Vaccination against shigellosis: is it the path that is difficult or is it the difficult that is the path?

Following several decades of research, there is not yet a convincing vaccine against shigellosis. It is still difficult, in spite of the breadth of strategies (i.e. live attenuated oral, killed oral, subunit parenteral) to select an optimal option. Two approaches are clearly emerging: (i) live attenuated deletion mutants based on rational selection of genes that are key in the pathogenic process, and (ii) conjugated detoxified polysaccharide parenteral vaccines, or more recently conjugated synthetic carbohydrates. Some of these approaches have already undergone phase I and II clinical trials with promising results, but important issues have also emerged, particularly the discrepancy between colonization and immunogenic potential of live attenuated vaccine candidates depending upon the population concerned (i.e. non endemic vs. endemic areas). Efforts are needed to definitely establish the proof of concept of these approaches, and thus the need for clinical trials which should also soon explore the possibility to associate different serotypes, in response to serotype specific protection against shigellosis. More basic research is also required to improve what we can still consider as first-generation vaccines, and to explore possible new paradigms including the search for cross-protective antigens.     

Aging: is oxidative stress a marker or is it causal?

Rapid developments in free radical biology and molecular technology have permitted exploration of the free radical theory of aging. Oxidative stress has also been implicated in the pathogenesis of a number of diseases. Studies have found evidence of oxidative damage to macromolecules (DNA, lipids, protein), and data in transgenic Drosophila melanogaster support the hypothesis that oxidative injury might directly cause the aging process. Additional links between oxidative stress and aging focus on mitochondria, leading to development of the mitochondrial theory of aging. However, despite the number of studies describing the association of markers of oxidative damage with advancing age, few, if any definitively link oxidative injury to altered energy production or cellular function. Although a causal role for oxidative stress in the aging process has not been clearly established, this does not preclude attempts to reduce oxidative injury as a means to reduce morbidity and perhaps increase the healthy, useful life span of an individual. This review highlights studies demonstrating enhanced oxidative stress with advancing age and stresses the importance of the balance between oxidants as mediators of disease and important components of signal transduction pathways.     

Metastatic melanoma, or is it?

A case of pigmented axillary nodes in the presence of malignant melanoma of the upper extremity points out the caution necessary in making intraoperative decisions based on gross appearance at the time of surgery. Had our preoperative plans been abandoned in consideration of the "obvious pathology," the patient might not have done as well. Histologic confirmation should always precede any altering of preoperative plans despite gross findings.     

Neutrophils and immunity: is it innate or acquired?

The neutrophil has long been considered a phagocytic cell with a short life-span whose major role is to destroy intruders to the body. Toll receptors and anti-infectious factors such as defensin, perforin and granzymes are newly discovered mechanisms used by neutrophils for the first line of defense against invaders. Moreover, subpopulations of neutrophils share specific functions like the synthesis of certain cytokines and chemokines, as well as the expression of immunoreceptors like the T cell receptor. A primary consequence of inflammation on neutrophils is a delay in their spontaneous programmed cell death. Hence, this multifunctional cell is also a necessary actor of the acquired immune response. Neutrophils have the capacity to degrade and process antigens as well as efficiently present antigenic peptides to lymphocytes. Neutrophil interactions with immune cells, in particular dendritic cells, lead to the formation of IL-12 and TNF-alpha deviating the immune response towards a Th1 phenotype. Thus, the neutrophil exhibits a cellular plasticity that explains its capacity to transdifferentiate depending on the local requirements of the immune response. The neutrophil is probably the most underappreciated immune cell among hematopoietic leukocytes, and many neutrophil functions remain to be unraveled.     

Targeted cancer therapy: What if the driver is just a messenger?

“Shoot the driver” is the paradigm of targeted cancer therapy. However, resistance to targeted inhibitors of signaling pathways is a major problem. In part, the redundancy of signaling networks can bypass targeted inhibitors and thereby reduce their biological effect. In this case, the driver turns out to be one of several potential messengers and is easily replaced. Cocktails of multiple targeted inhibitors are an obvious solution. This is limited, however, by the lack of potent inhibitors and may also produce increased toxicity. Therefore, we explored the direct blockade of a key biological activity downstream from multiple converging oncogenic signals. Specifically, several oncogenic signaling pathways, including AKT, MAPK and PIM kinase signals, converge on the activation of cap-dependent translation. In cancer cells, aberrant activation of cap-dependent translation favors the increased expression of short-lived oncoproteins like c-MYC, MCL1, CYCLIND1 and the PIM kinases. Intriguingly, cancer cells are especially sensitive to even temporary reductions in these proteins. We will discuss our findings concerning translational inhibitor therapy in cancer.Key words: targeted therapy, cancer, lymphoma, translation, eIF4ETargeted cancer therapies are designed to block selected pathways or molecules that are required for tumor cell survival. The most successful examples are inhibitors of the BCR-ABL fusion protein that drive chronic myeloid leukemia (CML). By shutting down the activity of a single molecule driving the growth of CML cells, imatinib and its successors dasatinib and nilotinib can produce complete and sustained remissions as single agents (reviewed in ref. ¹). However, it has proven difficult to translate this success to other cancers. In metastatic melanoma, for example, the BRAF inhibitor vemurafenib produces high response rates in patients whose tumors bear the BRAF-V600E activating mutation (reviewed in ref. ²). Excitement generated by these results is well-deserved, as they have opened a new treatment paradigm in a disease with few available options and a grim prognosis. Unfortunately, however, resistance to the drug generally is seen within a few months. Median progression-free survival resulting from vemurafenib for treatment-naïve metastatic melanoma patients with V600E was about six months,³ compared with imatinib in CML, which kept 93% of patients progression-free at five years.⁴ The success of TKIs in CML is still the exception and not the rule in targeted cancer therapeutics.Resistance to targeted inhibitors is an emerging problem with multiple causes and potential solutions. While mechanisms of resistance to BRAF inhibition in melanoma remain to be elucidated, they are well-described for some targets in other cancers. Drugs against the epidermal growth factor receptor (EGFR) kinase, for example, are bypassed both by activation of downstream mediators, most prominently KRAS, and by signaling through parallel pathways like the MET oncogene (reviewed in ref. ⁵). Resistance mechanisms are varied and complex in some ways, but most boil down to the same idea: evolution has provided multiple routes to the crucial endpoints that allow sustained growth and proliferation of cancer cells. Cocktails of multiple inhibitors have been proposed to prevent or thwart resistance (reviewed in ref. ⁶), and this approach appears highly promising based on some recent preclinical studies. In prostate cancer, for example, combined blockade of androgen receptor signaling and the PI3K/AKT/mTOR pathway showed potent synergy in model systems.⁷ Unfortunately, similar approaches against many other cancers are currently limited by the availability of potent, selective inhibitors and a need to identity which combinations will be effective. Moreover, combined toxicities from simultaneous use of multiple inhibitors will pose limits on the number and intensity of drugs that can be used.An alternative and potentially complementary approach is to directly target the downstream biological processes that are activated by signaling pathways and that cancer cells rely on. Experiences with inhibitors of EGFR, BRAF, and other signaling molecules suggest that most tumors can reliably activate parallel or downstream messengers to thwart efficacy. So inhibiting a signaling intermediate (i.e., a messenger) allows resistance if the biological effects can be achieved via an alternate route. Ultimately, tumor cells don''t depend on the messenger, often a kinase, and, instead, require a downstream biological function. This opens the possibility that targeting the critical effect directly may be an effective cancer therapy and could overcome the problem of redundant messengers.Several inherent properties of signaling pathways are relevant to targeted therapies, their side effects and mechanisms of resistance.⁸ For example, signaling pathways frequently converge on key activities. As explained above, this redundancy can produce resistance to targeted agents. Often redundant pathways are induced via feedback mechanisms, which provide robust signals and can also bypass selective inhibitors. Signals also diverge, however, and seemingly parallel pathways therefore also produce pleiotropic and non-overlapping effects. It is possible not all activities are required by cancer cells. Hence, an upstream block may produce toxicities, in part, by blocking activities that are not strictly required by tumor cells as much as by normal tissues. Finally, unlike metabolic pathways, where a limiting substrate is passed down, signaling cascades amplify signals toward a key activity, and the initial signal or message is both energetically “cheap” and infinitely recurrent. Accordingly, it is conceivable that direct block of the downstream effects provide an alternative or complementary approach to targeting upstream signaling molecules.Multiple oncogenic signals, including the PI3K, MAPK/ERK and PIM kinase pathways converge on the activation of cap-dependent translation, the process by which most capped mRNAs are translated into proteins (reviewed in ref. ⁹). Signaling pathways control the availability of the cap-binding protein eIF4E that is the limiting component of the multimeric translation complex eIF4F, which also includes scaffolds (eIF4G) and RNA helicase activities (eIF4A).^10–12 The complex ultimately mediates loading of mRNAs onto ribosomes. Availability of the eIF4E factor is especially important for mRNAs with long and structured 5′ UTRs. These include, in particular, short-lived cell cycle regulators and oncoproteins. Hence, regulation of eIF4E via upstream signals provides an immediate level of expression control that directly controls levels of proteins, including c-MYC, cyclin D1, BCL2, MCL1 and PIM1.^13–17 Cancer cells require continuous expression of these proteins. For example, even brief loss of MYC expression produces widespread cell death in several cancers but only produces reversible cell cycle arrest in normal tissues.¹⁸ Hence, the increased requirement for the continuous translation of oncoproteins in cancer cells may provide a therapeutic window for inhibitors of capdependent translation.We recently investigated the therapeutic potential of directly blocking capdependent translation in non-Hodgkin lymphoma (NHL).^15,16,19 Rapalogs, inhibitors of mTORC1, the upstream activator of cap-dependent translation, have been extensively studied in NHL in clinical trials (reviewed in ref. ²⁰). However, their activity has, overall, been modest, and our results implicate mTORC1-independent activation of translation by PIM kinases as one mechanism of rapalog resistance in NHLs.¹⁶ The PIM family kinases are active upon expression and do not require activating modifications. They have been known for some time to be able to promote phosphorylation of 4E-BP1 in a manner resistant to rapamycin.^21,22 We now report expression of PIM1 and/or PIM2 in more than 60% of diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) and more than 75% of mantle cell and small lymphocytic lymphomas. Our study found PIM expression (either PIM1 or PIM2) was associated with worse time to event and overall survival in FL, while another recent report points to PIM2 as a driver of aggressive disease in activated B-cell type DLBCL.²³ In addition, two recent studies of chromosomal translocations mediated by activation-induced deaminase (AID) identified PIM1 as a frequent target.^24,25 In sum, expression of PIM kinases is common in NHLs, may be associated with a more aggressive clinical course and exemplifies how the redundancy of messaging molecules can bypass the clinical activity of selective signaling inhibitors.In experimental systems, we found that direct blockade of cap-dependent translation was highly effective against lymphomas with redundant PI3K/AKT and PIM signals.¹⁶ Briefly, using both a constitutively active mutant 4E-BP1 allele that blocks eIF4E activity and a small molecule inhibitor of the eIF4A helicase, silvestrol, we were able to completely restore rapalog sensitivity in lymphomas engineered to express PIM2 kinase activity. Mechanistically, we found that silvestrol dramatically reduced the translation of critical oncoproteins, including c-MYC, cyclin D1 and MCL1. Interestingly, silvestrol also blocked the translation of both PIM kinases themselves. Moreover, consistent with prior reports, silvestrol treatment at an effective dose was well-tolerated in animals, and we observed no frank toxicity.¹⁹ Hence, blocking cap-dependent translation disrupts upstream signaling molecules, the PIM kinases and also key oncoproteins commonly considered “undruggable” oncoproteins, including c-MYC.Silvestrol worked dramatically better than inhibition of the upstream kinases. Briefly, we tested the SuperGen Inc. PIM kinase inhibitors SGI-1776 and SGI-1773 side by side with silvestrol in a panel of these PIM-expressing human NHL cell lines. Notably, SGI-1776 is the only PIM inhibitor that has entered clinical trials, although these had to be discontinued due to cardiac toxicity of the compound (SuperGen press release, 2010). In any case, silvestrol showed in vitro potency at IC₅₀ of less than 10 nM in all cases, and the PIM kinase inhibitors were 100 to 1,000 times less active. These results highlight some problems associated with the “inhibitor cocktail” approach and indicate a complementary strategy that includes direct blockade of a key biological activity, in this instance, cap-dependent translation of oncoproteins.Silvestrol is not the only means to block cap-dependent translation, and others are reviewed in reference ⁹. These include antisense oligonucleotides against eIF4E and peptide inhibitors of eIF4F complex formation, though neither has entered clinical trials. Silvestrol is the most well-studied of several compounds that emerged from library screens for ability to disrupt the function of the eIF4F subunit eIF4A, an RNA helicase required for its ability to promote mRNA translation. A plant-derived flavagline, silvestrol has shown activity against a variety of tumor types and can be given to mice at high enough concentrations for antitumor activity without major toxicity. The drug shows activity as a single agent against human breast and prostate cancer cell lines in xenograft experiments in nude mice.²⁶ This produced mild transient impairment of hepatic synthetic function but no toxicities producing morbidity or mortality. In genetically defined murine tumor models, silvestrol showed potent synergy with chemotherapy when used against tumors bearing translational activation due to loss of Pten or overexpression of eIf4e.¹⁹ Originally isolated from Aglaia silvestris, silvestrol has a complex structure that has proved difficult to chemically synthesize in quantity. For this reason, the parent compound is not an ideal clinical drug candidate. Efforts are underway by Drs. Pelletier (McGill) and Porco (Boston University) to develop analogs with more efficient synthesis profiles and that retain its biochemical properties. In sum, cap-dependent translation is a promising drug target alternate to mTORC1 and upstream kinase inhibitors.     

Phylloclimate or the climate perceived by individual plant organs: what is it? How to model it? What for?

This review introduces the emergence of a new research topic, phylloclimate, located at the crossroads between ecophysiology and canopy microclimate research. Phylloclimate corresponds to the physical environment actually perceived by each individual aerial organ of a plant population, and is described by physical variables such as spectral irradiance, temperature, on-leaf water and features of around-organ air (wind speed, temperature, humidity, etc.). Knowing the actual climate in which plant organs grow may enable advances in the understanding of plant-environment interactions, as knowing surface temperature instead of air temperature enabled advances in the study of canopy development. Characterizing phylloclimate variables, using experimental work or modeling, raises many questions such as the choice of suitable space- and time-scale as well as the ability to individualize plant organs within a canopy. This is of particular importance when aiming to link phylloclimate and function-structure plant models. Finally, recent trends and challenging questions in phylloclimate research are discussed, as well as the possible applications of phylloclimate results.