Excessive Collateral Damage Research Articles

Molecular and temporal control of restimulation-induced cell death (RICD) in T lymphocytes

For effective adaptive immunity, T lymphocytes must rapidly expand and contract in an antigen-specific manner to effectively control invading pathogens and preserve immunological memory, without sustaining excessive collateral damage to host tissues. Starting from initial antigen encounter, carefully calibrated programmed cell death pathways are critical for maintaining homeostasis over distinct phases of the T cell response. Restimulation-induced cell death (RICD), a self-regulatory apoptosis pathway triggered by re-engagement of the T cell receptor (TCR), is particularly important for constraining effector T cell expansion to preclude overt immunopathology; indeed, genetic disorders affecting key molecules involved in RICD execution can manifest in excessive lymphoproliferation, malignancy, and autoimmunity. Herein we review our current knowledge of how RICD sensitivity is ultimately regulated over the course of an immune response, including recent revelations on molecules that tune RICD by enforcing resistance or promoting susceptibility in expanding versus mature effector T cells, respectively. Detailed dissection of the molecular and temporal control of RICD also illuminates novel therapeutic strategies for correcting abnormal T cell responses noted in various immune disorders by ultimately tuning RICD sensitivity.

PD-1 mediated regulation of CD8 +T cells in the murine submandibular gland

Abstract Immune checkpoints, including programmed cell death protein 1 (PD-1), restrain the immune response, maintain self-tolerance, and prevent excessive collateral damage. Immune checkpoint blockade has revolutionized cancer immunotherapy by unleashing exhausted immune cells to kill tumor cells, but can lead to immune related adverse events (IrAEs). Patients receiving PD-1 blockade can develop IrAEs including vitiligo, colitis, endocrinopathies, and sicca syndrome, an autoimmune disease characterized by the accumulation of immune cells in the salivary and lacrimal glands, leading to deterioration and dysfunction. Because PD-1 is highly expressed on CD8 +T cells in the naive submandibular gland (SMG), we hypothesized that loss of PD-1 is likely to disrupt the previously reported hyporesponsive microenvironment of the SMG, leaving patients particularly susceptible to IrAEs affecting this gland. Our data indicates that genetic loss of PD-1 results in increased CD8 +T cell number and frequency in the SMG of naïve C57BL/6 animals. To characterize this expanding population, we recently collaborated with the ImmGen T consortium to perform single-cell RNA-seq on SMG T cells of wildtype and PD-1 knockout mice. Preliminary analysis indicates that the expanding CD8 +T cells in the salivary gland of PD-1 deficient animals have a unique phenotype. These cells exhibit exhaustion characteristics including expression of transcription factors Nr4a2and Tox, and inhibitory receptors LAG-3 and TIGIT. Altogether, our data provide insights for IrAE prevention and treatments in patients receiving PD-1 blockade. This work was supported by NIH research Grants AI46709 (L.B.), AI-072073 (ImmGen T), and a Brown University Presidential Award (S.B.). This work was supported by NIH research Grants AI46709 (L.B.), AI-072073 (ImmGen T), and a Brown University Presidential Award (S.B.).

Controlling Laser Irradiation with Tissue Temperature Feedback Enhances Photothermal Applications: Ex-Vivo Evaluation on Bovine Liver

Achieving repeatable and successful results without causing excessive collateral damage is of paramount importance for photothermal laser applications. Predetermined laser parameters cannot ensure patient safety and treatment success due to variance between optical and thermal characteristics among subjects. Controlling laser irradiation with tissue temperature feedback is the current gold standard for various photothermal treatments (PTT) which are rate processes described by the Arrhenius temperature integral. This study establishes the validity of our low-cost design that makes tissue surface temperature control during photothermal laser applications more accessible in resource limited clinical environments. We demonstrated the practical performance and potential of our system with ex-vivo bovine liver irradiation using an ytterbium fiber laser (λ=1071 nm) with two independent variables: laser power (3.4 W, 6.8 W and 10.2 W) and target surface temperature (55 °C, 65 °C and 75 °C). Our system efficiently maintained tissue surface temperatures at target values in all laser power groups. In contrast, fixed-dose application groups displayed a high final temperature range and variation in the control experiment. Temperature–time responses of samples varied significantly, in agreement with a wide range of optical and thermal coefficients. Long exposure duration groups (lower power, higher target temperature) displayed more radical differences suggesting a dominance of optical and thermal characteristics over the response. The low-cost surface-temperature-controlled medical laser system we have developed is capable of ensuring the success and reproducibility of PTT modalities and patient safety.

The role of immune checkpoint PD-1 in the submandibular gland

Abstract Immune checkpoints play an important role in restraining the immune response, maintaining self-tolerance and preventing excessive collateral damage. Immune checkpoint blockade has revolutionized cancer immunotherapy but can lead to immune related adverse events (IrAEs). Patients receiving programmed cell death protein 1 (PD-1) blockade can develop IrAEs including vitiligo, colitis, endocrinopathies, and Sicca Syndrome, an autoimmune disease affecting the salivary and lacrimal glands. After immune cell activation, PD-1 is upregulated, and PD-1 signaling results in reduced proliferation, cytokine production, and cytotoxicity. PD-1 is expressed on infiltrating lymphocytes of the submandibular gland (SMG) including natural killer (NK) and T cells, and recognizes the ligand, PD-L1. Hyporesponsive immune cells in the SMG are thought to retain the integrity of this delicate tissue, while inadvertently contributing to viral latency. We therefore hypothesized that signaling through coinhibitory receptors, including PD-1, may impact these hyporesponsive effector cells. Our preliminary data indicate that genetic loss of PD-1 results in increased CD8+ T cell number and frequency in the SMG, with greater proliferation status. Given the interplay between self-tolerance and chronic viral control, we also examined the role of PD-1 during latent murine cytomegalovirus (MCMV) infection in the SMG. Interestingly, an increased infiltration of CD8+ T cells in the SMG was also observed during MCMV latency period. These studies of PD-1 on SMG immune cells should provide insights for prevention and treatments of IrAEs and chronic viral infections. This work was supported by the following NIH research Grants: R01 AI46709 (L.B.), R01 AI122217 (L.B.) and a Brown University Presidential Award (S.B.).

Restimulation-Induced Cell Death (RICD): Methods for Modeling, Investigating, and Quantifying RICD Sensitivity in Primary Human T Cells via Flow Cytometric Analysis.

When the body mounts an immune response against a foreign pathogen, the adaptive arm of the immune system relies upon clonal expansion of antigen-specific T cell populations to exercise acquired effector and cytotoxic functions to clear it. However, T cell expansion must be modulated to effectively combat the perceived threat without inducing excessive collateral damage to host tissues. Restimulation-induced cell death (RICD) is an apoptotic program triggered in activated T cells when an abundance of antigen and IL-2 are present, imposing a negative feedback mechanism that constrains the growing T cell population. This autoregulatory process can be detected via increases in caspase activation, Annexin V binding, and loss of mitochondrial membrane potential. However, simple changes in T cell viability through flow cytometric analysis can reliably measure RICD sensitivity in response to T-cell receptor (TCR) restimulation. This protocol describes the in vitro polyclonal activation, expansion, and restimulation of human primary T cells isolated from donor peripheral blood mononuclear cells (PBMC). This simple procedure allows for accurate quantification of RICD via flow cytometry. We also describe strategies for interrogating the role of specific proteins and pathways that may alter RICD sensitivity. This straightforward protocol provides a quick and dependable tool to track RICD sensitivity in culture over time while probing critical factors that control the magnitude and longevity of an adaptive immune response. Graphic abstract: In-vitro simulation of restimulation-induced cell death in activated human T cells.

Intratumoral Thermal Reading During Photo‐Thermal Therapy by Multifunctional Fluorescent Nanoparticles

The tremendous development of nanotechnology is bringing us closer to the dream of clinical application of nanoparticles in photothermal therapies of tumors. This requires the use of specific nanoparticles that must be highly biocompatible, efficient light‐to‐heat converters and fluorescent markers. Temperature reading by the heating nanoparticles during therapy appears of paramount importance to keep at a minimum the collateral damage that could arise from undesirable excessive heating. In this work, this thermally controlled therapy is possible by using Nd3+ ion‐doped LaF3 nanocrystals. Because of the particular optical features of Nd3+ ions at high doping concentrations, these nanoparticles are capable of in vivo photothermal heating, fluorescent tumor localization and intratumoral thermal sensing. The successful photothermal therapy experiments here presented highlight the importance of controlling therapy parameters based on intratumoral temperature measurements instead of on the traditionally used skin temperature measurements. In fact, significant differences between intratumoral and skin temperatures do exist and could lead to the appearance of excessive collateral damage. These results open a new avenue for the real application of nano­particle‐based photothermal therapy at clinical level.

Sustainability Assessment of Certified Tropical Forestry using Emergy Analysis

Sustainable forestry production systems, such as certified forestry, re- duced impact logging or sustainable forest management, have become an important part of tropical countries’ strategy to oversee their forest re- sources. Particularly, certification of forest products has become a widely adopted practice as a response to a growing international concern over the ecological and economic sustainability of harvesting natural tropical forests because of the benefits on reducing excessive and unnecessary construction of forest roads, reducing excessive use of heavy machinery, diminishing excessive collateral damage, and improving the inefficient milling processes and wasting of residues. To accomplish the aim of this study, which is to compare conventional and certified forestry we used a systems analysis tool that could integrate various benefits of certified forestry including, stand age, wood harvest, soil losses and economic gain or loss. We found that the affected area under uncertified logging practices is twice the area of certified forestry because conventional prac- tices require more roads, more logging decks and more skid trails. Also, the rate of soil erosion between uncertified logging (154 Mg/ha/yr) and certified logging (66 Mg/ha/yr) is different. Based on these data, we es- timated that the total emergy removed from certified forests was 40% less than the forest under conventional forestry. The fact that certification does a better job of preserving forest while removing wood was also demonstrated through the Environmental Loading Ratio (ELR). We also found that certification leaves forests with a 30% better chance to recover after logging because less wood is removed. But forestry using certifica- tion showed a lower Emergy Yield Ratio (EYR) because the lower yield and the extra cost of certification. Certification is also favored for the higher price for certified wood in international markets. Then, when Bo- livia exports uncertified wood, it obtains a positive benefit, but lower than using uncertified wood domestically (EYR = 20.4). In other words the worst thing the country could do is certify wood for their domestic market and sell uncertified wood abroad.

Targeting the hypoxia-adenosinergic signaling pathway to improve the adoptive immunotherapy of cancer

The recent approval by the FDA of cancer vaccines and drugs that blockade immunological negative regulators has further enhanced interest in promising approaches of the immunotherapy of cancer. However, the disappointingly short life extension has also underscored the need to better understand the mechanisms that prevent tumor rejection and survival even after the blockade of immunological negative regulators. Here, we describe the implications of the "metabolism-based" immunosuppressive mechanism, where the local tissue hypoxia-driven accumulation of extracellular adenosine triggers suppression via A2 adenosine receptors on the surface of activated immune cells. This molecular pathway is of critical importance in mechanisms of immunosuppression in inflamed and cancerous tissue microenvironments. The protection of tumors by tumor-generated extracellular adenosine and A2 adenosine receptors could be the misguided application of the normal tissue-protecting mechanism that limits excessive collateral damage to vital organs during the anti-pathogen immune response. The overview of the current state of the art regarding the immunosuppressive effects of extracellular adenosine is followed by a historical perspective of studies focused on the elucidation of the physiological negative regulators that protect tissues of vital organs from excessive collateral damage, but, as a trade-off, may also weaken the anti-pathogen effector functions and negate the attempts of anti-tumor immune cells to destroy cancerous cells.

The Principle of Ambituity and the Prohibition against Excessive Collateral Damage to the Environment during Armed Conflict

This article aims to clarify the legal basis of the protection of the environment during armed conflict in general, and of the prohibition against excessive collateral damage to the environment in particular. It is submitted that the legal basis for the conventional and customary rules which protect the (intrinsic value of the) environment during armed conflict cannot be deduced from the four fundamental principles of the law of armed conflict: the principles of military necessity, distinction, proportionality and humanity. Rather, the specific obligations relating to environmental protection in times of armed conflict flow from the fundamental principle of ambituity. Similar to the principle of humanity, the principle of ambituity, which qualifies as a general principle of law in the sense of Article 38(1)(c) ICJ Statute, provides for an absolute limitation to the necessities of war. As such the principle of ambituity may be used to interpret existing conventional or customary rules of international law during armed conflict, to supplement, or under exceptional circumstances to modify or set aside these rules. With regard to the prohibition against excessive collateral damage to the environment during armed conflict, it is submitted that this prohibition flows from a customary rule which emerged in the 1990s, rather than from Articles 51 and 52 of Additional Protocol I, and which complements Articles 35 and 55 AP I (i.e. for States Parties to AP I). This article argues that any military action which causes collateral damage to the environment must first be assessed under this relatively new customary prohibition; and subsequently, if no breach can be established and if applicable, by reference to Articles 35(3) and 55 AP I. In order to enhance the scope of this prohibition and provide better protection for the environment against collateral damage it is suggested that further investigations should be conducted into the consequences of warfare on the environment.

Endonasal procedures in endocanalicular dacryocystorhinostomy

Dear Editor, First of all, thank you for your critiques and valuable comments. Diode laser, as being one of the most portable and least expensive of the lasers available for endocanalicular dacryocystorhinostomy (ECL DCR), is the most commonly used laser for ECL DCR [1–3]. It can ablate bone and the soft tissues without causing excessive collateral damage [3]. We have been also performing diode laser ECL DCR, since the beginning of 2006 [4, 5]. Initially, our surgical procedure was simply the application of the laser energy until the probe was visualized by nasal endoscopy, and then the enlargement of the neo-ostium by additional laser applications [4]. During the follow-up of those initial cases, we recognized that synechia between the lateral surface of the middle turbinate (MT) and the medial surface of the lateral wall of the nose was a common finding in the failures [4]. We conducted a retrospective study comparing those two groups of patients who underwent diode laser ECL DCR with and without endonasal procedures [4]. There were three differences between the surgical procedures of two groups: (1) initial partial anterior inferior third middle turbinate resection (MTR) before laser application, (2) endonasal enlargement of the neo-ostium, after obtaining the initial fistula by laser application, and (3) antibioticsoaked cotton pledget packed in the sac at the end. The anatomical success rates increased from 71% to 93% by adding the endonasal procedures. The synechia between the MT and the lateral nasal wall was the most common finding in the failures encountered in cases without endonasal procedures. Routine MTR is a controversial procedure. Some favor MT preservation, while some practice MTR routinely [6]. However, none of the surgeons provided any scientific rationale for their approaches [6, 7]. Partial MTR was reported to be a safe procedure without any adverse effect on nasal physiology [6]. There has been considerable debate as to whether the ostium size made during dacryocystorhinostomy plays a role on the outcomes. Linberg et al. [8] reported that there is no statistically valid correlation between the size of the neo-ostium and the final size of the healed intranasal ostium. On the other hand, Rosen et al. [1] showed that the endocanalicular approach usually creates smaller neo-ostia than the external and endonasal approach, and this is correlated with lower success rates of ECL DCR. In our study, we concluded that endonasal interventions enabled us to obtain larger neo-ostium without excessive laser energy based on our outcomes, but the neo-ostium size was not documented, and this was one of our drawbacks [4]. After gaining more experience with ECL DCR, and recognizing the high synechia incidence in cases in which no endonasal procedures were applied, we abandoned the performance of ECL DCR without endonasal procedures after the middle of 2008. We conducted a prospective randomized comparative study on 91 patients with primary All the authors have full control of all primary data, and agree to allow Graefe's Archive for Clinical and Experimental Ophthalmology to review their data upon request. H. Basmak (*) :H. Gursoy Department of Ophthalmology, Eskisehir Osmangazi University Medical Faculty, Eskisehir, Turkey e-mail: hbasmak@ogu.edu.tr

Intracellular pathogens: living in an extreme environment

The phylogenetic tree segregates into three major groups, which probably descended from a common ancestor: the bacteria, the archaea, and the eucarya (1–3). While bacteria and archaea lack a true nucleus and intracellular organelles, eucarya possess these. The origin of the ancestor remains elusive, but it emerged about 4 billion years ago. The bacteria probably started life 3.5 billion years ago and eukaryotes emerged 1.75 billion years thereafter. Sometime in between archaea appeared. Eukaryotes began as unicellular organisms but soon also developed multicellular forms. Bacteria and archaea had sufficient time to exploit all niches of the abiotic environment, ranging from ice cold glacial lakes to fiery geysers – from the highest mountain peaks to the deepest sea beds. They also quickly developed forms of mutualism by forming colonies composed of mixed populations. With the evolution of eucarya, new environments could be exploited. Today prokaryotes are the most prevalent form of life on earth, making up the majority of our biomass. It has been estimated that more than 10 prokaryotic organisms live on our globe. Thus, archaea and bacteria are far more prevalent than eucarya. Even human beings comprise more prokaryotic cells than mammalian cells, which range on the order of 10 human cells and 10 prokaryotes mostly concentrated in our intestinal system (4–7).

Peroperative fiducial placement for postoperative stereotactic Cyberknife radiosurgery

Radiotherapy is often the treatment used for inoperable early-stage lung cancer as well as pulmonary metastases. In the last decade, efforts were done to increase local control with high-dose radiation without excessive collateral damage. Initially used for cranial lesions, stereotactic radiosurgery enlarged extracranial indications; the Cyberknife radiosurgery system needs metal (gold) markers for image guidance. For lung tumours, fiducials are inserted computed tomography (CT)-guided or bronchoscopy-guided. We describe four patients where fiducial placement was done during surgery.

Hypoxia-dependent anti-inflammatory pathways in protection of cancerous tissues

The evolutionarily selected tissue-protecting mechanisms are likely to be triggered by an event of universal significance for all surrounding cells. Such an event could be damage to blood vessels, which would result in local tissue hypoxia. It is now recognized that tissue hypoxia can initiate the tissue-protecting mechanism mediated by at least two different biochemical pathways. The central message of this review is that tumor cells are protected from immune damage in hypoxic and immunosuppressive tumor microenvironments due to the inactivation of anti-tumor T cells by the combined action of these two hypoxia-driven mechanisms. Firstly, tumor hypoxia-produced extracellular adenosine inhibits anti-tumor T cells via their G(s)-protein-coupled and cAMP-elevating A2A and A2B adenosine receptors (A2AR/A2BR). Levels of extracellular adenosine are increased in tumor microenvironments due to the changes in activities of enzymes involved in adenosine metabolism. Secondly, TCR-activated and/or tumor hypoxia-exposed anti-tumor T cells may be inhibited in tumor microenvironments by Hypoxia-inducible Factor 1alpha (HIF-1alpha) Hence, HIF-1alpha activity in T cells may contribute to the tumor-protecting immunosuppressive effects of tumor hypoxia. Here, we summarize the data that support the view that protection of hypoxic cancerous tissues from anti-tumor T cells is mediated by the same mechanism that protects normal tissues from the excessive collateral damage by overactive immune cells during acute inflammation.

Association of H2-Blocker Therapy and Higher Incidence of Necrotizing Enterocolitis: A Case of Excessive Collateral Damage?

In this issue of Pediatrics Electronic Edition, Guillet et al1 report the association of H2-blocker therapy with higher incidence of stage II or greater necrotizing enterocolitis (NEC) in very low birth weight neonates using a case-control design. The finding is not necessarily surprising given the link between sepsis and NEC in preterm neonates2–4 and the association of antacid therapy with nosocomial infections that has been well known for at least 15 to 20 years in adult intensive care literature.5–12 Ironically, as … Address correspondence to Sanjay Patole, MD, DCH, FRACP, Department of Neonatal Paediatrics, King Edward Memorial Hospital for Women, University of Western Australia, Perth 6008, Western Australia. E-mail: sanjay.patole{at}health.wa.gov.au

Cutting edge: Physiologic attenuation of proinflammatory transcription by the Gs protein-coupled A2A adenosine receptor in vivo.

The A2A adenosine receptor plays a critical role in the physiologic immunosuppressive pathway that protects normal tissues from excessive collateral damage by overactive immune cells and their proinflammatory cytokines. In this study, we examine and clarify the mechanism of tissue protection by extracellular adenosine using A2AR-deficient mice and show that the A2AR inhibits TLR-induced transcription of proinflammatory cytokines in vivo. The observed increase in proinflammatory cytokines mRNA in A2AR-deficient mice was associated with enhanced activity of the NF-kappaB transcription factor. These observations provide the genetic in vivo evidence for attenuation of proinflammatory transcriptional activity of NF-kappaB by a "metabokine" adenosine and point to the need to re-evaluate the regulation of other transcription factors in hypoxic and adenosine-rich microenvironments of inflamed normal tissues and solid tumors.

Physiological control of immune response and inflammatory tissue damage by hypoxia-inducible factors and adenosine A2A receptors.

Immune cell-mediated destruction of pathogens may result in excessive collateral damage to normal tissues, and the failure to control activated immune cells may cause immunopathologies. The search for physiological mechanisms that downregulate activated immune cells has revealed a critical role for extracellular adenosine and for immunosuppressive A2A adenosine receptors in protecting tissue from inflammatory damage. Tissue damage-associated deep hypoxia, hypoxia-inducible factors, and hypoxia-induced accumulation of adenosine may represent one of the most fundamental and immediate tissue-protecting mechanisms, with adenosine A2A receptors triggering "OFF" signals in activated immune cells. In these regulatory mechanisms, oxygen deprivation and extracellular adenosine accumulation serve as "reporters," while A2A adenosine receptors serve as "sensors" of excessive tissue damage. The A2A receptor-triggered generation of intracellular cAMP then inhibits activated immune cells in a delayed negative feedback manner to prevent additional tissue damage. Targeting A2A adenosine receptors may have important clinical applications.