Cytokine-induced killer cells: A prospective vector for effective smuggling of oncolytic virus to tumor cells

The concept of using viruses to kill tumors has long been established, but the field has suffered great setbacks and “bottle neck” in target efficiency. The problem with using systemic virotherapy is that the immune system and tumor microenvironment could seek, sabotage and destroy virus, which allows only a tiny fraction of viruses to find their way to tumors. In our prospect, cytokine-induced killer (CIK) cells can be a prospective sheltering agent. The tumor-selective viruses encapsuled in CIK cells can be safely and efficiently delivered to tumor cells, attaining a synergy of tumor killing by both CIK cells and tumor-selective viruses. For successful delivery, the viruses should have high infectious ability to CIK cells, and the replication of viruses should be strictly modulated by cell vehicles. Ad5F35 chimeric adenovirus can be satisfactory agents if their replication can be driven by promoter of CD40 ligands. Moreover, ensuring absolute safety, either CIK cells or viral passengers can be engineered to express certain therapeutic genes to further enhance tumoricidal effect.     

The emergence of combinatorial strategies in the development of RNA oncolytic virus therapies

Oncolytic viruses (OVs) represent an exciting new biological approach to cancer therapy. In particular, RNA viruses have emerged as potent agents for oncolytic virotherapy because of their capacity to specifically target and destroy tumour cells while sparing normal cells and tissues. Several barriers remain in the development of OV therapy, including poor penetration into the tumour mass, inefficient virus replication in primary cancers, and tumour-specific resistance to OV-mediated killing. The combination of OVs with cytotoxic agents, such as small molecule inhibitors of signalling or immunomodulators, as well as stealth delivery of therapeutic viruses have shown promise as novel experimental strategies to overcome resistance to viral oncolysis. These agents complement OV therapy by unblocking host pathways, delivering viruses with greater efficiency and/or increasing virus proliferation at the tumour site. In this review, we summarize recent development of these concepts, the potential obstacles, and future prospects for the clinical utilization of RNA OVs in cancer therapy.     

Resistance to Co-Occurring Phages Enables Marine Synechococcus Communities To Coexist with Cyanophages Abundant in Seawater

Recent reports documenting very high viral abundances in seawater have led to increased interest in the role of viruses in aquatic environments and a resurgence of the hypothesis that viruses are significant agents of bacterial mortality. Synechococcus spp., small unicellular cyanobacteria that are important primary producers at the base of the marine food web, were used to assess this hypothesis. We isolated a diverse group of Synechococcus phages that at times reach titers of between 10³ and 10⁴ cyanophages per ml in both inshore and offshore waters. However, despite their diversity and abundance, we present evidence in support of the hypothesis that lytic phages have a negligible effect in regulating the densities of marine Synechococcus populations. Our results indicate that these bacterial communities are dominated by cells resistant to their co-occurring phages and that these viruses are maintained by scavenging on the relatively rare sensitive cells in these communities.     

A Mechanistic Tumor Penetration Model to Guide Antibody Drug Conjugate Design

Antibody drug conjugates (ADCs) represent novel anti-cancer modalities engineered to specifically target and kill tumor cells expressing corresponding antigens. Due to their large size and their complex kinetics, these therapeutic agents often face heterogeneous distributions in tumors, leading to large untargeted regions that escape therapy. We present a modeling framework which includes the systemic distribution, vascular permeability, interstitial transport, as well as binding and payload release kinetics of ADC-therapeutic agents in mouse xenografts. We focused, in particular, on receptor dynamics such as endocytic trafficking mechanisms within cancer cells, to simulate their impact on tumor mass shrinkage upon ADC administration. Our model identified undesirable tumor properties that can impair ADC tissue homogeneity, further compromising ADC success, and explored ADC design optimization scenarios to counteract upon such unfavorable intrinsic tumor tissue attributes. We further demonstrated the profound impact of cytotoxic payload release mechanisms and the role of bystander killing effects on tumor shrinkage. This model platform affords a customizable simulation environment which can aid with experimental data interpretation and the design of ADC therapeutic treatments.     

Critical components of testicular function and sensitivity to disruption

Toxic agents can interfere with the male reproductive system at many targets. Radiation and cancer chemotherapeutic drugs represent one class of toxins the sterilizing effects of which can be analyzed qualitatively and quantitatively in terms of testicular cell kinetics. The cells most sensitive to killing by these agents are the rapidly dividing, differentiating spermatogonia. Cells past the DNA-synthetic stages, including spermatocytes, spermatids, and nongerminal cells, are generally resistant. The slow cycling stem spermatogonia show an intermediate sensitivity, but appear to be the critical targets for the resulting long-term oligo- or azoospermia and infertility. The extent of recovery of spermatogenesis and the duration of infertility can be predicted on the basis of stem cell survival alone, independent of the antineoplastic agent used. When murine stem cells are killed, regeneration of their number and repopulation of the seminiferous epithelium begin almost immediately. In man, recovery can be delayed for years after exposure to agents that kill stem cells. This is a result of the regulation of stem cell regeneration and differentiation in man, the mechanisms of which are unknown. This regulation can explain quantitative differences in interspecies sensitivities to toxic agents. For example, man is much more sensitive than the mouse to reduction in sperm count by radiation at short times after exposure, but not when sufficient recovery times are allowed.     

The secret garden's gardeners

The role of the microbial fauna in our gut for health and well-being is undisputed. Now, scientists are discovering that gut viruses also play a crucial role in modulating our risk for a wide range of diseases.Research has shown that the microbiota—the population of micro-organisms inhabiting the gut—has a profound influence on health in both humans and animals. However, most studies have largely ignored the viral population of the gut—the virome—although it is much larger, both in number of organisms and in genetic diversity. This is because the virome was thought to be less important for health and immunity, as it mainly comprises bacteriophages that only affect bacteria. However, researchers are beginning to realize that the viruses present might well be important in human health, as they manipulate the microbiota, swapping genetic virulence factors among bacteria, and through interaction with the host immune system.There are two distinct categories of virus in the gut: phages, which infect bacteria, and viruses that target host cells. Although these two categories are apparently independent of each other, there is a relationship between them, as indicated by growing evidence that the microbiota as a whole, including phages, has a crucial role in protecting against bacterial and viral infections [1].The phage and bacteria populations of the gut have an intricate relationship, which raises the potential therapeutic use of phages to treat a variety of conditions caused by bacteria in the gut, especially those involving chronic inflammation. The first step, however, is to explore and analyse the phage populations in the gut in terms of diversity and number, along with their interactions with their bacterial targets. This has proven to be a major challenge, given the enormous difficulties in identifying, isolating and amplifying genetic material from the phage population.Nevertheless, researchers from the Weizmann Institute of Science and Tel Aviv University in Israel have made substantial progress by indirectly identifying phages through clustered regularly interspaced short palindromic repeats (CRISPRs) in their bacterial hosts [2]. CRISPRs function as a prokaryotic adaptive immune system against genetic invaders such as phages by recognizing foreign DNA and then silencing its expression in a manner analogous to RNA interference (RNAi) in eukaryotes. Short segments of the foreign DNA, known as spacers, are incorporated into the bacterial genome to provide the memory of past exposures to enable recognition of phage DNA.The phage and bacteria populations of the gut have an intricate relationship, which raises the potential therapeutic use of phages to treat a variety of conditions…The Israeli study reconstructed the CRISPR bacterial immune system in the human gut microbiomes of 124 European individuals, and from that identified 991 phages targeted in at least one of the individuals. Of these phages, 78% were present in at least two individuals and some turned out to be the same ones that had already been identified in Japanese and American people. This global distribution of particular phages was a surprise, given that in other ecological niches, notably seawater, where phages are highly abundant, there is great genetic diversity among the populations, even over short distances.The Israeli team further succeeded in deducing the bacterial hosts of 130 of the phages, which allowed them to study the associated phage–bacteria interactions. It turned out that a subset of the phages had developed closer associations with their host bacteria as lysogenized prophages after fusing their DNA with the bacterial chromo-some or as plasmids. Rotem Sorek, a specialist in microbial warfare at the Weizmann Institute of Science and co-author of the Israel study, commented that this behaviour allows bacteria to take advantage of the phage by incorporating and transmitting genes that provide vital functions and occasionally aid pathogenesis. “There are clear instances of phages ‘helping'' pathogenic bacteria to attack humans,” Sorek said. “The toxins of the Cholera, Diphtheria and Shigella (disenteria) are all carried by phages that are integrated into the bacterial genome.”Horizontal gene transfer among bacteria has long been known to increase the adaptability of several potentially virulent bacterial species, but it is only recently that the mechanisms involving prophages have begun to be elucidated. A significant advance was made in a Japanese study from the University of Miyazaki, inspired by the observation that many sequenced bacterial genomes contain multiple prophages carrying a wide range of genes involved in virulence, and that these often seem to contain genetic defects [3]. The team analysed a virulent strain of Escherichia coli, known as O157, which contains 18 prophages that encode various genes involved in the production of virulence factors, including two potent cytotoxins: Shiga toxins 1 and 2. Most of the prophages they identified contained multiple genetic defects, yet they seemed to be capable of transporting virulence elements between not only members of the same strain but also different E. coli strains.The conclusion from their study was that defective prophages in close proximity within E. coli cells were still capable of recombining to yield a new phage that was released from the cell and could infect other cells nearby in the gut. It seems that these defective prophages were not just evolutionary leftovers, but were important components of the bacterial genome, conferring additional adaptive flexibility through horizontal gene transfer. Many other bacteria contain multiple prophages with genetic defects, so it is probable that this mechanism is not confined to E. coli.Other studies have focused on the composition of phage virus populations outside bacteria in the gut, as part of initiatives to compare and contrast the virome and bacteriome in response to individual genetic variation and environmental factors such as diet. One might imagine that phage and bacteria populations should be closely correlated, but it turns out that there are significant differences in the level of variation between individuals, as well as over time within the same individual. A study at the Washington University School of Medicine, USA, on monozygotic twins, found that in contrast to bacteriomes, viromes tended to be unique to individuals and less varied over time in response to changes in diet or other factors. By contrast, the bacterial population changed much more with diet and was also quite similar between twins.“There are clear instances of phages ‘helping'' pathogenic bacteria to attack humans”…Given that there is a direct relationship between the bacteria of the gut and the immune system of the individual—which is not the case for phage viruses—these findings make a degree of sense. Furthermore, as noted by Jeffrey Gordon, co-author of the Washington University study, there is not a one-to-one relationship between bacteria and the phages they host: “It''s been shown in other environments that you can have several different viruses capable of infecting the same bacterial host, while the specificity of each virus is usually quite narrow, typically extending only to a few strains within a species-level phylotype,” he explained. “This leads to a greater genetic diversity in the virome. Furthermore, a viral genome is enriched for genes involved in genome replication and virion assembly. Thus, the functional composition of the virome and the microbiome are quite different.”The situation is different for non-phage viruses in the gut that have a direct relationship with the human or animal host. The main research interest here is the three-way relationship between the virus, the bacteriome and the host''s immune system. Research on this front has already led to a new understanding of the role played by the entire microbiome in immunity. Often, the microbiome provides protection against viruses, but in some cases it can encourage their propagation. This is relevant in the context of human immunodeficiency virus (HIV), for example, given that the virus infects immune cells such as helper T cells, macrophages and dendritic cells, the activity and production of which in turn are related to the microbiota. An important question is whether the course of HIV and its possible development into symptomatic acquired immunodeficiency syndrome (AIDS) might be encouraged by the microbiota, if it stimulates production of such immune cells. Whilst this has yet to be established for AIDS, there is evidence that it is the case in monkeys carrying the related simian immunodeficiency virus (SIV), which infects at least 45 species of African primates. Unlike HIV in humans, SIV is usually non-pathogenic, as many primates evolved to coexist with the virus; but it does cause AIDS in rhesus macaque monkeys.Another Washington University School of Medicine study, this time looking at the link between viruses, the bacteriome and host immunity, began with the insight that animals developing AIDS experience immune hyperactivation, including higher levels of inflammatory chemokines, cytokines and activated T cells. This observation suggested that excessive inflammation is an important factor in progression to AIDS, presumably because it increases the number of cells vulnerable to HIV and SIV infection. The US team investigated whether there were any corresponding changes in the virome, finding that whilst it remains unchanged in uninfected animals, including rhesus macaque monkeys that had not succumbed to AIDS, its diversity increases significantly in infected macaques with full-blown AIDS [4].Often, the microbiome provides protection against viruses, but in some cases it can encourage their propagationThe team is following up by probing the relationship between enteric viruses and AIDS and how the immune system is stimulated. The animals suffer from progressive damage to their intestinal walls, which could increase the absorption of viruses that in turn stimulate the immune system, promoting replication of SIV and possibly encouraging more opportunistic viral infection, thereby creating a vicious circle. “This is one of several possible scenarios that we are actively investigating,” commented Scott Handley, lead author of the study. “What is uncertain is what is causing the damage to the intestinal wall. It could be the viruses which expand in the enteric virome during SIV infection, or some other factor, which could be immune-mediated or some other microbe or microbial product in the enteric microbiome.”Handley''s team has identified some of the viruses involved, including both common ones and some previously undiscovered. “Many of the viruses we identified have been associated with [gastrointestinal] disease in one form or another,” Handley said. “It is true that many of the viruses we identified are common; however, we identified at least 32 novel subtypes of these viruses never seen before.” He added that it remains to be established whether SIV infection encourages opportunistic infection by these viruses or not: “We don''t really have a good handle on what viruses would be considered ‘commensal'' viruses in any animal, including research primates. So whether they are already there when infection with SIV occurs, or are just more susceptible to opportunistic infection, is unclear.”Handley further commented that this work could lead to new therapeutic targets for treating HIV infection and preventing AIDS, and he is investigating whether the same expansion of the enteric virome occurs in humans infected with HIV. “In addition, we are interested to see if vaccination can reverse the enteric virome expansion,” he explained. “We would also like to better understand if the viruses we have identified were already circulating in these primates, or are succumbing to opportunistic infections.”Handley argued that his team''s work and that of others provides a compelling case for devoting more resources to studying the role of viruses in the gut, which would require further advances in laboratory and analytical techniques. “One challenge with studying the viral members of a microbiome is that there are no well-defined marker sequences,” he said. “Therefore, we are largely dependent on random shotgun sequencing approaches, which are less efficient and more expensive. Not only do you have to gather much more data, the computational analysis required is much more complex than the well-established techniques developed for studying the bacterial microbiome. While we know there is a great deal of interest in studying the virome, current techniques and technology tend to limit the number of labs that can participate in these efforts. We are very interested in developing new tools and techniques to help alleviate this issue.”An important question is whether the course of HIV […] might be encouraged by the microbiota, if it stimulates production of such immune cellsWork undertaken so far has already shown that probiotic or prebiotic treatments that provide or encourage beneficial gut bacteria can benefit patients infected with HIV. Improvements in intestinal health could reduce the leakage of all antigens, including viral ones, through the intestinal wall. More generally, a better understanding of how phages, viruses and bacteria in the gut interact could lead to new therapies that manipulate the microbiome to restore intestinal health in sufferers of a variety of conditions, including those involving chronic inflammation.     

DNA tumor viruses -- the spies who lyse us

Identifying the molecular lesions that are 'mission critical' for tumorigenesis and maintenance is one of the burning questions in contemporary cancer biology. In addition, therapeutic strategies that trigger the lytic and selective death of tumor cells are the unfulfilled promise of cancer research. Fortunately, viruses can provide not only the necessary 'intelligence' to identify the critical players in the cancer cell program but also have great potential as lytic agents for tumor therapy. Recent studies with DNA viruses have contributed to our understanding of critical tumor targets (such as EGFR, PP2A, Rb and p53) and have an impact on the development of novel therapies, including oncolytic viral agents, for the treatment of cancer.     

Differential Effects of Lovastatin on Cisplatin Responses in Normal Human Mesothelial Cells versus Cancer Cells: Implication for Therapy

The cancer killing efficacy of standard chemotherapeutic agents such as cisplatin (CDDP) is limited by their side effects to normal tissues. Therefore, research efforts optimizing the safety and efficacy of those agents are clinically relevant. We did screen for agents that specifically protect normal human mesothelial cells against CDDP without reducing the cancer cell killing efficacy. Lovastatin was identified from the screen. Lovastatin at a pharmacologically relevant concentration strongly arrested the proliferation of normal cells, whereas cancer cells were less affected. CDDP-induced DNA damage response was not activated and normal cells showed enhanced tolerance to CDDP when normal cells were treated with the combination of CDDP and lovastatin. We demonstrate that interfering with protein geranylgeranylation is involved in the lovastatin-mediated CDDP protective effect in normal cells. In contrast to normal cells, in cancer cells lovastatin did not change the CDDP-induced response, and cancer cells were not protected by lovastatin. Furthermore, lovastatin at the pharmacological relevant concentration per se induced DNA damage, oxidative stress and autophagy in cancer cells but not in normal mesothelial cells. Therefore, our data suggest that lovastatin has a potential to improve the therapeutic index of cisplatin-based therapy.     

A translational study “case report” on the small molecule “energy blocker” 3-bromopyruvate (3BP) as a potent anticancer agent: from bench side to bedside

The small alkylating molecule, 3-bromopyruvate (3BP), is a potent and specific anticancer agent. 3BP is different in its action from most currently available chemo-drugs. Thus, 3BP targets cancer cells’ energy metabolism, both its high glycolysis (“Warburg Effect”) and mitochondrial oxidative phosphorylation. This inhibits/ blocks total energy production leading to a depletion of energy reserves. Moreover, 3BP as an “Energy Blocker”, is very rapid in killing such cells. This is in sharp contrast to most commonly used anticancer agents that usually take longer to show a noticeable effect. In addition, 3BP at its effective concentrations that kill cancer cells has little or no effect on normal cells. Therefore, 3BP can be considered a member, perhaps one of the first, of a new class of anticancer agents. Following 3BP’s discovery as a novel anticancer agent in vitro in the Year 2000 (Published in Ko et al. Can Lett 173:83–91, 2001), and also as a highly effective and rapid anticancer agent in vivo shortly thereafter (Ko et al. Biochem Biophys Res Commun 324:269–275, 2004), its efficacy as a potent anticancer agent in humans was demonstrated. Here, based on translational research, we report results of a case study in a young adult cancer patient with fibrolamellar hepatocellular carcinoma. Thus, a bench side discovery in the Department of Biological Chemistry at Johns Hopkins University, School of Medicine was taken effectively to bedside treatment at Johann Wolfgang Goethe University Frankfurt/Main Hospital, Germany. The results obtained hold promise for 3BP as a future cancer therapeutic without apparent cyto-toxicity when formulated properly.     

Effects of antibiotic resistance alleles on bacterial evolutionary responses to viral parasites

Antibiotic resistance has wide-ranging effects on bacterial phenotypes and evolution. However, the influence of antibiotic resistance on bacterial responses to parasitic viruses remains unclear, despite the ubiquity of such viruses in nature and current interest in therapeutic applications. We experimentally investigated this by exposing various Escherichia coli genotypes, including eight antibiotic-resistant genotypes and a mutator, to different viruses (lytic bacteriophages). Across 960 populations, we measured changes in population density and sensitivity to viruses, and tested whether variation among bacterial genotypes was explained by their relative growth in the absence of parasites, or mutation rate towards phage resistance measured by fluctuation tests for each phage. We found that antibiotic resistance had relatively weak effects on adaptation to phages, although some antibiotic-resistance alleles impeded the evolution of resistance to phages via growth costs. By contrast, a mutator allele, often found in antibiotic-resistant lineages in pathogenic populations, had a relatively large positive effect on phage-resistance evolution and population density under parasitism. This suggests costs of antibiotic resistance may modify the outcome of phage therapy against pathogenic populations previously exposed to antibiotics, but the effects of any co-occurring mutator alleles are likely to be stronger.     

“Targeting the absence” and therapeutic engineering for cancer therapy

The Gotham Prize was awarded to Alex Varshavsky for “Targeting the absence”, a strategy employing negative targets of cancer therapy. This is a brilliant example of therapeutic engineering: designing a sequence of events that leads to the selective killing of one type of cell, while sparing all others. A complex molecular device (Varshavsky’s Demon) examines DNA, recognizes the present target in normal cells and kills cancer cells. The strategy is limited by the delivery (transfection or infection) of DNA-based devices into each cell of our body. How can we overcome this limitation? Can therapeutic engineering be applied to small drugs? Can each small molecule reach a cell separately and, once in a cell, exert orchestrated action governed by cellular context? Here I describe how a combination of small drugs can acquire a demonic power to check, choose and selectively kill. The cytotoxicity is restricted to cells lacking (or having) one of the targets. For example, in the presence of a normal target, one drug can cancel the cytotoxic action of another drug. And by increasing a number of targets, we can increase the precision and power of such ‘restrictive’ combinations. Here I discuss restrictive combinations of currently available drugs that could be tested in clinical trials. Could then these combinations cure cancer today? And what does ‘cure’ really mean? This article suggests the answer.     

Biofilms in vitro and in vivo: do singular mechanisms imply cross-resistance?

Microbial biofilm has become inexorably linked with man's failure to control them by antibiotic and biocide regimes that are effective against suspended bacteria. This failure relates to a localized concentration of biofilm bacteria, and their extracellular products (exopolymers and extracellular enzymes), that moderates the access of the treatment agent and starves the more deeply placed cells. Biofilms, therefore, typically present gradients of physiology and concentration for the imposed treatment agent, which enables the less susceptible clones to survive. Such clones might include efflux mutants in addition to genotypes with modifications in single gene products. Clonal expansion following subeffective treatment would, in the case of many antibiotics, lead to the emergence of a resistant population. This tends not to occur for biocidal treatments where the active agent exhibits multiple pharmacological activity towards a number of specific cellular targets. Whilst resistance development towards biocidal agents is highly unlikely, subeffective exposure will lead to the selection of less susceptible clones, modified either in efflux or in their most susceptible target. The latter might also confer resistance to antibiotics where the target is shared. Thus, recent reports have demonstrated that sublethal concentrations of the antibacterial and antifungal agent triclosan can select for resistant mutants in Escherichia coli and that this agent specifically targets the enzyme enoyl reductase that is involved in lipid biosynthesis. Triclosan may, therefore, select for mutants in a target that is shared with the anti-E. coli diazaborine compounds and the antituberculosis drug isoniazid. Although triclosan may be a uniquely specific biocide, sublethal concentrations of less specific antimicrobial agents may also select for mutations within their most sensitive targets, some of which might be common to therapeutic agents. Sublethal treatment with chemical antimicrobial agents has also been demonstrated to induce the expression of multidrug efflux pumps and efflux mutants. Whilst efflux does not confer protection against use concentrations of biocidal products it is sufficient to confer protection against therapeutic doses of many antibiotics. It has, therefore, been widely speculated that biocide misuse may have an insidious effect, contributing to the evolution and persistence of drug resistance within microbial communities. Whilst such notions are supported by laboratory studies that utilize pure cultures, recent evidence has strongly refuted such linkage within the general environment where complex, multispecies biofilms predominate and where biocidal products are routinely deployed. In such situations the competition, for nutrients and space, between community members of disparate sensitivities far outweighs any potential benefits bestowed by the changes in an individual's antimicrobial susceptibility.     

Restriction-modification systems and bacteriophage invasion: Who wins?

The success of a phage that infects a bacterial cell possessing a restriction-modification (R-M) system depends on the activities of the host methyltransferase and restriction endonuclease, and the number of susceptible sites in the phage genome. However, there is no model describing this dependency and linking it to observable parameters such as the fraction of surviving cells under excess phage, or probability of plating at low amount of phages. We model the phage infection of a cell with a R-M system as a pure birth process with a killing state. We calculate the transitional probabilities and the stationary distribution for this process. We generalize the model developed for a single cell to the case of multiple identical cells invaded by a Poisson-distributed number of phages. The R-M enzyme activities are assumed to be constant, time-dependent, or random. The obtained results are used to estimate the ratio of the methyltransferase and endonuclease activities from the observed fraction of surviving cells.     

Characterization of Pseudomonas aeruginosa phage KPP21 belonging to family Podoviridae genus N4‐like viruses isolated in Japan

Bacteriophages (phages) belonging to the family Podoviridae genus N4‐like viruses have been used as therapeutic agent in phage therapy against Pseudomonas aeruginosa infections. P. aeruginosa phage KPP21 was isolated in Japan, and phylogenetically investigated the phages belonging to this viral genus. Morphological and genetic analyses confirmed that phage KPP21 belongs to the family Podoviridae genus N4‐like viruses. Moreover, phylogenetic analyses based on putative DNA polymerase and major virion protein showed that P. aeruginosa phages belonging to the genus N4‐like viruses are separated into two lineages and that phage KPP21 is in the same clade as phage LUZ7.     

Target cell recognition structures in LDCC and ODCC

Cytotoxic T lymphocyte effector cells specific for a defined class I antigen can kill target cells displaying a wide range of different class I proteins in the presence of certain lectins and oxidizing agents. However, optimal lysis of the target cell (TC) still requires interaction of the CTL with the TC class I proteins. This raises the question of how the lectin or oxidizing agent alters the system in such a way that an "inappropriate" CTL-TC interaction takes place, in a class I-dependent manner. In this study we show that if papain-sensitive molecules are cleared from the TC surface and are allowed to regenerate in the presence of tunicamycin, the cells still serve as targets in direct, class I antigen-specific CTL killing, but not in LDCC or ODCC. Target cells treated in this way display N-linked carbohydrate-less class I proteins, and presumably other N-linked carbohydrate-less, papain-sensitive molecules as well. We present data showing that both types of molecules are important in nonspecific lytic reactions.     

An integrated agent-mathematical model of the effect of intercellular signalling via the epidermal growth factor receptor on cell proliferation

We have previously developed Epitheliome, a software agent representation of the growth and repair characteristics of epithelial cell populations, where cell behaviour is governed by a number of simple rules. In this paper, we describe how this model has been extended to incorporate an example of a molecular 'mechanism' behind a rule-in this case, how signalling by both endogenous and exogenous ligands of the epidermal growth factor receptor (EGFR) can impact on the proliferation of cell agents. We have developed a mathematical model representing release of endogenous ligand by cells, three-dimensional diffusion of the secreted molecules through a volume of cell culture medium, ligand-receptor binding, and bound receptor internalization and trafficking. Information relating to quantities of molecular species associated with each cell agent is frequently exchanged between the agent and signalling models, and the ratio of bound to free receptors determines cell cycle progression and hence the proliferative behaviour of the cell agents. We have applied this integrated model to examine the effect of plating density on tissue growth via autocrine/paracrine signalling. This predicts that cell growth is dependent on the concentration of exogenous ligand, but where this is limited, then growth becomes dependent on cell density and the availability of endogenous ligand. We have further modified the calcium concentration of the medium to modulate the formation of intercellular bonds between cells and shown that the increased propensity for cells to form colonies in physiological calcium does not result in significantly different patterns of receptor occupancy. In conclusion, our approach demonstrates that by combining agent-based and mathematical modelling paradigms, it is possible to probe the complex feedback relationship between the behaviour of individual cells and their interaction with one another and their environment.     

Effects of busulfan on murine spermatogenesis: cytotoxicity, sterility, sperm abnormalities, and dominant lethal mutations

The alkylating agent busulfan (Myleran) adversely affects spermatogenesis in mammals. We treated male mice with single doses of busulfan in order to quantitate its cytotoxic action on spermatogonial cells for comparison with effects of other chemotherapeutic agents, to determine its long-term effects on fertility, and to assess its possible mutagenic action. Both stem cell and differentiating spermatogonia were killed and, at doses above 13 mg/kg, stem cell killing was more complete than that of differentiating spermatogonia. Azoospermia at 56 days after treatment, which is a result of stem cell killing, was achieved at doses of over 30 mg/kg; this dose is below the LD50 for animal survival, which was over 40 mg/kg. Busulfan is the only antineoplastic agent studied thus far that produces such extensive damage to stem, as opposed to differentiating, spermatogonia. The duration of sterility following busulfan treatment depended on the level of stem cell killing and varied according to quantitative predictions based on stem cell killing by other cytotoxic agents. The return of fertility after a sterile period did not occur unless testicular sperm count reached 15% of control levels. Dominant lethal mutations, measured for assessment of possible genetic damage, were not increased, suggesting that stem cells surviving treatment did not propagate a significant number of chromosomal aberrations. Sperm head abnormalities remained significantly increased at 44 weeks after busulfan treatment, however, the genetic implications of this observation are not clear. Thus, we conclude that single doses of busulfan can permanently sterilize mice at nonlethal doses and cause long-term morphological damage to sperm produced by surviving stem spermatogonia.     

Rad51 and BRCA2--New molecular targets for sensitizing glioma cells to alkylating anticancer drugs

First line chemotherapeutics for brain tumors (malignant gliomas) are alkylating agents such as temozolomide and nimustine. Despite growing knowledge of how these agents work, patients suffering from this malignancy still face a dismal prognosis. Alkylating agents target DNA, forming the killing lesion O(6)-alkylguanine, which is converted into DNA double-strand breaks (DSBs) that trigger apoptosis. Here we assessed whether inhibiting repair of DSBs by homologous recombination (HR) or non-homologous end joining (NHEJ) is a reasonable strategy for sensitizing glioma cells to alkylating agents. For down-regulation of HR in glioma cells, we used an interference RNA (iRNA) approach targeting Rad51 and BRCA2, and for NHEJ we employed the DNA-PK inhibitor NU7026. We also assessed whether inhibition of poly(ADP)ribosyltransferase (PARP) by olaparib would enhance the killing effect. The data show that knockdown of Rad51 or BRCA2 greatly sensitizes cells to DSBs and the induction of cell death following temozolomide and nimustine (ACNU). It did not sensitize to ionizing radiation (IR). The expression of O(6)-methylguanine-DNA methyltransferase (MGMT) abolished all these effects, indicating that O(6)-alkylguanine induced by these drugs is the primary lesion responsible for the formation of DSBs and increased sensitivity of glioma cells following knockdown of Rad51 and BRCA2. Inhibition of DNA-PK only slightly sensitized to temozolomide whereas a significant effect was observed with IR. A triple strategy including siRNA and the PARP inhibitor olaparib further improved the killing effect of temozolomide. The data provides evidence that down-regulation of Rad51 or BRCA2 is a reasonable strategy for sensitizing glioma cells to killing by O(6)-alkylating anti-cancer drugs. The data also provide proof of principle that a triple strategy involving down-regulation of HR, PARP inhibition and MGMT depletion may greatly enhance the therapeutic effect of temozolomide.     

The return of the phage

Phages have been used to treat infectious diseases since their discovery nearly a century ago. Modern sequencing and genetic engineering technologies now enable researchers to vastly expand the use of phages as general drug delivery vehicles....it is only in the past five years that the regulatory guidelines for the approval of phage products—both in therapy and food safety—have been createdOver the past decade, bacteriophages have occasionally stirred public and media interest because of their potential as biological weapons against bacterial infections. Such reports have tended to come from Russian or Georgian laboratories, whereas Western research institutes and companies have usually found that phages do not live up to their promise. More than a decade later, however, the view of bacteriophages is set to change. Spurred on by advances in sequencing and other molecular techniques, research into phages has yielded its first applications. Not only are phages proving effective as therapeutic agents, but they are also playing a role in food safety and as delivery vehicles for drugs against a wide range of diseases.Interest in phages as therapeutic agents emerged almost immediately after their discovery nearly a century ago (Twort, 1915; d''Hérelle, 1917). This interest evaporated quickly in the West after the discovery of penicillin, but phage research was kept alive in the old Soviet Union and continued after its collapse in the 1990s. Ongoing studies there, although not always conforming to the most rigorous standards, provided the only evidence of the therapeutic potential of phages.Eventually, especially in the light of the increasing threat from drug-resistant bacteria, Western researchers turned to exploring phages again. However, it is only in the past five years that the regulatory guidelines for the approval of phage products—in both therapy and food safety—have been created. Previously, the US Food and Drug Administration (FDA) had lacked the appropriate regulatory measures; it took them four years to approve the first phage product for use in food safety in 2006. ListShield^TM is a cocktail of several phages that target Listeria monocytogenes, contaminants in meat and poultry products. Approvals for other food safety products have followed with greater speed (Sulakvelidze, 2011). Moreover, in 2008, the FDA approved the first phase 1 clinical trial of phages. This again involved a cocktail of eight phages to target various bacteria including Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, in venous leg ulcers. This trial eventually established the safety of the phage preparation and cleared the way for more phage therapy trials (www.clinicaltrials.gov).The recent acceptance in the West of phages as anti-pathogenic agents was preceded by their use for diagnostic purposes to identify bacteria...The recent acceptance in the West of phages as anti-pathogenic agents was preceded by their use for diagnostic purposes to identify bacteria, according to Martin Loessner from the Institute of Food, Nutrition and Health in Zürich, Switzerland. “It then became possible to [...] harness the specificity of phage for applications such as recognition of the host cell, and also for reporter phage, which is a genetically modified phage with a gene so [you] can easily see the phage''s impact on the target cell,” he explained. “Later on we figured why not go and revisit the idea of using phages against pathogens.”This approach turned out to be highly successful against key food pathogens, Loessner said, because of the way phages work: “[T]he phage has been very finely tuned through zillions of generations in the evolutionary arms race, and is highly specific.” This specificity is important for targeting the few bacteria that cause food poisoning while sparing the bacteria in fermented food—such as soft cheeses—that are harmless and contribute flavour. “The phage is also immune to development of resistance by the host bacteria, because if not it would have become extinct a long time ago,” Loessner said.It is bacterial toxins that cause food poisoning rather than bacteria themselves, so phages are used as a preventive measure to stop the growth of bacteria such as Listeria in the first place. As such, it is important to bombard food products with a large number of phages to ensure that virtually all target bacteria are eradicated. “I always have this magic number of 10⁸, or 100 million per gram of food,” Loessner said. “In 1 g of food there are often only 500 target bacteria, so there is not enough to amplify the phage and you need really high numbers to kill the bacteria in one round of infection.” He added that, in his view, phages would soon become the main treatment for preventing bacterial contamination. “Phage in the near future will be the number one [treatment against] Listeria and Salmonella. It''s becoming number one already, especially in the US.”In Europe, the use of phages in food safety therapy is being held back by the requirement that foods treated with them are labelled as containing viruses, which means they are likely to meet consumer resistance, as happened with foods containing or made from genetically modified organisms. Loessner commented that education is required to raise awareness that the properly controlled use of phages involves minimal risk and could greatly enhance food safety. However, he also emphasized that the use of phages should represent an extra level of protection, not replace existing quality control measures....because phage lysins are often specific to a single bacterial genus, they would allow the specific targeting of pathogenic bacteriaThe ability of phages to target specific bacteria while leaving others alone also has great potential for treating bacterial infections, particularly in the light of increasing antibiotic resistance. Such treatments would not necessarily involve the phage themselves, but rather the use of their lysins—the enzymes that weaken the bacterial cell wall to allow newly formed viruses to exit the host cell. Lysins can be administered as antibiotics, at least for gram-positive bacteria that lack a separate outer membrane around the cell wall. Moreover, because phage lysins are often specific to a single bacterial genus, they would allow the specific targeting of pathogenic bacteria. “The fact that phage lysins leave the commensal microflora undisturbed is particularly significant,” commented Olivia McAuliffe, Senior Research Officer at the Teagasc Food Research Centre in Cork, Ireland. “Most of the antibiotics used clinically have broad-activity spectra and treatment with these antibiotics can have devastating effects on the normal flora, in particular for those taking long-term antibiotic courses.”Phages also have another great advantage over most conventional antibiotics in being potent against both dividing and non-dividing cells. “Because most antibiotics target pathways such as protein synthesis, DNA replication, and cell wall biosynthesis, they can only act when the cells are actively growing,” McAuliffe added. “Because lysins are enzymes, they will chew away the peptidoglycan in both viable and non-viable cells, dividing and non-dividing cells. This would be particularly important in the case of slow-growing organisms that cause infection, an example being Mycobacterium species.”This specificity of phages and their lysins is particularly important for treating chronic conditions resulting from persistent bacterial infection, particularly in the respiratory system or digestive tract. Broad-spectrum antibiotics also attack harmless and beneficial commensal bacteria, and can even worsen the condition by encouraging the growth of resistant bacteria. This is the case with Clostridium difficile, a cause of secondary infections and a major nosocomial (hospital-acquired) antibiotic-resistant pathogen, according to McAuliffe. It is a Gram-positive, rod-shaped, spore-forming bacterium that is the most serious and common cause of diarrhoea and other intestinal disease when competing bacteria in the gut flora have been wiped out by antibiotics. The bacterium and its spores, which form in aerobic conditions outside the body, are widespread in the environment and are present in the guts of 3% of healthy individuals and 66% of infants, according to the UK''s Health Protection Agency. Clostridium spreads readily on the hands of healthcare staff and visitors in hospitals. The ability of the bacteria to form spores resistant to heat, drying and disinfectants, which then adhere to surfaces, enables them to persist in the hospital environment.Because Clostridium is resistant to most conventional antibiotics, it has for some years usually been treated with metronidazole, which exploits the fact that Clostridium is anaerobic during infection. Metronidazole has proven particularly appealing as it has relatively little impact on human cells or commensal aerobic bacteria in the gut as it does not work in the presence of oxygen. But metronidazole does not always work, and physicians have therefore been using vancomycin, a stronger but more toxic antibiotic, as a last resort. Moreover, even in cases where antibiotics seem to eliminate Clostridium and cure the associated diarrhoea, infection recurs in as many as 20% of hospital patients (Kelly & LaMont, 2008). About one-fifth of these 20%, or 4% of the total number of patients succumbing to Clostridium, end up with a long-term infection that at present is difficult to eradicate.This is where phages step in, because they are well tolerated by patients and their specificity means that they will not target other gut bacteria. Clostridium phages have already been demonstrated to work selectively and there is the possibility of extracting lysins against Clostridium from the phage itself; an avenue being pursued by Aidan Coffey''s group at the Department of Biological Sciences at the Cork Institute of Technology in Bishoptown, Ireland.There is also growing interest in using phages to tackle various other infections that are resistant to existing drugs—for example, in wounds that fail to heal, which are a major risk for diabetics. The application of phages in such cases is not new—before penicillin it was often the only option—but the difference now is that modern molecular techniques for isolating bacterial strains from biopsies and matching them to phages greatly increases efficiency. One clinical trial, organized by the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences, is currently recruiting patients to evaluate the use of phage preparations against a range of drug-resistant bacteria, including MRSA (methicillin-resistant Staphylococcus aureus), Enterococcus, Escherichia, Citrobacter, Enterobacter, Klebsiella, Shigella and Salmonella. The intention is to isolate bacterial strains from each patient and to identify matching phages from the Institute''s bacteriophage collection in Wrocław.Although the potential of phages or their lysins to combat bacterial pathogens, whether in food or those causing infectious diseases, has long been recognized, more recent work has identified new applications as delivery vehicles for vaccines or cytotoxic drugs to treat cancer. These applications do not exploit the phage''s natural targeting of bacteria, but make use of their ability to carry surface ligands that attract them to specific host cells.Even though phages do not attack human cells, they elicit an immune response and can be used as vectors to carry an engineered antigen on their surface to vaccinate against viral or bacterial disease. This approach has been tested in rabbits with a DNA vaccine against hepatitis B (Clark et al, 2011). The study compared the phage DNA vaccine with Engerix B—a commercially available vaccine based on a homologous recombinant protein—and found that the phage vaccine produced a significantly higher antibody response more quickly, as well as being potentially cheaper to produce and stable at a wider range of temperatures. This hepatitis B vaccine is now being developed by the UK biotech firm BigDNA in Edinburgh, Scotland, which has been granted a European patent, pending future clinical trials in humans.Modified phages could also serve as nanoparticles to deliver cytotoxic drugs straight to tumour cells, bypassing healthy cellsModified phages could also serve as nanoparticles to deliver cytotoxic drugs straight to tumour cells, bypassing healthy cells. Phages are a promising candidate vehicle because they can be readily engineered both to display appropriate ligands for targeting tumour cells specifically, and to carry a cytotoxic payload that is only released inside the target. One Israeli group has developed a technology for manufacturing phage nanoparticles that in principle can be used to target drugs to either tumour cells or pathogens (Bar et al, 2008). The group chose one particular phage family, known as filamentous phages, because of their small size and the relative ease of engineering them. Filamentous phages comprise just 10 genes with a sheath of several thousand identical α-helical coat proteins in a helical array assembled around a single-stranded circular DNA molecule. The Israeli scientists combine genetic modification and chemical engineering to create a phage that is able to attach to its target cell and release cytotoxic molecules. “Genetic engineering makes it possible to convert the phage to a targeted particle by displaying a target-specifying molecule on the phage coat,” explained Itai Benhar from Tel-Aviv University, the lead author of the paper. “Genetic engineering also makes it possible to design a drug-release mechanism. Finally chemical engineering makes it possible to load the particle with a large payload of cargo.”The group has used the same approach to target two bacteria species, Staphylococcus aureus and Escherichia coli, with the antibiotic chloramphenicol, which was first developed in 1949 but has raised concerns over its toxicity. According to the Israeli group, the phage nanoparticle loaded with the drug was 20,000 times more potent against both bacteria than the drug administered on its own. Just as importantly, the phage particles do not affect other cells. The overall advantage of the phage-based delivery approach is that it can deliver highly effective and toxic drugs in a safe way. The other point is that this and other methods in which phages are engineered to reach specific targets have nothing directly to do with the natural ability of phage viruses to attack bacteria. “The phage''s natural ability to infect bacteria is totally irrelevant to their application for targeting non-bacterial cells,” said Benhar. “In fact, they are not relevant for targeting bacteria either in this case, since the chemical modification we subject the phages to renders them non-infective.”However, the phage nanoparticles retain their immunogenic effect, which is a problem if the objective is merely to deliver a drug to the target while minimizing all other impacts. “Phages are immunogenic, and although we found a way to reduce their immunogenicity we did not totally eliminate it,” Benhar said. The other challenge is that, as the particles carry the payload drug on their surface, the physical and chemical properties change every time a new drug is loaded. Although the payload itself is inert until it reaches the target, the varying characteristics could alter the host response and therefore affect regulatory approval for each new phage construct, as safety would have to be demonstarted in each case.The use of phages is no longer confined to directly attacking infectious bacteria, but has vastly expanded in terms of methods, applications and the diseases that can be tackledNevertheless, this approach holds great promise as a novel way of delivering not just new drugs but also existing ones that are effective but too toxic for healthy cells. This is exactly the most exciting aspect of recent therapeutic phage research. The use of phages is no longer confined to directly attacking infectious bacteria, but has vastly expanded in terms of methods, applications and the diseases that can be tackled.