FAGUS CRENATA SEEDS

The spatial distribution of tree seeds is influenced by the behavior of seed dispersers and seed-killing agents. In this study we evaluated the relative importance of multiple seed-killing agents on changes in the spatial pattern of Fagus crenata seeds. The dispersal of seeds and causes of seed death following dispersal were investigated on a 0.6- ha study plot in northern Japan. Seeds were collected using seed traps (n = 110) in the autumn of 1995, as well as directly from the AO layer of the soil, including leaf litter, intermediate, and humus layers (n = 110), in the spring of 1996. A total of 44075 seeds were disseminated into the seed traps, including 17 964 mature seeds; 15% of these were viable the following spring, indicating a large decrease in the abundance of viable seeds during the post-dispersal seed stage. Spatial association of undamaged seeds changed from a positive to independent association between the different dispersal stages, indicating rearrangement of the spatial pattern of seeds through time. During this period, numbers of living seeds generally decreased beneath nonspecific adults compared to those located away from the adults. This change resulted from the spatially different behavior of several biotic and abiotic killing agents. Rodents and fungi attacked the seeds, depending on seed densities and presence of nonspecific adult crowns, respectively, but desiccation was independent of both seed density and nonspecific crowns. The multiple killing agents with different spatial behavior jointly influenced the distributions of recruits entering tree populations.

Proteases commit microbicide

White blood cells use proteases to kill their targets, according to new results from Emer Reeves, Hiu Lu, Anthony Segal (University College, London, England), and colleagues. The findings contradict previously accepted notions that reactive oxygen species (ROSs) are the killing agents. Figure Neutrophils kill engulfed microbes (white) using proteases. ROSs like superoxide are produced by NADPH oxidase activity when white blood cells, primarily neutrophils, phagocytose pathogens. For decades it has been thought that high concentrations of the toxic ROSs resulting from this respiratory burst are directly responsible for killing microorganisms. But the new results prove otherwise. Segal's group showed that mice deficient in protease activity have increased sensitivity to microbes. Additionally, purified neutrophils from the protease-deficient mice are impaired in their antimicrobial activity in vitro. “Our results are conclusive,” says Segal. “By taking out the proteases, we do not interfere with the respiratory burst, but do interfere with killing.” The proteases are activated through increases in both pH and hypertonicity in the phagocytotic vacuoles. These changes arise when NADPH oxidase activity drives electrons into the vacuole. To compensate for the charge transfer across the membrane, K+ also enters. The resulting hypertonic conditions release the cationic proteases from their bound state on an anionic proteoglycan matrix. K+ entry also results in alkaline conditions that stimulate protease activity. The proteases are then free to bind to and digest the target microbes. ▪ Reference: Reeves, E., et al. 2002. Nature. 416:291–297. [PubMed]

Assembly and activation of the neutrophil NADPH oxidase in granule membranes.

Phagocyte activation is accompanied by assembly of an NADPH oxidase that reduces oxygen to form a number of reactive species. These oxygen radicals can eradicate invading microorganisms, regulate the function of other immune reactive cells, and cause damage to "innocent bystander" cells. It is generally assumed that the NADPH oxidase is activated exclusively in the plasma membrane. In neutrophils, this assumption does not fit with the subcellular localization of the membrane component of the oxidase, which is stored in granule compartments. It has now become increasingly evident that oxidants are also produced in an intracellular compartment that we identify as the specific granules. Myeloperoxidase is stored in another granule subset, the azurophil granules, and participates in the processing of the oxidative metabolites. We suggest that neutrophil activation is accompanied by fusion between azurophil and specific granules, allowing these peroxidase-dependent reactions to take place. The presented data suggest a requisite role for neutrophil oxidants complementing their function as microbial killing agents. Signaling capabilities of the oxidants, affecting for example, the state of protein phosphorylation, regulation of transcription factors, and induction of apoptosis, are discussed.

Influence of trap type, pheromone quantity and trapping location on capture of the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae).

Field experiments were conducted in order to evaluate the effect of trap design, pheromone quantity and trapping location on the capture of Pectinophora gossypiella in two cotton fields located in Central Greece. In the first field, three commercially available gossyplure-baited trap types were compared: two adhesive, Delta and Pherocon II, and the Funnel trap, on which moths are captured with the addition of a killing agent (DDVP strip). In the second field, Delta traps with 1, 2, 3 or 4 gossyplure lures were compared. Significant differences were noted among trap types. The Funnel traps caught approx. 4.2 and 29.9 times more moths compared to the Delta and the Pherocon II traps, respectively. The correlation of captures among the three trap types was positive and significant only until mid-July. On the other hand, throughout the entire trapping period (June–September), this correlation was significant only between the two adhesive trap types. No significant differences were noted among traps with different pheromone quantity. In addition, the correlation of captures among traps with different lure numbers was positive and significant during the entire trapping period. For both experimental fields, no significant differences were recorded between centrally located and peripheral traps, although a wider range of captures was recorded in the peripheral traps.

Bi-specific antibodies in cancer therapy.

In the 1980s monoclonal antibodies promised an era of “magic” bullet therapies that could specifically kill tumor cells while leaving healthy tissues intact. But the need to covalently couple the killing agent (toxin, radioisotopes, etc.) limited their clinical potential. Now, new bispecific antibodies (BsAbs, or bi-functional antibodies) have been developed. The prototypic BsAb consists of two different specific binding sites, with one directed to the target antigen on the surface of tumor cells, and the other to a trigger molecule.