Killing Strategies Research Articles

Multifunctional host defense peptides: intracellular‐targeting antimicrobial peptides

There is widespread acceptance that cationic antimicrobial peptides, apart from their membrane-permeabilizing/disrupting properties, also operate through interactions with intracellular targets, or disruption of key cellular processes. Examples of intracellular activity include inhibition of DNA and protein synthesis, inhibition of chaperone-assisted protein folding and enzymatic activity, and inhibition of cytoplasmic membrane septum formation and cell wall synthesis. The purpose of this minireview is to question some widely held views about intracellular-targeting antimicrobial peptides. In particular, I focus on the relative contributions of intracellular targeting and membrane disruption to the overall killing strategy of antimicrobial peptides, as well as on mechanisms whereby some peptides are able to translocate spontaneously across the plasma membrane. Currently, there are no more than three peptides that have been convincingly demonstrated to enter microbial cells without the involvement of stereospecific interactions with a receptor/docking molecule and, once in the cell, to interfere with cellular functions. From the limited data currently available, it seems unlikely that this property, which is isolated in particular peptide families, is also shared by the hundreds of naturally occurring antimicrobial peptides that differ in length, amino acid composition, sequence, hydrophobicity, amphipathicity, and membrane-bound conformation. Microbial cell entry and/or membrane damage associated with membrane phase/transient pore or long-lived transitions could be a feature common to intracellular-targeting antimicrobial peptides and mammalian cell-penetrating peptides that have an overrepresentation of one or two amino acids, i.e. Trp and Pro, His, or Arg. Differences in membrane lipid composition, as well as differential lipid recruitment by peptides, may provide a basis for microbial cell killing on one hand, and mammalian cell passage on the other.

Design of Antibacterial Surfaces and Interfaces: Polyelectrolyte Multilayers as a Multifunctional Platform

The adhesion and proliferation of bacteria on abiotic surfaces pose challenges related to human infection, including subsequent formation of antibiotic-resistant biofilms in both healthcare and industrial applications. Although the design of antibacterial materials is a longstanding effort, the surface properties that modulate adhesion of viable bacteria—the critical first step in biofilm formation—have been difficult to decouple. This partial and limited success is due chiefly to two factors. First, bacteria cells exhibit multiple, complex adhesion mechanisms that vary with bacteria strain, rapid genetic mutations within a given strain, and mutable environmental stimuli such as nutrient levels and fluid velocities. Second, there exist only a limited number of studies that systematically characterize or vary the physical, chemical, and mechanical properties of potential antimicrobial materials. Here, we briefly review the dominant strategies for antimicrobial material surface design, including the advantages and limitations of approaches developed via synthetic and natural polymers. We then consider polyelectrolyte multilayers (PEMs) as a versatile materials platform to adopt and integrate these strategies, as well as to elucidate the individual contributions of tunable material properties that limit viable bacteria adhesion. Together, these findings suggest that PEMs can be tailored to leverage the key advantages of bacterial adhesion resistance, contact killing, and biocide leaching strategies for a wide range of antimicrobial surface applications.

Switch from perforin‐expressing to perforin‐deficient CD8+ T cells accounts for two distinct types of effector cytotoxic T lymphocytes in vivo

Although CD8(+) cytotoxic T lymphocytes (CTL) exhibit both Fas ligand (FasL) -based and perforin-based lytic activities, the accepted hallmark of a fully active CTL remains its perforin killing machinery. Yet the origin, rationale for possessing both a slow-acting (FasL) and a fast-acting (perforin) killing mechanism has remained enigmatic. Here we have investigated perforin expression in CTL directly involved in acute tumour (i.e. leukaemias EL4 and L1210) allograft rejection occurring within the peritoneal cavity. We show that at the height of the immune response, the majority of conjugate-forming CD8(+) CTL express high levels of perforin messenger RNA and protein, and kill essentially via perforin. Later however, coinciding with complete rejection, fully cytocidal CTL emerge which exhibit a stark decrease in perforin and now kill preferentially via constitutively expressed FasL. Although late in emergence, and persistent, these powerful CTL are neither effector-memory nor memory CTL. This finding has implications for the monitoring of anti-transplant responses in clinical settings, based on assessing perforin expression in graft infiltrating CD8(+) T cells. The results show that as the immune response progresses in vivo, targeted cellular suicide mainly prunes high perforin-expressing CD8(+) cells, resulting in the gradual switch in effector CTL, from mostly perforin-based to largely Fas/FasL-based killers. Hence, two kinds of CD8(+) CTL have two killing strategies.

Release of HMGB1 in Response to Proapoptotic Glioma Killing Strategies: Efficacy and Neurotoxicity

In preparation for a phase I clinical trial using a combined cytotoxic/immunotherapeutic strategy with adenoviruses (Ad) expressing Flt3L (Ad-Flt3L) and thymidine kinase (Ad-TK) to treat glioblastoma (GBM), we tested the hypothesis that Ad-TK+GCV would be the optimal tumor-killing agent in relation to efficacy and safety when compared with other proapoptotic approaches. The efficacy and neurotoxicity of Ad-TK+GCV was compared with Ads encoding the proapoptotic cytokines [tumor necrosis factor-alpha, tumor necrosis factor-related apoptosis-inducing factor (TRAIL), and Fas ligand (FasL)], alone or in combination with Ad-Flt3L. In rats bearing small GBMs (day 4), only Ad-TK+GCV or Ad-FasL improved survival. In rats bearing large GBMs (day 9), the combination of Ad-Flt3L with Ad-FasL did not improve survival over FasL alone, whereas Ad-Flt3L combined with Ad-TK+GCV led to 70% long-term survival. Expression of FasL and TRAIL caused severe neuropathology, which was not encountered when we used Ad-TK+/-Ad-Flt3L. In vitro, all treatments elicited release of high mobility group box 1 protein (HMGB1) from dying tumor cells. In vivo, the highest levels of circulating HMGB1 were observed after treatment with Ad-TK+GCV+Ad-Flt3L; HMGB1 was necessary for the therapeutic efficacy of AdTK+GCV+Ad-Flt3L because its blockade with glycyrrhizin completely blocked tumor regression. We also showed the killing efficacy of Ad-TK+GCV in human GBM cell lines and GBM primary cultures, which also elicited release of HMGB1. Our results indicate that Ad-TK+GCV+Ad-Flt3L exhibit the highest efficacy and safety profile among the several proapoptotic approaches tested. The results reported further support the implementation of this combined approach in a phase I clinical trial for GBM.

Structural Determinants of Antimicrobial and Antiplasmodial Activity and Selectivity in Histidine-rich Amphipathic Cationic Peptides

Designed histidine-rich amphipathic cationic peptides, such as LAH4, have enhanced membrane disruption and antibiotic properties when the peptide adopts an alignment parallel to the membrane surface. Although this was previously achieved by lowering the pH, here we have designed a new generation of histidine-rich peptides that adopt a surface alignment at neutral pH. In vitro, this new generation of peptides are powerful antibiotics in terms of the concentrations required for antibiotic activity; the spectrum of target bacteria, fungi, and parasites; and the speed with which they kill. Further modifications to the peptides, including the addition of more hydrophobic residues at the N terminus, the inclusion of a helix-breaking proline residue or using D-amino acids as building blocks, modulated the biophysical properties of the peptides and led to substantial changes in toxicity to human and parasite cells but had only a minimal effect on the antibacterial and antifungal activity. Using a range of biophysical methods, in particular solid-state NMR, we show that the peptides are highly efficient at disrupting the anionic lipid component of model membranes. However, we also show that effective pore formation in such model membranes may be related to, but is not essential for, high antimicrobial activity by cationic amphipathic helical peptides. The information in this study comprises a new layer of detail in the understanding of the action of cationic helical antimicrobial peptides and shows that rational design is capable of producing potentially therapeutic membrane active peptides with properties tailored to their function.

Cloning, expression, purification and functional characterization of the oligomerization domain of Bcr–Abl oncoprotein fused to the cytoplasmic transduction peptide

Protein-based cellular therapeutics have been limited by getting molecules into cells and the fact that many proteins require accurate cellular localization for function. Cytoplasmic transduction peptide (CTP) is a newly designed transduction peptide that carries molecules across the cell membrane with a preference to localize in the cytoplasmic compartment and is, therefore, applicable for cytoplasmic targeting. The Bcr–Abl fusion protein, playing major causative role in chronic myeloid leukemia (CML), is a cytoplasmic oncoprotein that contains an N-terminus oligomerization domain (OD) mediating homodimerization of Bcr–Abl proteins, and an intact OD in Bcr–Abl is required both for the activation of its transforming activity and tyrosine kinase. Therefore, disrupting Bcr–Abl oligomerization represents a potential therapeutic strategy for inhibiting Bcr–Abl oncogenicity. In this study, we explored the possible homodimerization-disrupting and tyrosine kinase inhibiting effect of the transduction of OD in Bcr–Abl positive K562 cells. By expressing in Escherichia coli a CTP-OD-HA fusion protein followed by Ni +–NTA affinity purification, immunoblot identification and enterokinase cleavage, we showed that the CTP-OD-HA protein was structurally and functionally active in that it potently transduced and primarily localized into the cytoplasmic compartment, heterodimerized with Bcr–Abl, and potently inhibited the phospho-tyrosine pathways of Bcr–Abl oncoprotein at a low concentration of 4 μM. These results delineate strategies for the expression and purification of therapeutic molecules for intracytoplasmic protein based therapeutics and the CTP-OD-HA-mediated killing strategy could be explored as a promising anti-leukemia agent or an adjuvant to the conventional therapeutic modalities in chronic myeloid leukemia, such as in vitro purging.

Imaging a dying tumor

When T cells come across a tumor, they kill off the outer layers and then move in, say Boissonnas et al. on page 345. The authors use intravital microscopy to show that this infiltration and obliteration of the tumor is antigen dependent. Figure 1 Solid tumors are destroyed (top) only when tumor cells (green) express antigen. Intravital microscopy has previously revealed the killing strategy of T cells in lymphoid tissue. The T cells use a search-and-destroy tactic, moving rapidly through the tissue while constantly scanning for targets. When an enemy is identified, they stop, kill the intruder, and then resume the hunt. The T cells' method of killing inside a solid nonlymphoid tumor environment, however, is less certain. “We knew that the immune system can reject tumors,” says senior author Sebastian Amigorena, “but we didn't really understand how T cells function inside the tumor.” Amigorena and his colleagues now show that the initial contact between anti-tumor T cells and their targets is at the tumor periphery. Once the peripheral cells are dead, the T cells attack the next layer and the next, thus gradually diminishing the tumor mass. This method of tumor destruction by T cells is dependent on antigen expression by the tumor cells. In tumors that do not express a cognate antigen, T cells are initially detected in the periphery but then drift away when they fail to find an appropriate target. The team now plans to use their imaging set-up to investigate whether anti-tumor T cells are doing the killing on their own or, as seen in lymphoid tissue, are recruiting other immune cells. Knowing what cells work against a tumor might lead to anti-tumor strategies that help these cells penetrate to a tumor's depths.

A moving superimposed finite element method for structural topology optimization

Level set methods are becoming an attractive design tool in shape and topology optimization for obtaining efficient and lighter structures. In this paper, a dynamic implicit boundary-based moving superimposed finite element method (s-version FEM or S-FEM) is developed for structural topology optimization using the level set methods, in which the variational interior and exterior boundaries are represented by the zero level set. Both a global mesh and an overlaying local mesh are integrated into the moving S-FEM analysis model. A relatively coarse fixed Eulerian mesh consisting of bilinear rectangular elements is used as a global mesh. The local mesh consisting of flexible linear triangular elements is constructed to match the dynamic implicit boundary captured from nodal values of the implicit level set function. In numerical integration using the Gauss quadrature rule, the practical difficulty due to the discontinuities is overcome by the coincidence of the global and local meshes. A double mapping technique is developed to perform the numerical integration for the global and coupling matrices of the overlapped elements with two different co-ordinate systems. An element killing strategy is presented to reduce the total number of degrees of freedom to improve the computational efficiency. A simple constraint handling approach is proposed to perform minimum compliance design with a volume constraint. A physically meaningful and numerically efficient velocity extension method is developed to avoid the complicated PDE solving procedure. The proposed moving S-FEM is applied to structural topology optimization using the level set methods as an effective tool for the numerical analysis of the linear elasticity topology optimization problems. For the classical elasticity problems in the literature, the present S-FEM can achieve numerical results in good agreement with those from the theoretical solutions and/or numerical results from the standard FEM. For the minimum compliance topology optimization problems in structural optimization, the present approach significantly outperforms the well-recognized ‘ersatz material’ approach as expected in the accuracy of the strain field, numerical stability, and representation fidelity at the expense of increased computational time. It is also shown that the present approach is able to produce structures near the theoretical optimum. It is suggested that the present S-FEM can be a promising tool for shape and topology optimization using the level set methods. Copyright © 2005 John Wiley & Sons, Ltd.

Feeding behaviour and bite force of sabretoothed predators

The feeding behaviour of extinct sabretoothed predators (machaeroidines, nimravids, barbourofelids, machairodonts and thylacosmilines) is investigated using beam theory. Because bite force applied along the mandible should be proportional to the external dimension of the mandibular corpus, patterns of variation in these dimensions at interdental gaps will reflect the adaptation of the jaw to specific loads, related to killing methods. Comparison of the mandibular force profiles of sabretooths to those of extant conical-toothed carnivorans of known feeding behaviour reveals that sabretooths had a powerful bite, as strong or stronger than extant felids of similar mandibular length. Loads exerted at the lower canine were better constrained in the sagittal plane than in extant conical-toothed carnivorans, indicating that prey was efficiently restrained when the sabre bite was delivered. The mandibular symphysis is generally better buttressed dorsoventrally in dirk-toothed sabretooths than in scimitar-toothed sabretooths, implying different killing strategies for the two ecomorphs: dirktooths delivered powerful sabre bites on prey they restrained with their forelimbs, while scimitartooths delivered slashing sabre bites and may have used their incisor battery to subdue their prey. The mandibular symphysis of Smilodon fatalis is less buttressed dorsoventrally than that of other dirk-toothed sabretooths, possibly as a consequence of the greater torsional stresses induced while feeding rapidly on carcasses in response to intense competition. The mandibular symphysis of Thylacosmilus atrox is better buttressed dorsoventrally in juveniles than in adults, suggesting that young marsupial sabretooths underwent an extended period of parental care as typically observed in modern felids and inferred for eutherian sabretooths. Finally, machaeroidines and the nimravid Nimravus brachyops are exceptional in exhibiting a degree of dorsoventral buttressing of the mandibular symphysis that is intermediate between advanced sabretooths and conical-toothed felids but similar to the extant Neofelis nebulosa, suggesting that the latter taxon may be close to the ancestral condition of a new sabretooth radiation. © 2005 The Linnean Society of London, Zoological Journal of the Linnean Society, 2005, 145, 393–426.

RNA molecules as anti-cancer agents

The inhibition of cancer growth and progression is one of the major challenges facing modern medicine. Despite significant progress in the development of therapies against cancer, only in a few cases are these therapies effective. Because cancer is a complex disease, agents that target a single oncogenic pathway have low efficacy, in addition to allowing the emergence of drug resistance. There is a need for specific therapy, which can affect a broad range of cancers, with minimal side effects. Here we summarize several novel anti-cancer strategies that answer the above criteria. These strategies utilize the multiple anti-proliferative effects of double stranded RNA (dsRNA): (1) the classical antisense RNA hybridizes with its target mRNA, leading to reduced levels of a specific oncoprotein; (2) short dsRNA of specific sequence, known as small inhibitory RNA (siRNA), can selectively and efficiently inhibit expression of specific oncogenes, expressed in cancer cells but not in normal cells. Shutting down the expression of cancer-promoting genes by siRNA has proven to be an effective approach against several cancers; (3) long dsRNA, frequently expressed in cells infected with viruses, activates mechanisms that efficiently kill the infected cells, thereby preventing spread of the virus. The dsRNA killing strategy (DKS), involving the in situ generation of dsRNA of sufficient length to induce antiviral defenses specifically in cancer cells, is a novel strategy developed in our laboratory. DKS has the potential to be applicable to a wide range of cancers. Thus dsRNA-based anti-cancer strategies could be powerful tools for cancer treatment.

Evolution of South American mammalian predators (Borhyaenoidea): anatomical and palaeobiological implications

The evolution of South American carnivorous marsupials, the borhyaenoids, has been investigated through the functional analysis of postcranial adaptive traits and palaeobiological data. There is evidence that the evolutionary history of Borhyaenoidea proceeded from a noncursorial ancestor. The locomotion and habits of the early Palaeocene Mayulestes ferox probably approached a generalized plesiomorphic pattern for marsupial locomotion, i.e. primarily terrestrial with secondary arboreal adaptations. An exceptionally rich early Miocene Patagonian fauna has yielded various morphological predator types, from scansorial ambusher to terrestrial, incipiently cursorial, taxa. The most specialized borhyaenoid was the powerful sabretooth Thylacosmilus atrox that survived until the late Pliocene. The evolution from a scansorial pattern towards a cursorial trend, illustrated by Borhyaena tuberata, and towards the probable development of postlactational parental care in Thylacosmilus, required by its dental specialization and killing strategy, suggests a modification of the selective pressures and predatory activities of the group over evolutionary time. © 2004 The Linnean Society of London, Zoological Journal of the Linnean Society, 2004, 140, 487–521.

Eliciting hyperacute rejection as a tumor killing strategy. Herpes amplicon vector transfer of the alpha(1,3)galactosyltransferase gene.

Eliciting hyperacute rejection as a tumor killing strategy. Herpes amplicon vector transfer of the alpha(1,3)galactosyltransferase gene.

Microwear on Canines and Killing Behavior in Large Carnivores: Saber Function in Smilodon fatalis

Canids and hyaenids display a high density of features of microwear on canine teeth that reflect their similarity in killing strategy. However, hyaenids tend to have a higher percentage of short and wide features (pits) because of the high percentage of bone in their diet. Canines of felids display relatively fewer features than canids and hyaenids, which suggest that felids apprehend prey by delivering deep, strong bites that result in less abrasion of the canines. I suggest that canids rely more heavily on their canines and incisors for feeding than do felids. Smilodon fatalis, a large saber-tooth cat from the Pleistocene of North America, is similar to living felids in having relatively few features of canines, but differs from cheetahs (Acinonyx jubatus) and African lions (Panthern leo) in having relatively more pits. S. fatalis showed no consistent similarity in features of microwear on canine teeth to six large carnivores (leopard, Panthera pardus; cheetah; African lion; spotted hyaena, Crocuta crocuta; African wild dog, Lycaon pictus; gray wolf, Canis lupus), which suggests that its killing behavior and possibly dietary preference differed from modern predators. S. fatalis may have avoided contact with bone during killing and feeding encounters, which caused fewer features to form on the upper canines.

The M/G/1 queue with negative customers

We derive expressions for the generating function of the equilibrium queue length probability distribution in a single server queue with general service times and independent Poisson arrival streams of both ordinary, positive customers and negative customers which eliminate a positive customer if present. For the case of first come first served queueing discipline for the positive customers, we compare the killing strategies in which either the last customer in the queue or the one in service is removed by a negative customer. We then consider preemptive-restart with resampling last come first served queueing discipline for the positive customers, combined with the elimination of the customer in service by a negative customer—the case of elimination of the last customer yields an analysis similar to first come first served discipline for positive customers. The results show different generating functions in contrast to the case where service times are exponentially distributed. This is also reflected in the stability conditions. Incidently, this leads to a full study of the preemptive-restart with resampling last come first served case without negative customers. Finally, approaches to solving the Fredholm integral equation of the first kind which arises, for instance, in the first case are considered as well as an alternative iterative solution method.

The M/G/1 queue with negative customers

We derive expressions for the generating function of the equilibrium queue length probability distribution in a single server queue with general service times and independent Poisson arrival streams of both ordinary, positive customers and negative customers which eliminate a positive customer if present. For the case of first come first served queueing discipline for the positive customers, we compare the killing strategies in which either the last customer in the queue or the one in service is removed by a negative customer. We then consider preemptive-restart with resampling last come first served queueing discipline for the positive customers, combined with the elimination of the customer in service by a negative customer—the case of elimination of the last customer yields an analysis similar to first come first served discipline for positive customers. The results show different generating functions in contrast to the case where service times are exponentially distributed. This is also reflected in the stability conditions. Incidently, this leads to a full study of the preemptive-restart with resampling last come first served case without negative customers. Finally, approaches to solving the Fredholm integral equation of the first kind which arises, for instance, in the first case are considered as well as an alternative iterative solution method.