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Archive for the ‘Nuclear Medicine’ Category


President Carter’s Status

Author: Larry H. Bernstein, MD, FCAP

 

 

Most Experts Not Surprised by Carter’s Status 

But early response does not mean ‘cure’

http://www.medpagetoday.com/HematologyOncology/SkinCancer/55076

 

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by Charles Bankhead
Staff Writer, MedPage Today

 

Former President Jimmy Carter’s announcement that he is free of metastatic melanoma surprised many people but, not most melanoma specialists contacted by MedPage Today.

With the evolution of modern radiation therapy techniques and targeted drugs, more patients with metastatic melanoma achieve complete and partial remissions, including remission of small brain metastases like the ones identified during the evaluation and initial treatment of Carter. However, the experts — none of whom have direct knowledge of Carter’s treatment or medical records — cautioned that early remission offers no assurance that the former president is out of the woods.

“If I had a patient of my own with four small brain mets undergoing [stereotactic radiation therapy], I would tell them that I fully expected the radiation to take care of those four lesions,” said Vernon K. Sondak, MD, of Moffitt Cancer Center in Tampa. “The fact that President Carter reports that it has done just that is not a surprise to me at all.

“I would also tell my patient that the focused radiation only treats the known cancer in the brain, and that if other small areas of cancer are present, they will likely eventually grow large enough to need radiation or other treatment as well, and that periodic brain scans will be required to monitor for this possibility.”

Carter also is being treated with the immune checkpoint inhibitor pembrolizumab (Keytruda), which is known to stimulate immune cells that then migrate to tumor sites to eradicate the lesions, noted Anna Pavlick, DO, of NYU Langone Medical Center in New York City.

“Melanoma is no longer a death sentence, and we are really changing what happens to patients,” said Pavlick. “It really is amazing.”

Carter’s melanoma story began to emerge in early August when he had surgery to remove what was described as “a small mass” from his liver. Following the surgery, Carter announced that his doctors had discovered four small melanoma lesions in his brain, confirming a suspicion the specialists had shared with him at the time of the surgery.

Carter subsequently underwent focused radiation therapy to eradicate the brain lesions and initiated a 12-week course of treatment with pembrolizumab. The radiation therapy-targeted therapy combination was a logical option for Carter, given observations that the PD-L1 inhibitor has synergy with radiation, noted Stergios Moschos, MD, of the University of North Carolina Lineberger Comprehensive Cancer Center at Chapel Hill.

“I have seen this in other patients with metastatic melanoma,” said Gary K. Schwartz, MD, of Columbia University Medical Center in New York City. “It is remarkable but absolutely possible within the realm of immunotherapy today.”

Although Carter’s announcement is undeniably good news, the optimism should be tempered by a long-term perspective, suggested Nagla Abdel Karim, MD, PhD, of the University of Cincinnati Medical Center.

“We do have similar stories; however, we would be careful to call it a ‘complete remission’ and ‘disease control’ and not a ‘cure,’ so far,” said Karim. “We would resume therapy and follow-up any autoimmune side effects. Most important is the quality of life, which he seems to enjoy, and we are very happy with that.”

Darrell S. Rigel, MD, also of NYU Langone Medical Center, represented the lone dissenter among specialists who responded to MedPage Today‘s request for comments.

“I’m happy for him, but it’s very unusual, especially in older men, who usually have a worse prognosis,” said Rigel. “He is on a new drug that may have a little more promise, but there is no definitive cure at this point.”

 

 

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Brain Cancer Vaccine in Development and other considerations

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

GEN News Highlights   Mar 3, 2016

Advanced Immunotherapeutic Method Shows Promise against Brain Cancer

http://www.genengnews.com/gen-news-highlights/advanced-immunotherapeutic-method-shows-promise-against-brain-cancer/81252433/

 

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The researchers induced a specific type of cell death in brain cancer cells from mice. The dying cancer cells were then incubated together with dendritic cells, which play a vital role in the immune system. The researchers discovered that this type of cancer cell killing releases “danger signals” that fully activate the dendritic cells. “We re-injected the activated dendritic cells into the mice as a therapeutic vaccine,” Professor Patrizia Agostinis explains. “That vaccine alerted the immune system to the presence of dangerous cancer cells in the body. As a result, the immune system could recognize them and start attacking the brain tumor.” [©KU Leuven Laboratory of Cell Death Research & Therapy, Dr. Abhishek D. Garg]

 

Scientists from KU Leuven in Belgium say they have shown that next-generation cell-based immunotherapy may offer new hope in the fight against brain cancer.

Cell-based immunotherapy involves the injection of a therapeutic anticancer vaccine that stimulates the patient’s immune system to attack the tumor. Thus far, the results of this type of immunotherapy have been mildly promising. However, Abhishek D. Garg and Professor Patrizia Agostinis from the KU Leuven department of cellular and molecular medicine believe they have found a novel way to produce more effective cell-based anticancer vaccines.

The researchers induced a specific type of cell death in brain cancer cells from mice. The dying cancer cells were then incubated together with dendritic cells, which play a vital role in the immune system. The investigators discovered that this type of cancer cell killing releases “danger signals” that fully activate the dendritic cells.

“We re-injected the activated dendritic cells into the mice as a therapeutic vaccine,” explains Prof. Agostinis. “That vaccine alerted the immune system to the presence of dangerous cancer cells in the body. As a result, the immune system could recognize them and start attacking the brain tumor.”

Combined with chemotherapy, this novel cell-based immunotherapy drastically increased the survival rates of mice afflicted with brain tumors. Almost 50% of the mice were completely cured. None of the mice treated with chemotherapy alone became long-term survivors.

“The major goal of any anticancer treatment is to kill all cancer cells and prevent any remaining malignant cells from growing or spreading again,” says Professor Agostinis. “This goal, however, is rarely achieved with current chemotherapies, and many patients relapse. That’s why the co-stimulation of the immune system is so important for cancer treatments. Scientists have to look for ways to kill cancer cells in a manner that stimulates the immune system. With an eye on clinical studies, our findings offer a feasible way to improve the production of vaccines against brain tumors.”

The team published its study (“Dendritic Cell Vaccines Based on Immunogenic Cell Death Elicit Danger Signals and T Cell–Driven Rejection of High-Grade Glioma”) in Science Translational Medicine.

 

Dendritic cell vaccines based on immunogenic cell death elicit danger signals and T cell–driven rejection of high-grade glioma

 

SLC7A11 expression is associated with seizures and predicts poor survival in patients with malignant glioma

 

Cortical GABAergic excitation contributes to epileptic activities around human glioma

 

Spherical Nucleic Acid Nanoparticle Conjugates as an RNAi-Based Therapy for Glioblastoma

Glioblastoma multiforme (GBM) is a neurologically debilitating disease that culminates in death 14 to 16 months after diagnosis. An incomplete understanding of how cataloged genetic aberrations promote therapy resistance, combined with ineffective drug delivery to the central nervous system, has rendered GBM incurable. Functional genomics efforts have implicated several oncogenes in GBM pathogenesis but have rarely led to the implementation of targeted therapies. This is partly because many “undruggable” oncogenes cannot be targeted by small molecules or antibodies. We preclinically evaluate an RNA interference (RNAi)–based nanomedicine platform, based on spherical nucleic acid (SNA) nanoparticle conjugates, to neutralize oncogene expression in GBM. SNAs consist of gold nanoparticles covalently functionalized with densely packed, highly oriented small interfering RNA duplexes. In the absence of auxiliary transfection strategies or chemical modifications, SNAs efficiently entered primary and transformed glial cells in vitro. In vivo, the SNAs penetrated the blood-brain barrier and blood-tumor barrier to disseminate throughout xenogeneic glioma explants. SNAs targeting the oncoprotein Bcl2Like12 (Bcl2L12)—an effector caspase and p53 inhibitor overexpressed in GBM relative to normal brain and low-grade astrocytomas—were effective in knocking down endogenous Bcl2L12 mRNA and protein levels, and sensitized glioma cells toward therapy-induced apoptosis by enhancing effector caspase and p53 activity. Further, systemically delivered SNAs reduced Bcl2L12 expression in intracerebral GBM, increased intratumoral apoptosis, and reduced tumor burden and progression in xenografted mice, without adverse side effects. Thus, silencing antiapoptotic signaling using SNAs represents a new approach for systemic RNAi therapy for GBM and possibly other lethal malignancies.

 

Rapid, Label-Free Detection of Brain Tumors with Stimulated Raman Scattering Microscopy

Surgery is an essential component in the treatment of brain tumors. However, delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology, SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally, we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue, SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct.

 

Neural Stem Cell–Mediated Enzyme/Prodrug Therapy for Glioma: Preclinical Studies

 

Magnetic Resonance Metabolic Imaging of Glioma

 

Exploiting the Immunogenic Potential of Cancer Cells for Improved Dendritic Cell Vaccines

Cancer immunotherapy is currently the hottest topic in the oncology field, owing predominantly to the discovery of immune checkpoint blockers. These promising antibodies and their attractive combinatorial features have initiated the revival of other effective immunotherapies, such as dendritic cell (DC) vaccinations. Although DC-based immunotherapy can induce objective clinical and immunological responses in several tumor types, the immunogenic potential of this monotherapy is still considered suboptimal. Hence, focus should be directed on potentiating its immunogenicity by making step-by-step protocol innovations to obtain next-generation Th1-driving DC vaccines. We review some of the latest developments in the DC vaccination field, with a special emphasis on strategies that are applied to obtain a highly immunogenic tumor cell cargo to load and to activate the DCs. To this end, we discuss the effects of three immunogenic treatment modalities (ultraviolet light, oxidizing treatments, and heat shock) and five potent inducers of immunogenic cell death [radiotherapy, shikonin, high-hydrostatic pressure, oncolytic viruses, and (hypericin-based) photodynamic therapy] on DC biology and their application in DC-based immunotherapy in preclinical as well as clinical settings.

Cancer immunotherapy has gained considerable momentum over the past 5 years, owing predominantly to the discovery of immune checkpoint inhibitors. These inhibitors are designed to release the brakes of the immune system that under physiological conditions prevent auto-immunity by negatively regulating cytotoxic T lymphocyte (CTL) function. Following the FDA approval of the anti-cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) monoclonal antibody (mAb) ipilimumab (Yervoy) in 2011 for the treatment of metastatic melanoma patients (1), two mAbs targeting programed death (PD)-1 receptor signaling (nivolumab and pembrolizumab) have very recently joined the list of FDA-approved checkpoint blockers (respectively, for the treatment of metastatic squamous non-small cell lung cancer and relapsed/refractory melanoma patients) (2, 3).

However, the primary goal of cancer immunotherapy is to activate the immune system in cancer patients. This requires the induction of tumor-specific T-cell-mediated antitumor immunity. Checkpoint blockers are only able to abrogate the brakes of a functioning antitumoral immune response, implying that only patients who have pre-existing tumor-specific T cells will benefit most from checkpoint blockade. This is evidenced by the observation that ipilimumab may be more effective in patients who have pre-existing, albeit ineffective, antitumor immune responses (4). Hence, combining immune checkpoint blockade with immunotherapeutic strategies that prime tumor-specific T cell responses might be an attractive and even synergistic approach. This relatively new paradigm has lead to the revival of existing, and to date disappointing (as monotherapies), active immunotherapeutic treatment modalities. One promising strategy to induce priming of tumor-specific T cells is dendritic cell (DC)-based immunotherapy.

Dendritic cells are positioned at the crucial interface between the innate and adaptive immune system as powerful antigen-presenting cells capable of inducing antigen-specific T cell responses (5). Therefore, they are the most frequently used cellular adjuvant in clinical trials. Since the publication of the first DC vaccination trial in melanoma patients in 1995, the promise of DC immunotherapy is underlined by numerous clinical trials, frequently showing survival benefit in comparison to non-DC control groups (68). Despite the fact that most DC vaccination trials differ in several vaccine parameters (i.e., site and frequency of injection, nature of the DCs, choice of antigen), DC vaccination as a monotherapy is considered safe and rarely associates with immune-related toxicity. This is in sharp contrast with the use of mAbs or cytokine therapies. Ipilumumab has, for instance, been shown to induce immune-related serious adverse events in up to one-third of treated melanoma patients (1). The FDA approval of Sipuleucel-T (Provenge), an autologous DC-enriched vaccine for hormone-resistant metastatic prostate cancer, in 2010 is really considered as a milestone in the vaccination community (9). After 15 years of extensive clinical research, Sipileucel-T became the first cellular immunotherapy ever that received FDA approval, providing compelling evidence for the substantial socio-economic impact of DC-based immunotherapy. DC vaccinations have most often been applied in patients with melanoma, prostate cancer, high-grade glioma, and renal cell cancer. Although promising objective responses and tumor-specific T cell responses have been observed in all these cancer-types (providing proof-of-principle for DC-based immunotherapy), the clinical success of this treatment is still considered suboptimal (6). This poor clinical efficacy can in part be attributed to the severe tumor-induced immune suppression and the selection of patients with advanced disease status and poor survival prognostics (6, 1012).

There is a consensus in the field that step-by-step optimization and standardization of the production process of DC vaccines, to obtain a Th1-driven immune response, might enhance their clinical efficacy (13). In this review, we address some recent DC vaccine adaptations that impact DC biology. Combining these novel insights might bring us closer to an ideal DC vaccine product that can trigger potent CTL- and Th1-driven antitumor immunity.

One factor requiring more attention in this production process is the immunogenicity of the dying or dead cancer cells used to load the DCs. It has been shown in multiple preclinical cancer models that the methodology used to prepare the tumor cell cargo can influence the in vivo immunogenic potential of loaded DC vaccines (1419). Different treatment modalities have been described to enhance the immunogenicity of cancer cells in the context of DC vaccines. These treatments can potentiate antitumor immunity by inducing immune responses against tumor neo-antigens and/or by selectively increasing the exposure/release of particular damage-associated molecular patterns (DAMPs) that can trigger the innate immune system (14, 1719). The emergence of the concept of immunogenic cell death (ICD) might even further improve the immunogenic potential of DC vaccines. Cancer cells undergoing ICD have been shown to exhibit excellent immunostimulatory capacity owing to the spatiotemporally defined emission of a series of critical DAMPs acting as potent danger signals (20, 21). Thus far, three DAMPs have been attributed a crucial role in the immunogenic potential of nearly all ICD inducers: the surface-exposed “eat me” signal calreticulin (ecto-CRT), the “find me” signal ATP and passively released high-mobility group box 1 (HMGB1) (21). Moreover, ICD-experiencing cancer cells have been shown in various mouse models to act as very potent Th1-driving anticancer vaccines, already in the absence of any adjuvants (21, 22). The ability to reject tumors in syngeneic mice after vaccination with cancer cells (of the same type) undergoing ICD is a crucial hallmark of ICD, in addition to the molecular DAMP signature (21).

Here, we review the effects of three frequently used immunogenic modalities and four potent ICD inducers on DC biology and their application in DC vaccines in preclinical as well as clinical settings (Tables (Tables11 and and2).2). Moreover, we discuss the rationale for combining different cell death-inducing regimens to enhance the immunogenic potential of DC vaccines and to ensure the clinical relevance of the vaccine product.

A list of prominent enhancers of immunogenicity and ICD inducers applied in DC vaccine setups and their associations with DAMPs and DC biology.
A list of preclinical tumor models and clinical studies for evaluation of the in vivo potency of DC vaccines loaded with immunogenically killed tumor cells.
The Impact of DC Biology on the Efficacy of DC Vaccines

Over the past years, different DC vaccine parameters have been shown to impact the clinical effectiveness of DC vaccinations. In the next section, we will elaborate on some promising adaptations of the DC preparation protocol.

Given the labor-intensive ex vivo culturing protocol of monocyte-derived DCs and inspired by the results of the Provenge study, several groups are currently exploiting the use of blood-isolated naturally circulating DCs (7678). In this context, De Vries et al. evaluated the use of antigen-loaded purified plasmacytoid DCs for intranodal injection in melanoma patients (79). This strategy was feasible and induced only very mild side effects. In addition, the overall survival of vaccinated patients was greatly enhanced as compared to historical control patients. However, it still remains to be determined whether this strategy is more efficacious than monocyte-derived DC vaccine approaches (78). By contrast, experiments in the preclinical GL261 high-grade glioma model recently showed that vaccination with tumor antigen-loaded myeloid DCs resulted in more robust Th1 responses and a stronger survival benefit as compared to mice vaccinated with their plasmacytoid counterparts (80).

In view of their strong potential to stimulate cytotoxic T cell responses, several groups are currently exploring the use of Langerhans cell-like DCs as sources for DC vaccines (8183). These so-called IL-15 DCs can be derived from CD14+monocytes by culturing them with IL-15 (instead of the standard IL-4). Recently, it has been shown that in comparison to IL-4 DCs, these cells have an increased capacity to stimulate antitumor natural killer (NK) cell cytotoxicity in a contact- and IL-15-dependent manner (84). NK cells are increasingly being recognized as crucial contributors to antitumor immunity, especially in DC vaccination setups (85, 86). Three clinical trials are currently evaluating these Langerhans cell-type DCs in melanoma patients (NCT00700167, NCT 01456104, and NCT01189383).

Targeting cancer stem cells is another promising development, particularly in the setting of glioma (87). Glioma stem cells can foster tumor growth, radio- and chemotherapy-resistance, and local immunosuppression in the tumor microenvironment (87, 88). Furthermore, glioma stem cells may express higher levels of tumor-associated antigens and MHC complex molecules as compared to non-stem cells (89, 90). A preclinical study in a rodent orthotopic glioblastoma model has shown that DC vaccines loaded with neuropsheres enriched in cancer stem cells could induce more immunoreactivity and survival benefit as compared to DCs loaded with GL261 cells grown under standard conditions (91). Currently there are four clinical trials ongoing in high-grade glioma patients evaluating this approach (NCT00890032, NCT00846456, NCT01171469, and NCT01567202).

With regard to the DC maturation status of the vaccine product, a phase I/II clinical trial in metastatic melanoma patients has confirmed the superiority of mature antigen-loaded DCs to elicit immunological responses as compared to their immature counterparts (92). This finding was further substantiated in patients diagnosed with prostate cancer and recurrent high-grade glioma (93, 94). Hence, DCs need to express potent costimulatory molecules and lymph node homing receptors in order to generate a strong T cell response. In view of this finding, the route of administration is another vaccine parameter that can influence the homing of the injected DCs to the lymph nodes. In the context of prostate cancer and renal cell carcinoma it has been shown that vaccination routes with access to the draining lymph nodes (intradermal/intranodal/intralymphatic/subcutaneous) resulted in better clinical response rates as compared to intravenous injection (93). In melanoma patients, a direct comparison between intradermal vaccination and intranodal vaccination concluded that, although more DCs reached the lymph nodes after intranodal vaccination, the melanoma-specific T cells induced by intradermal vaccination were more functional (95). Furthermore, the frequency of vaccination can also influence the vaccine’s immunogenicity. Our group has shown in a cohort-comparison trial involving relapsed high-grade glioma patients that shortening the interval between the four inducer DC vaccines improved the progression-free survival curves (58, 96).

Another variable that has been systematically studied is the cytokine cocktail that is applied to mature the DCs. The current gold standard cocktail for DC maturation contains TNF-α, IL-1β, IL-6, and PGE2 (97, 98). Although this cocktail upregulates DC maturation markers and the lymph node homing receptor CCR7, IL-12 production by DCs could not be evoked (97, 98). Nevertheless, IL-12 is a critical Th1-driving cytokine and DC-derived IL-12 has been shown to associate with improved survival in DC vaccinated high-grade glioma and melanoma patients (99, 100). Recently, a novel cytokine cocktail, including TNF-α, IL-1β, poly-I:C, IFN-α, and IFN-γ, was introduced (101, 102). The type 1-polarized DCs obtained with this cocktail produced high levels of IL-12 and could induce strong tumor-antigen-specific CTL responses through enhanced induction of CXCL10 (99). In addition, CD40-ligand (CD40L) stimulation of DCs has been used to mature DCs in clinical trials (100, 103). Binding of CD40 on DCs to CD40L on CD4+ helper T cells licenses DCs and enables them to prime CD8+ effector T cells.

A final major determinant of the vaccine immunogenicity is the choice of antigen to load the DCs. Two main approaches can be applied: loading with selected tumor antigens (tumor-associated antigens or tumor-specific antigens) and loading with whole tumor cell preparations (13). The former strategy enables easier immune monitoring, has a lower risk of inducing auto-immunity, and can provide “off-the-shelf” availability of the antigenic cargo. Whole tumor cell-based DC vaccines, on the other hand, are not HLA-type dependent, have a reduced risk of inducing immune-escape variants, and can elicit immunity against multiple tumor antigens. Meta-analytical data provided by Neller et al. have demonstrated enhanced clinical efficacy in several tumor types of DCs loaded with whole tumor lysate as compared to DCs pulsed with defined tumor antigens (104). This finding was recently also substantiated in high-grade glioma patients, although this study was not set-up to compare survival parameters (105).

Toward a More Immunogenic Tumor Cell Cargo

The majority of clinical trials that apply autologous whole tumor lysate to load DC vaccines report the straightforward use of multiple freeze–thaw cycles to induce primary necrosis of cancer cells (8, 93). Freeze–thaw induced necrosis is, however, considered non-immunogenic and has even been shown to inhibit toll-like receptor (TLR)-induced maturation and function of DCs (16). To this end, many research groups have focused on tackling this roadblock by applying immunogenic modalities to induce cell death.

Immunogenic Treatment Modalities

Tables Tables11 and and22 list some frequently applied treatment methods to enhance the immunogenic potential of the tumor cell cargo that is used to load DC vaccines in an ICD-independent manner (i.e., these treatments do not meet the molecular and/or cellular determinants of ICD). Immunogenic treatment modalities can positively impact DC biology by inducing particular DAMPs in the dying cancer cells (Table (Table1).1). Table Table22 lists the preclinical and clinical studies that investigated their in vivo potential. Figure Figure11 schematically represents the application and the putative modes of action of these immunogenic enhancers in the setting of DC vaccines.

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A schematic representation of immunogenic DC vaccines. Cancer cells show enhanced immunogenicity upon treatment with UV irradiation, oxidizing treaments, and heat shock, characterized by the release of particular danger signals and the (increased) production of tumor (neo-)antigens. Upon loading onto DCs, DCs undergo enhanced phagocytosis and antigen uptake and show phenotypic and partial functional maturation. Upon in vivo immunization, these DC vaccines elicit Th1- and cytotoxic T lymphocyte (CTL)-driven tumor rejection.

Ultraviolet Irradiation ….

Oxidation-Inducing Modalities

In recent years, an increasing number of data were published concerning the ability of oxidative stress to induce oxidation-associate molecular patterns (OAMPs), such as reactive protein carbonyls and peroxidized phospholipids, which can act as DAMPs (28, 29) (Table (Table1).1). Protein carbonylation, a surrogate indicator of irreversible protein oxidation, has for instance been shown to improve cancer cell immunogenicity and to facilitate the formation of immunogenic neo-antigens (30, 31).

One prototypical enhancer of oxidation-based immunogenicity is radiotherapy (21,23). In certain tumor types, such as high-grade glioma and melanoma, clinical trials that apply autologous whole tumor lysate to load DC vaccines report the random use of freeze–thaw cycles (to induce necrosis of cancer cells) or a combination of freeze–thaw cycles and subsequent high-dose γ-irradiation (8, 18) (Table (Table2).2). However, from the available clinical evidence, it is unclear which of both methodologies has superior immunogenic potential. In light of the oxidation-based immunogenicity that is associated with radiotherapy, we recently demonstrated the superiority of DC vaccines loaded with irradiated freeze–thaw lysate (in comparison to freeze–thaw lysate) in terms of survival advantage in a preclinical high-grade glioma model (18) (Table (Table2).2). ….

Heat Shock Treatment

Heat shock is a term that is applied when a cell is subjected to a temperature that is higher than that of the ideal body temperature of the organisms of which the cell is derived. Heat shock can induce apoptosis (41–43°C) or necrosis (>43°C) depending on the temperature that is applied (110). The immunogenicity of heat shock treated cancer cells largely resides within their ability to produce HSPs, such as HSP60, HSP70, and HSP90 (17, 32) (Table (Table1).1). …

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Figure 2

A schematic representation of immunogenic cell death (ICD)-based DC vaccines. ICD causes cancer cells to emit a spatiotemporally defined pattern of danger signals. Upon loading of these ICD-undergoing cancer cells onto DCs, they induce extensive phagocytosis and antigen uptake. Loaded DCs show enhanced phenotypic and functional maturation and immunization with these ICD-based DC vaccines instigates Th1-, Th17-, and cytotoxic T lymphocyte (CTL)-driven antitumor immunity in vivo.
Inducers of Immunogenic Cell Death

Immunogenic cell death is a cell death regimen that is associated with the spatiotemporally defined emission of immunogenic DAMPs that can trigger the immune system (20, 21, 113). ICD has been found to depend on the concomitant induction of reactive oxygen species (ROS) and activation of endoplasmatic reticulum (ER) stress (111). Besides the three DAMPs that are most crucial for ICD (ecto-CRT, ATP, and HMGB1), other DAMPs such as surface-exposed or released HSPs (notably HSP70 and HSP90) have also been shown to contribute to the immunogenic capacity of ICD inducers (20, 21). The binding of these DAMPs to their respective immune receptors (CD91 for HSPs/CRT, P2RX7/P2RY2 for ATP, and TLR2/4 for HMGB1/HSP70) leads to the recruitment and/or activation of innate immune cells and facilitates the uptake of tumor antigens by antigen-presenting cells and their cross-presentation to T cells eventually leading to IL-1β-, IL-17-, and IFN-γ-dependent tumor eradiation (22). This in vivo tumor rejecting capacity induced by dying cancer cells in the absence of any adjuvant, is considered as a prerequisite for an agent to be termed an ICD inducer. …

Although the list of ICD inducers is constantly growing (113), only few of these immunogenic modalities have been tested in order to generate an immunogenic tumor cell cargo to load DC vaccines (Tables (Tables11 and and2).2). Figure Figure22 schematically represents the preparation of ICD-based DC vaccines and their putative modes of action.

Radiotherapy

Ionizing X-ray or γ-ray irradiation exerts its anticancer effect predominantly via its capacity to induce DNA double-strand breaks leading to intrinsic cancer cell apoptosis (114). The idea that radiotherapy could also impact the immune system was derived from the observation that radiotherapy could induce T-cell-mediated delay of tumor growth in a non-irradiated lesion (115). This abscopal (ab-scopus, away from the target) effect of radiotherapy was later explained by the ICD-inducing capacity (116). Together with anthracyclines, γ-irradiation was one of the first treatment modalities identified to induce ICD. …

Shikonin

The phytochemical shikonin, a major component of Chinese herbal medicine, is known to inhibit proteasome activity. It serves multiple biological roles and can be applied as an antibacterial, antiviral, anti-inflammatory, and anticancer treatment. …

High-hydrostatic pressure

High-hydrostatic pressure (HHP) is an established method to sterilize pharmaceuticals, human transplants, and food. HHP between 100 and 250 megapascal (MPa) has been shown to induce apoptosis of murine and human (cancer) cells (121123). While DNA damage does not seem to be induced by HHP <1000 MPa, HHP can inhibit enzymatic functions and the synthesis of cellular proteins (122). Increased ROS production was detected in HHP-treated cancer cell lines and ER stress was evidenced by the rapid phosphorylation of eIF2α (42).  …

Oncolytic Viruses

Oncolytic viruses are self-replicating, tumor selective virus strains that can directly lyse tumor cells. Over the past few years, a new oncolytic paradigm has risen; entailing that, rather than utilizing oncolytic viruses solely for direct tumor eradication, the cell death they induce should be accompanied by the elicitation of antitumor immune responses to maximize their therapeutic efficacy (128). One way in which these oncolytic viruses can fulfill this oncolytic paradigm is by inducing ICD (128).

Thus far, three oncolytic virus strains can meet the molecular requirements of ICD; coxsackievirus B3 (CVB3), oncolytic adenovirus and Newcastle disease virus (NDV) (Table (Table1)1) (113). Infection of tumor cells with these viruses causes the production of viral envelop proteins that induce ER stress by overloading the ER. Hence, all three virus strains can be considered type II ICD inducers (113). …

Photodynamic therapy

Photodynamic therapy (PDT) is an established, minimally invasive anticancer treatment modality. It has a two-step mode of action involving the selective uptake of a photosensitizer by the tumor tissue, followed by its activation by light of a specific wavelength. This activation results in the photochemical production of ROS in the presence of oxygen (129131). One attractive feature of PDT is that the ROS-based oxidative stress originates in the particular subcellular location where the photosensitizer tends to accumulate, ultimately leading to the destruction of the tumor cell (132). …

Combinatorial Regimens

In DC vaccine settings, cancer cells are often not killed by a single treatment strategy but rather by a combination of treatments. In some cases, the underlying rationale lies within the additive or even synergistic value of combining several moderately immunogenic modalities. The combination of radiotherapy and heat shock has, for instance, been shown to induce higher levels of HSP70 in B16 melanoma cells than either therapy alone (16). In addition, a combination therapy consisting of heat shock, γ-irradiation, and UV irradiation has been shown to induce higher levels of ecto-CRT, ecto-HSP90, HMGB1, and ATP in comparison to either therapy alone or doxorubicin, a well-recognized inducer of ICD (57). ….

Triggering antitumor immune responses is an absolute requirement to tackle metastatic and diffusely infiltrating cancer cells that are resistant to standard-of-care therapeutic regimens. ICD-inducing modalities, such as PDT and radiotherapy, have been shown to be able to act as in situ vaccines capable of inducing immune responses that caused regression of distal untreated tumors. Exploiting these ICD inducers and other immunogenic modalities to obtain a highly immunogenic antigenic tumor cell cargo for loading DC vaccines is a highly promising application. In case of the two prominent ICD inducers, Hyp-PDT and HHP, preclinical studies evaluating this relatively new approach are underway and HHP-based DC vaccines are already undergoing clinical testing. In the preclinical testing phase, more attention should be paid to some clinically driven considerations. First, one should consider the requirement of 100% mortality of the tumor cells before in vivo application. A second consideration from clinical practice (especially in multi-center clinical trials) is the fact that most tumor specimens arrive in the lab in a frozen state. This implies that a significant number of cells have already undergone non-immunogenic necrosis before the experimental cell killing strategies are applied. ….

 

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Methylation and cancer epigenomics

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

2015 DNA methylation research grant winners

https://www.diagenode.com/landingPages/display/dna-methylation-grant-applications

 

Persistency of Transgene Expression Mediated by Lentiviral Gene Delivery in Pluripotent Cell Lines

Suleiman Alhaji
Ph.D. Student, University Putra, Malaysia

My general objectives are to (1) determine the duration of reporter gene expression from pluripotent cell lines transduced by lentivirus and (2) to assess the epigenetic effects on the provirus. More specifically, I plan to (1) produce LV carrying Green Fluorescent Protein (GFP) reporter gene, (2) obtain and maintain the required cell lines including establishing primary mouse fibroblast as a control, 3) measure the duration of GFP expression from pluripotent and control cell lines transduced with the lentivirus, (4) exclude the loss of the integrated provirus as the factor for GFP silencing in transduced non-pluripotent cell lines, and (5) study the effects of epigenetics on the GFP gene and the regulatory sequence of the provirus.

Transgene integration by lentiviral (LV) vector in the host cell’s genome would theoretically generate a prolonged or permanent transgene expression. However, several citations have reported a decline in transgene expression in early progenitor cells and stem cells transduced by LV. We hypothesized that prolonged transgene expression can be achieved if the transgene is introduced into the cells before epigenetic markers are established in the genome, i.e during the pluripotency period. Therefore, the proposed study seeks to determine if this phenomenon would occur in pluripotent cell lines, focusing on mouse induced pluripotent stem (iPS) cells as the target cell, in a gene therapy context.

Two epigenetic analyses that will be performed on the promoter and transgene of the provirus are DNA methylation and chromatin modification. For DNA methylation profiling, the genomic DNA of the cell will be treated with bisulfite prior to PCR and sequencing of the proviral DNA. The cells may also be analyzed for 5-hmC using 5-hmC monoclonal antibody (C15200200-200) or hMeDIP Kit (C02010031) to assess the level of hydroxymethylation. We may also consider using Diagenode’s MethylCap kit (C02020010) to fractionate the methylated DNA by CpG density.

For the chromatin modification analysis, the cells will be treated with trichostatin A (TSA) before chromatin immunoprecipitation (ChIP) analysis by chromatin IP – bisulfite – sequencing (ChIP-Bis-Seq) and as well Combining chromatin IP and DNA methylation profiles in one assay using the Premium Bisulfite kit, Diagenode. We may also perform pull-down methylated DNA analysis by using specific antibodies such as (1) H3K4 monoclonal antibody (C1541065) (2) H3K4 polyclonal antibody (C15410037) (3) H3K9 polyclonal antibody (C15410004). Beads only will be used as a control.

 

Epigenome-wide methylation pattern discovery for irradiated skin in radiotherapy

Maxwell Johnson, Ph.D.
Research Fellow, University of Southern California, Department of Plastic and Reconstructive Surgery

Radiotherapy is utilized in neoadjuvant, definitive, and palliative treatment of a wide variety of cancers. From a reconstructive perspective, however, irradiated fields pose significant challenges as the tissue is often stiff, brittle, and heals poorly. Little is known about the mechanism by which irradiation produces these changes. The objective of our research is to reveal the epigenetic changes that occur in irradiated skin in order to identify potential targets for therapy. We aim to translate our findings into interventional studies in both cell culture and a mouse model to assess their efficacy in vitro and in vivo.

We have access to a bank of paired samples of irradiated and non-irradiated tissues from patients who have undergone reconstructive procedures after cancer treatment. We have used the Illumina Infinium Human Methylation450 BeadChip array to assess epigenome-wide methylation status of eight paired samples, and have identified a signature methylation pattern for irradiated skin. We would like to utilize the Diagenode Premium WGBS Kit for bisulfite sequencing of additional paired samples for two purposes. First, we would like to confirm the findings of our BeadChip array utilizing a more robust method of assessing epigenome-wide methylation status. Second, we would like to assess methylation status at loci that are not evaluated by the BeadChip array. Using this information, we plan to identify loci with the greatest change in methylation status between irradiated and non-irradiated samples. By comparing these loci to literature, we intend to identify genes that are known to have an effect on wound healing. We then plan to design interventional studies to assess the effects of modulating the expression of these genes and/or supplementing gene products in cell culture and a mouse model. We would like to utilize the Diagenode Bisulfite Kit to confirm methylation status at target genes in these additional studies.

 

Next-generation sequencing based methylome study of primary breast tumours

Rajbir Batra, Ph.D. Researcher
Cancer Research UK Cambridge Institute, University of Cambridge

Breast cancer is one of the leading causes of cancer death in women, and is unanimously considered a heterogeneous disease displaying distinct therapeutic responses and outcomes. While recent advances have led to the refinement of the molecular classification of the disease, the epigenetic landscape has received less attention.

We are delighted to win the DNA methylation research grant award and intend to use it to conduct a next-generation sequencing based methylome study of primary breast tumours. DNA methylation markers will also be investigated in Patient Derived Tumour Xenografts (PDTXs) and in circulating tumour DNA (ctDNA) to identify potential prognostic and predictive methylation biomarkers in breast cancer.

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New Imaging Application for Parkinson’s

Larry H. Bernstein, Md, FCAP, Curator

LPBI

 

Brain imaging technology offers new approach for studying Parkinsonian syndromes

http://www.bioopticsworld.com/articles/pt/2016/01/brain-imaging-technology-offers-new-approach-for-studying-parkinsonian-syndromes.html

 

file

 

The prefrontal cortex, highlighted in red, is responsible for high-level functions like memory, attention and problem solving.
Credit: Life Science Databases

http://www.bioopticsworld.com/content/bow/en/articles/pt/2016/01/brain-imaging-technology-offers-new-approach-for-studying-parkinsonian-syndromes/_jcr_content/leftcolumn/article/headerimage.img.jpg

Using a portable device, researchers have identified differences in brain activation patterns associated with postural stability in people with Parkinsonian syndromes and healthy adults. The findings describe the critical role of the prefrontal cortex in balance control and may have implications with respect to detecting and treating Parkinsonian symptoms in the elderly.

The BioOptics World take on this story:

A newly developed portable device that employs functional near-infrared (fNIR) spectroscopy (a light-based technique to monitor changes in blood oxygenation in the brain) can identify differences in brain activation patterns associated with postural stability in people with Parkinson’s disease and healthy adults. The development allows scientists to better understand the role of the brain’s prefrontal cortex during standing and walking.

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Brain imaging technology offers new approach for studying Parkinsonian syndromes
 Drexel University

Parkinson’s disease is a neurological disorder that arises when brain cells that control movement die, leaving many patients in the late stages of the disease unable to take a few steps before falling. Parkinsonian syndromes, which are common in older adults, are conditions that do not rise to a Parkinson’s diagnosis but encompass many symptoms of the disease, like rigidity, tremor and difficulty walking.

Past attempts to compare brain activity and stability in people with Parkinsonian syndromes have been limited, because neuroimaging tools could only be used when a study participant was lying flat, rather than walking or standing. In these cases, the person undergoing the brain scan could only imagine that he or she was performing the tasks.

A portable system created by researchers in Drexel’s School of Biomedical Engineering and Health Systems overcomes this challenge. It has allowed scientists, for the first time, to better understand the role of the brain’s prefrontal cortex during standing and walking.

The device employs functional near-infrared, or fNIR, spectroscopy, which uses light to monitor changes in blood oxygenation in the brain as individuals perform tasks, take tests or receive stimulation. The prefrontal cortex is the area responsible for higher-level processing, such as memory, attention, problem solving and decision-making. When a person is learning a new skill, for instance, neural activity is greater in this region.

Unlike fMRI (functional magnetic resonance imaging), the fNIR system is fully portable: Participants wear a headband, allowing them to talk and move around while a computer collects data in real time.

“Postural instability is a major risk factor for older adults. If we can monitor the cognitive component of staying balanced, then this could eventually lead to better treatment options for people with Parkinsonian syndromes or even Parkinson’s disease,” said Meltem Izzetoglu, PhD, an assistant research professor of biomedical engineering at Drexel who co-authored the study.

Researchers at Albert Einstein College of Medicine used the fNIR technology to compare 126 healthy adults to 117 individuals with mild Parkinson’s symptoms and 26 with more severe symptoms. While wearing the fNIR headband, the participants were asked to stand and look straight ahead while counting for 10 seconds. They then walked on a mat that captured their gait speed, pace and stride length. The fNIR system recorded their brain oxygen levels during the entire testing period.

The researchers found that those with Parkinsonian symptoms demonstrated significantly higher prefrontal oxygenation levels to maintain stability when standing than participants with mild and no symptoms.

“In fact, brain activity in the frontal brain region was nearly twice as large,” said Jeannette R. Mahoney, PhD, assistant professor of neurology at Einstein and the study’s lead author.

“This initial study allowed us to measure brain activity in real-time, in a realistic setting. It shows that there are indeed differences in the prefrontal cortex of healthy and Parkinsonian syndrome patients, and those differences relate to their performance in maintaining stability while standing,” Izzetoglu said. “It opens up new fields of research.”

In an upcoming clinical trial, the researchers will use a computerized cognitive training program and the fNIR system to identify how cognitive training affects brain activation during walking.

The portable technology could aid in diagnosing Parkinsonian syndromes or developing interventions.

“Our goal is to be able to intervene with Parkinsonian symptoms and develop novel remediation in the not-so-distant future to improve elders’ quality of life,” Mahoney said.

Jeannette R. Mahoney, Roee Holtzer, Meltem Izzetoglu, Vance Zemon, Joe Verghese, Gilles Allali. The role of prefrontal cortex during postural control in Parkinsonian syndromes a functional near-infrared spectroscopy study. Brain Research, 2015; DOI:10.1016/j.brainres.2015.10.053

Drexel University. “Brain imaging technology offers new approach for studying Parkinsonian syndromes.” ScienceDaily. ScienceDaily, 18 December 2015. <www.sciencedaily.com/releases/2015/12/151218113326.htm>.
New imaging test gives physicians better tool to diagnose Parkinson’s disease
August 25, 2011    Source:  Northwestern Memorial Hospital
Summary:
Physicians now have an objective test to evaluate patients for Parkinsonian syndromes, such as Parkinson’s disease. DaTscan™ is the only FDA-approved imaging agent for assessment of movement disorders. Until now, there were no definitive tests to identify the disease, forcing physicians to rely on clinical examinations to make a diagnosis. This technology allows doctors to differentiate Parkinson’s from other movement disorders.

Until now, there were no definitive tests to identify the disease, forcing physicians to rely on clinical examinations to make a diagnosis. This technology allows doctors to differentiate Parkinson’s from other movement disorders.

“The scan by itself does not make the diagnosis of Parkinson’s but it allows us to identify patients who have loss of dopamine, the major chemical responsible for the symptoms, from those who have no dopamine deficiency,” said Tanya Simuni, MD, a neurologist at Northwestern Memorial and director of Northwestern’s Parkinson’s Disease and Movement Disorders Center. “This is a very important step in being able to accurately identify and treat movement disorders and hopefully allow us to better understand these diseases over time.”

Parkinson’s disease is a neurodegenerative disorder that afflicts nearly 1.5 million Americans, with an additional 50,000 to 60,000 new cases identified each year. People with Parkinson’s lack dopamine in the brain, which leads to tremor, slowness of movement, muscle stiffness and balance problems. Clinical examinations, particularly early in the disease when symptoms are slight, can be inconclusive or lead to misdiagnosis of another movement disorder, such as essential tremor, which share similar symptoms to Parkinson’s, but require different treatment.

Developed by GE Healthcare, DaTscan is a substance used to detect the presence of dopamine transporters (DaT) in the brain. A patient is injected with the contrast agent and then undergoes a single-photon emission computed tomography (SPECT) scan. The test captures detailed pictures of the brain’s dopamine system and can provide visual evidence of the presence of dopamine transporters. Scans of patients with Parkinson’s disease or another parkinsonian syndrome will show very low dopamine levels. A SPECT scan examines brain function, rather than structure, and can show change in the brain’s chemistry.

“In Parkinson’s patients the brain’s anatomy remains largely normal, unlike other conditions such as stroke, where damage to the brain is visible,” explained Simuni, who is also an associate professor of neurology at Northwestern University Feinberg School of Medicine. “DaTscan attaches to dopamine neurons which illuminate on the SPECT scan; the more light areas that exist, the more healthy dopamine brain cells remain. If the areas of the brain that should show dopamine remain dark, it may indicate the patient has some type of parkinsonian syndrome.”

An accurate clinical diagnosis for patients with neurodegenerative movement disorders, such as Parkinson’s, can take up to six years. While symptoms often mimic Parkinson’s, other movement disorders, such as essential tremor, occur in different areas of the brain and do not involve the dopamine system.

“Even though they may appear similar, other movement disorders require different management. DaTscan allows us to confirm our diagnosis earlier and start the correct course of treatment sooner,” said Simuni. “We are hopeful that this will lead to improved quality of life for these patients with better long term outcomes, as well as protection from unnecessary treatments initiated because of misdiagnosis.”

While Simuni does not believe it is necessary for every patient to confirm their Parkinson’s diagnosis with DaTscan, she does see it as a valuable tool for patients with uncertain syndromes, or those who have not responded to treatment. She also sees it as a means for improving Parkinson’s research by ensuring those enrolled in studies actually have the disease. DaTscan is already being used by the Michael J. Fox Foundation for its landmark biomarkers study, the Parkinson’s Progression Markers Initiative (PPMI), to validate that the subjects have Parkinson’s disease. Northwestern is one of the 14 U.S. medical centers enrolling for the PPMI, which is among the first clinical trials using DaTscan in this way.

“Currently, we are not able to say with certainty that those enrolled in Parkinson’s studies have the disease,” said Simuni. “With the addition of DaTscan, we can be much more confident in the status of research subjects in both the control and experimental groups. By having a better understanding of these populations, we should be able to have clearer outcomes and hopefully that will translate sooner into treatments and eventually a cure.”

Researchers are also hopeful that DaTscan will prove to be useful in following the progression of Parkinson’s throughout a patient’s lifetime. “The disease is clinically measured at certain points of time to help physicians understand its development,” said Simuni. “A lot of questions about how Parkinson’s disease progresses can be answered if DaTscan is able to show us changes in the brain’s chemistry over time.”

 Northwestern Memorial Hospital. “New imaging test gives physicians better tool to diagnose Parkinson’s disease.” ScienceDaily. ScienceDaily, 25 August 2011. <www.sciencedaily.com/releases/2011/08/110825105029.htm>.
Abnormal oscillation in the brain causes motor deficits in Parkinson’s disease
November 1, 2011    National Institute for Physiological Sciences
Summary:   Scientists have shown that the ‘oscillatory’ nature of electrical signals in subcortical nuclei, the basal ganglia, causes severe motor deficits in Parkinson’s disease, by disturbing the information flow of motor commands.

The research group headed by Professor Atsushi Nambu (The National Institute for Physiological Sciences) and Professor Masahiko Takada (Primate Research Institute,Kyoto University) has shown that the ‘oscillatory’ nature of electrical signals in subcortical nuclei, the basal ganglia, causes severe motor deficits in Parkinson’s disease, by disturbing the information flow of motor commands. The group also found that chemical inactivation of the subthalamic nucleus (a structure of the basal ganglia) in parkinsonian monkeys improved the motor impairments by reducing the ‘oscillations.’

The results of this study were reported in European Journal of Neuroscience,November 2011 issue.

A member of the research group, Assistant Professor Yoshihisa Tachibana, succeeded to record electrical signals in monkey basal ganglia neurons under unanesthetized conditions. The group found that neurons in the parkinsonian basal ganglia showed abnormal ‘oscillatory’ activity, which was rarely seen in normal subjects. The abnormal rhythm was completely eliminated by systemic administration of a dopamine precursor (L-DOPA), which is clinically used for human parkinsonian patients. The group considered that loss of dopamine induced the ‘oscillations’ in the basal ganglia and that the following disturbances in information flow of motor commands impaired motor performances.

Abnormal neuronal oscillations were already reported in parkinsonian patients and animal models, but this report has provided the direct evidence that ‘oscillations’ are associated with motor abnormalities. Moreover, it was also shown that the injection of a chemical inhibitor, muscimol, into the subthalamic nucleus silenced the oscillatory signals, and eventually reversed parkinsonian motor signs.

Professor Nambu claims, “By investigating the ‘oscillatory’ nature of electrical signals in the basal ganglia, we can advance our understanding of the pathophysiology of Parkinson’s disease. We improved motor deficits by means of infusion of the chemical inhibitor (muscimol) into the subthalamic nucleus to silence the ‘oscillatory’ signals in the brain structure. This may provide us important clues to developing new treatments for Parkinson’s disease.”

This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Japan Intractable Diseases Research Foundation and Hori Information Science Promotion Foundation to Y. Tachibana and A. Nambu, and NIH grants (NS-47085 and NS-57236) to H. Kita.

Yoshihisa Tachibana, Hirokazu Iwamuro, Hitoshi Kita, Masahiko Takada, Atsushi Nambu. Subthalamo-pallidal interactions underlying parkinsonian neuronal oscillations in the primate basal ganglia.European Journal of Neuroscience, 2011; 34 (9): 1470 DOI:10.1111/j.1460-9568.2011.07865.x

National Institute for Physiological Sciences. “Abnormal oscillation in the brain causes motor deficits in Parkinson’s disease.” ScienceDaily. ScienceDaily, 1 November 2011. <www.sciencedaily.com/releases/2011/11/111101095306.htm>.
Mental picture of others can be seen using fMRI, finds new study 
March 5, 2013
Source:  Cornell University
Summary:
It is possible to tell who a person is thinking about by analyzing images of his or her brain. Our mental models of people produce unique patterns of brain activation, which can be detected using advanced imaging techniques according to a new study.

“When we looked at our data, we were shocked that we could successfully decode who our participants were thinking about based on their brain activity,” said Spreng, assistant professor of human development in Cornell’s College of Human Ecology.

Understanding and predicting the behavior of others is a key to successfully navigating the social world, yet little is known about how the brain actually models the enduring personality traits that may drive others’ behavior, the authors say. Such ability allows us to anticipate how someone will act in a situation that may not have happened before.

To learn more, the researchers asked 19 young adults to learn about the personalities of four people who differed on key personality traits. Participants were given different scenarios (i.e. sitting on a bus when an elderly person gets on and there are no seats) and asked to imagine how a specified person would respond. During the task, their brains were scanned using functional magnetic resonance imaging (fMRI), which measures brain activity by detecting changes in blood flow.

They found that different patterns of brain activity in the medial prefrontal cortex (mPFC) were associated with each of the four different personalities. In other words, which person was being imagined could be accurately identified based solely on the brain activation pattern.

The results suggest that the brain codes the personality traits of others in distinct brain regions and this information is integrated in the medial prefrontal cortex (mPFC) to produce an overall personality model used to plan social interactions, the authors say.

The study, “Imagine All the People: How the Brain Creates and Uses Personality Models to Predict Behavior,” published online March 5 in the journal Cerebral Cortex and was coauthored by Demis Hassabis, University College London, Andrie Rusu, Vrije Univesiteit, Clifford Robbins, Harvard University, Raymond Mar, York University, and Daniel L. Schacter, Harvard University

Demis Hassabis, R. Nathan Spreng, Andrei A. Rusu, Clifford A. Robbins, Raymond A. Mar, and Daniel L. Schacter. Imagine All the People: How the Brain Creates and Uses Personality Models to Predict Behavior.Cerebral Cortex, 2013; DOI: 10.1093/cercor/bht042

Cornell University. “Mental picture of others can be seen using fMRI, finds new study.” ScienceDaily. ScienceDaily, 5 March 2013. <www.sciencedaily.com/releases/2013/03/130305091000.htm>.

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Studying Alzheimer’s biomarkers in Down syndrome

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

NIH supports new studies to find Alzheimer’s biomarkers in Down syndrome

Groundbreaking initiative will track dementia onset, progress in Down syndrome volunteers

http://www.nih.gov/news-events/news-releases/nih-supports-new-studies-find-alzheimers-biomarkers-down-syndrome

 

The National Institutes of Health has launched a new initiative to identify biomarkers and track the progression of Alzheimer’s in people with Down syndrome. Many people with Down syndrome have Alzheimer’s-related brain changes in their 30s that can lead to dementia in their 50s and 60s. Little is known about how the disease progresses in this vulnerable group. The NIH Biomarkers of Alzheimer’s Disease in Adults with Down Syndrome Initiative will support teams of researchers using brain imaging, as well as fluid and tissue biomarkers in research that may one day lead to effective interventions for all people with dementia.

The studies will be funded by the National Institute on Aging (NIA) and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), both part of NIH. The institutes are jointly providing an estimated $37 million over five years to two highly collaborative projects, which enlist a number of leading researchers to the effort. To advance Alzheimer’s research worldwide, the teams will make their data and samples freely available to qualified researchers.

“This is the first large-scale Alzheimer’s biomarker endeavor to focus on this high-risk group,” said Laurie Ryan, Ph.D., chief of the Dementias of Aging Branch in NIA’s Division of Neuroscience, which leads NIH research on Alzheimer’s.  “Much like the long-established Alzheimer’s Disease Neuroimaging Initiative, the goal of this initiative is to develop biomarker measures that signal the onset and progression of Alzheimer’s in people with Down syndrome. Hopefully, one day, we will also use these biomarkers to determine the effectiveness of promising treatments.”

The link between Alzheimer’s and Down syndrome is well-known. People with Down syndrome are born with an extra copy of chromosome 21, which contains the amyloid precursor protein gene. This gene plays a role in the production of harmful amyloid plaque, sticky clumps that build up outside neurons in Alzheimer’s disease. Having three copies of this gene is a known risk factor for early-onset Alzheimer’s that can occur in people in their 30s, 40s and 50s. By middle age, most but not all adults with Down syndrome develop signs of Alzheimer’s, and a high percentage go on to develop symptoms of dementia as they age into their 70s.

The initiative establishes funding for two research teams that will pool data and standardize procedures, increase sample size, and collectively analyze data that will be made widely available to the research community. The teams will employ an array of biomarkers to identify and track Alzheimer’s-related changes in the brain and cognition for over 500 Down syndrome volunteers, aged 25 and older. The measures include:

  • Positron emission tomography (PET) scans that track levels of amyloid and glucose (energy used by brain cells); MRI of brain volume and function; and levels of amyloid and tau in cerebrospinal fluid and blood;
  • Blood tests to identify biomarkers in blood, including proteins, lipids and markers of inflammation;
  • Blood tests to collect DNA for genome-wide association studies that identify the genetic factors that may confer risk, or protect against, developing Alzheimer’s;
  • Evaluations of medical conditions and cognitive and memory tests to determine levels of function and monitor any changes;
  • For the first time in people with Down syndrome, PET brain scans that detect levels of tau, the twisted knots of protein within brain cells that are a hallmark Alzheimer’s disease.

Aside from earlier onset, Alzheimer’s in people with Down syndrome is similar to Alzheimer’s in others. The first symptom may be memory loss, although people with Down syndrome initially tend to show behavior changes and problems with walking.

“Over the past 30 years, the average lifespan of people with Down syndrome has doubled to 60 years—a  bittersweet achievement when faced with the possibility of developing Alzheimer’s,” said Melissa Parisi, M.D., Ph.D., chief of the NICHD Intellectual and Developmental Disabilities Branch, which leads NIH’s Down syndrome research. “There is much to learn about Alzheimer’s in Down syndrome, and we’re hopeful that these new projects will provide some answers. One mystery we hope to solve is whether or not the disease progresses at a faster rate in this group.”

Parisi noted that research into Alzheimer’s in Down syndrome is a key focus of the National Plan to Address Alzheimer’s Disease(link is external), which calls for improved care for specific populations that are unequally burdened by the disease, including people with Down syndrome, and for increased research that may lead to possible Alzheimer’s therapies.

Benjamin Handen, Ph.D., Department of Psychiatry, University of Pittsburgh, heads a team that involves investigators and data from: Banner Alzheimer’s Institute, Phoenix; Cambridge University, England; Alzheimer’s Disease Cooperative Study, San Diego; Laboratory of Neuro Imaging, University of Southern California, Los Angeles. Nicole Schupf, Ph.D., Columbia University Medical Center, New York City, leads a team involving investigators at: University of California, Irvine; Kennedy Krieger Institute/Johns Hopkins University, Baltimore; Massachusetts General Hospital/Harvard University, Boston; and the University of North Texas Health Sciences Center, Fort Worth.

Learn more about this topic at https://www.nia.nih.gov/alzheimers/publication/alzheimers-disease-people-down-syndrome.

About the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD): The NICHD sponsors research on development, before and after birth; maternal, child, and family health; reproductive biology and population issues; and medical rehabilitation. For more information, visit the Institute’s website at http://www.nichd.nih.gov.

About the National Institute on Aging: The NIA leads the federal government effort conducting and supporting research on aging and the health and well-being of older people. It provides information on age-related cognitive change and neurodegenerative disease specifically at its Alzheimer’s Disease Education and Referral (ADEAR) Center at www.nia.nih.gov/alzheimers.

About the National Institutes of Health (NIH): NIH, the nation’s medical research agency, includes 27 Institutes and Centers and is a component of the U.S. Department of Health and Human Services. NIH is the primary federal agency conducting and supporting basic, clinical, and translational medical research, and is investigating the causes, treatments, and cures for both common and rare diseases. For more information about NIH and its programs, visit www.nih.gov.

 

 

NATIONAL PLAN TO ADDRESS ALZHEIMER’S DISEASE: 2015 UPDATE

pdf-document/national-plan-address-alzheimer%E2%80%99s-disease-2015-update (58 PDF pages)

Introduction

Vision Statement

National Alzheimer’s Project Act

Alzheimer’s Disease and Related Dementias

The Challenges

Framework and Guiding Principles

Goals as Building Blocks for Transformation

2015 Update

 

The Connection between Down Syndrome and Alzheimer’s Disease

Many, but not all, people with Down syndrome develop Alzheimer’s disease when they get older. Alzheimer’s is an irreversible, progressive brain disorder that slowly destroys memory and thinking skills and, eventually, the ability to carry out simple tasks.

Alzheimer’s disease is the most common cause of dementia among older adults. Dementia is the loss of cognitive functioning—thinking, remembering, and reasoning—and behavioral abilities to such an extent that it interferes with a person’s daily life and activities.

People with Down syndrome are born with an extra copy of chromosome 21, which carries the APP gene. This gene produces a specific protein called amyloid precursor protein (APP). Too much APP protein leads to a buildup of protein clumps called beta-amyloid plaques in the brain. By age 40, almost all people with Down syndrome have these plaques, along with other protein deposits, called tau tangles, which cause problems with how brain cells function and increase the risk of developing Alzheimer’s dementia.

However, not all people with these brain plaques will develop the symptoms of Alzheimer’s. Estimates suggest that 50 percent or more of people with Down syndrome will develop dementia due to Alzheimer’s disease as they age into their 70s.

Alzheimer’s Disease Symptoms

Many people with Down syndrome begin to show symptoms of Alzheimer’s disease in their 50s or 60s. But, like in all people with Alzheimer’s, changes in the brain that lead to these symptoms are thought to begin at least 10 years earlier. These brain changes include the buildup of plaques and tangles, the loss of connections between nerve cells, the death of nerve cells, and the shrinking of brain tissue (called atrophy).

The risk for Alzheimer’s disease increases with age, so it’s important to watch for certain changes in behavior, such as:

  • increased confusion
  • short-term memory problems (for example, asking the same questions over and over)
  • reduction in or loss of ability to do everyday activities

Other possible symptoms of Alzheimer’s dementia are:

  • seizures that begin in adulthood
  • problems with coordination and walking
  • reduced ability to pay attention
  • behavior and personality changes, such as wandering and being less social
  • decreased fine motor control
  • difficulty finding one’s way around familiar areas

Currently, Alzheimer’s disease has no cure, and no medications have been approved to treat Alzheimer’s in people with Down syndrome.

Down Syndrome and Alzheimer’s Disease Research

Alzheimer’s can last several years, and symptoms usually get worse over time.  Scientists are working hard to understand why some people with Down syndrome develop dementia while others do not. They want to know how Alzheimer’s disease begins and progresses, so they can develop drugs or other treatments that can stop, delay, or even prevent the disease process.

Research in this area includes:

  • Basic studies to improve our understanding of the genetic and biological causes of brain abnormalities that lead to Alzheimer’s
  • Observational research to measure cognitive changes in people over time
  • Studies of biomarkers (biological signs of disease), brain scans, and other tests that may help diagnose Alzheimer’s—even before symptoms appear—and show brain changes as people with Down syndrome age
  • Clinical trials to test treatments for dementia in adults with Down syndrome. Clinical trials are best the way to find out if a treatment is safe and effective in people.

 

Alzheimers Disease Neuroimaging Initiative (ADNI)

A public-private partnership, the purpose of ADNI is to develop a multisite, longitudinal, prospective, naturalistic study of normal cognitive aging, mild cognitive impairment (MCI), and early Alzheimer’s disease as a public domain research resource to facilitate the scientific evaluation of neuroimaging and other biomarkers for the onset and progression of MCI and Alzheimer’s disease.

Dr. Laurie Ryan of the NIA gives a brief overview of ADNI in this video:

https://youtu.be/0rBVe0Fwnik

Dr. Thomas Obisesan of Howard University, an ADNI study participant, and a study companion describe ADNI and what it’s like to be involved in the study

https://youtu.be/rK1yWvvHHl8

Learn more about this topic at https://www.nia.nih.gov/alzheimers/publication/alzheimers-disease-people-down-syndrome.

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Brain Science

Larry H Bernstein, MD, FCAP, Curator

LPBI

 

A Protein Atlas of the Brain

http://www.biosciencetechnology.com/news/2015/11/protein-atlas-brain

What looks like an island is actually a schematic representation of a mouse brain. Researchers have now analyzed the mouse brain proteome and summarized the data in an atlas. (Image:  MPI of Biochemistry/ K. Scharma )

http://www.biosciencetechnology.com/sites/biosciencetechnology.com/files/bt1511_mpi_proteome.jpg

What looks like an island is actually a schematic representation of a mouse brain. Researchers have now analyzed the mouse brain proteome and summarized the data in an atlas. (Image: MPI of Biochemistry/ K. Scharma )

 

Just as in the Middle Ages when there were still many uncharted areas on Earth, researchers today are aware that there is still a great deal to learn about cells in our microcosm. But instead of sextants and compasses, researchers nowadays use modern methods such as mass spectrometry to look into the world of protein molecules. Neuroscientists are focussed particularly on resolving brain complexity with its billions of specialized cells. To understand the brain’s functions, scientists from the Max Planck Institutes of Biochemistry in Martinsried and Experimental Medicine in Göttingen have for the first time quantified the entire set of proteins ‒ the proteome ‒ in the adult mouse brain. The information about which proteins and how many of them are found in the various cell types and regions has been summarized in a protein atlas.

The brain consists of hundreds of billions of interconnected cells which communicate with one another. Different cell types specialize in different functions. Nerve cells transmit and process stimuli from outside; distinct glial cells supply them with nutrients, regulate the flow of blood in the brain, help in isolating nerve fibres and perform tasks in the immune system.

Cells are comprised of proteins which are the functional building blocks. They act as small molecular machines and give the cell its structure. The information for synthesis of protein molecules is encoded in DNA and RNA; biomolecules which have been extensively examined in the brain. “Up to now, however, it was not known which and how many proteins are produced in the different, highly specialized cells or even how the numbers of proteins in the individual regions differ”, explains neuroscientist Mikael Simons. “To examine this, we needed modern measuring and analysis methods in order to be able to record and evaluate these enormous numbers of proteins.” Together with protein research specialists, a team headed by Matthias Mann in Martinsried, the scientists further developed the mass spectrometry technology for in-depth profiling of brain proteins in a rapid, reproducible and a quantitative fashion.

They were able to show that there are around 13,000 different proteins in the adult mouse brain. The quantity of proteins in the different cell types and brain regions, and how they differ from one another can now be found in the recently established protein atlas at http://www.mousebrainproteome.com. The protein data presented there from five different cell types and ten regions in the mouse brain constitute the most comprehensive collection to date.

This deep proteome investigation should serve as a rich resource for analyses of brain development and function. “Surprisingly, only 10 per cent of all proteins are cell type-specific”, explains Kirti Sharma, lead author of the study. “These cell-specific proteins are mostly found on the surface of the cell.” The large majority – 90 per cent of all proteins – are found in all cell types. As in a satellite view of previously uncharted landscapes, the researchers have created a protein atlas based on the most comprehensive data collection that should help in the development of new treatments for alleviating brain diseases.

Source: Max Planck Institute

 

 

New Computational Strategy Finds Brain Tumor-shrinking Molecules

http://www.biosciencetechnology.com/news/2015/11/new-computational-strategy-finds-brain-tumor-shrinking-molecules

 

These are MRI renderings of mouse brain tumors. Tumors treated with SKOG102 (lower panels) shrank by about half compared to tumors treated with a control (upper panels). (Credit: UC San Diego Health)

http://www.biosciencetechnology.com/sites/biosciencetechnology.com/files/bt1511_ucsandiego_braintumor.jpg

These are MRI renderings of mouse brain tumors. Tumors treated with SKOG102 (lower panels) shrank by about half compared to tumors treated with a control (upper panels). (Credit: UC San Diego Health)

 

Patients with glioblastoma, a type of malignant brain tumor, usually survive fewer than 15 months following diagnosis. Since there are no effective treatments for the deadly disease, University of California, San Diego researchers developed a new computational strategy to search for molecules that could be developed into glioblastoma drugs. In mouse models of human glioblastoma, one molecule they found shrank the average tumor size by half. The study is published October 30 byOncotarget.

The newly discovered molecule works against glioblastoma by wedging itself in the temporary interface between two proteins whose binding is essential for the tumor’s survival and growth. This study is the first to demonstrate successful inhibition of this type of protein, known as a transcription factor.

“Most drugs target stable pockets within proteins, so when we started out, people thought it would be impossible to inhibit the transient interface between two transcription factors,” said first author Igor Tsigelny, Ph.D., research scientist at UC San Diego Moores Cancer Center, as well as the San Diego Supercomputer Center and Department of Neurosciences at UC San Diego. “But we addressed this challenge and created a new strategy for drug design — one that we expect many other researchers will immediately begin implementing in the development of drugs that target similar proteins, for the treatment of a variety of diseases.”

Transcription factors control which genes are turned “on” or “off” at any given time. For most people, transcription factors labor ceaselessly in a highly orchestrated system. In glioblastoma, one misfiring transcription factor called OLIG2 keeps cell growth and survival genes “on” when they shouldn’t be, leading to quick-growing tumors.

In order to work, transcription factors must buddy up, with two binding to each other and to DNA at same time. If any of these associations are disrupted, the transcription factor is inhibited.

In this study, Tsigelny and team aimed to disrupt the OLIG2 buddy system as a potential treatment for glioblastoma. Based on the known structure of related transcription factors, study co-author Valentina Kouznetsova, Ph.D., associate project scientist at UC San Diego, developed a computational strategy to search databases of 3D molecular structures for those small molecules that might engage the hotspot between two OLIG2 transcription factors. The team used the Molecular Operation Environment (MOE) program produced by the Chemical Computing Group in Montreal, Canada and high-performance workstations at the San Diego Supercomputer Center to run the search.

With this approach, the researchers identified a few molecules that would likely fit the OLIG2 interaction. They then tested the molecules for their ability to kill glioblastoma tumors in the Moores Cancer Center lab of the study’s senior author, Santosh Kesari, M.D., Ph.D.. The most effective of these candidate drug molecules, called SKOG102, shrank human glioblastoma tumors grown in mouse models by an average of 50 percent.

“While the initial pre-clinical findings are promising,” Kesari cautioned, “it will be several years before a potential glioblastoma therapy can be tested in humans. SKOG102 must first undergo detailed pharmacodynamic, biophysical and mechanistic studies in order to better understand its efficacy and possible toxicity.”

To this end, SKOG102 has been licensed to Curtana Pharmaceuticals, which is currently developing the inhibitor for clinical applications. Kesari is a co-founder, has an equity interest in and is chair of the scientific advisory board for Curtana Pharmaceuticals. Co-authors Rajesh Mukthavaram, Ph.D., and Wolfgang Wrasidlo, Ph.D., also own stock in Curtana Pharmaceuticals.

his research was funded, in part, by the National Institutes of Health, Voices Against Brain Cancer Foundation, Christopher and Bronwen Gleeson Family Trust and American Brain Tumor Association Drug Discovery Grant.

Source: University of California San Diego

Musical Rhythms in the Brain

http://www.biosciencetechnology.com/news/2015/10/musical-rhythms-brain

 

Researchers at Max Planck Institute for Empirical Aesthetics in Frankfurt and of New York University have identified how brain rhythms are used to process music, a finding that also contributes to a better understanding of the auditory system. Furthermore, the study suggests that musical training can enhance the functional role of brain rhythms.

The paper, which appears in the journal Proceedings of the National Academy of Sciences, points to a newfound role the brain’s cortical oscillations play in the detection of musical sequences. The term “cortical oscillations” refers to the rhythmic electrical activity generated spontaneously and in response to stimuli by neural tissue in the central nervous system. The importance of brain oscillations in sensory-cognitive processes has become increasingly evident.

“We’ve isolated the rhythms in the brain that match rhythms in music,” explains Keith Doelling, lead author. “Specifically, our findings show that the presence of these rhythms enhances our perception of music and of pitch changes.”

 Headbanging is most common in the rock, punk and heavy metal music genres. Scientist have now identified how rhythms inside the human brain are used to process music. (Image: Wikimedia / Małgorzata Miłaszewska)

http://www.biosciencetechnology.com/sites/biosciencetechnology.com/files/bt1510_maxplanck_music.jpg

Headbanging is most common in the rock, punk and heavy metal music genres. Scientist have now identified how rhythms inside the human brain are used to process music. (Image: Wikimedia / Małgorzata Miłaszewska)

 

Previous research has shown that brain rhythms very precisely synchronize with speech, enabling us to parse continuous streams of speech — in other words, how we can isolate syllables, words, and phrases from speech, which is not, when we hear it, marked by spaces or punctuation.

However, it has not been clear what role such cortical brain rhythms, or oscillations, play in processing other types of complex sounds, such as music.

To address these questions, the researchers conducted three experiments using magnetoencephalography (MEG), which allows the tiny magnetic fields generated by brain activity to be measured. The study’s subjects were asked to detect short pitch distortions in 13-second clips of classical piano music (by Bach, Beethoven, Brahms) that varied in tempo — from half a note to eight notes per second. The study’s authors divided the subjects into musicians (those with at least six years of musical training and who were currently practicing music) and non-musicians (those with two or fewer years of musical training and who were no longer involved in it).

Not surprisingly, the study found that musicians have more potent oscillatory mechanisms than non-musicians do. “What this shows is we can be trained, in effect, to make more efficient use of our auditory-detection systems,” observes study co-author David Poeppel, director of the Max Planck Institute for Empirical Aesthetics. “Musicians, through their experience, are simply better at this type of processing.”

For music that is faster than one note per second, both musicians and non-musicians showed cortical oscillations that synchronized with the note rate of the clips. The researchers therefore conclude that these oscillations were effectively employed by everyone to process the sounds they heard, although musicians’ brains synchronized more to the musical rhythms. Only musicians, however, showed oscillations that synchronized with unusually slow clips. This difference, the researchers say, may suggest that non-musicians are less able to process the music as a continuous melody rather than as individual notes. Moreover, musicians are able to detect pitch distortions much more accurately — as evidenced by corresponding cortical oscillations.

Thus, brain rhythms appear to play a role in parsing and grouping sound streams into ‘chunks’ that are then analyzed as speech or music, the scientists add.

Source: Max Planck Institute

 

 

New Three-Minute Test Detects Lewy Body Disease

http://www.biosciencetechnology.com/news/2015/10/new-three-minute-test-detects-lewy-body-disease

Lewy Body disease is the second most common type of progressive dementia, according to the Mayo Clinic, and affects approximately 1.3 million Americans.

The Lewy Body Dementia Association says the disease is widely accepted to be highly underdiagnosed and is the most frequently misdiagnosed form of dementia.

The new test, called the “Lewy Body Composite Risk Score” (LBCRS), developed by James E. Galvin, M.D., M.P.H., professor of clinical biomedical science at FAU, is a simple, one page-survey, that includes yes or no questions for a clinician to complete.  The structured questions look at six non-motor features that are present in patients with LBD, but are much less common in other forms of dementia.  The tool helps clinician assess whether a patient has rest tremor, postural instability, rigidity, or bradykensia, without having to grade each extremity.

Bioscience Bulletin: Potential Alzheimer’s Test, and Stress Linked to Stroke

http://www.biosciencetechnology.com/news/2015/10/bioscience-bulletin-potential-alzheimers-test-cancer-drug-overestimated-and-stress-linked-stroke

N.J. Researchers Closing in on Alzheimer’s, Parkinson’s Tests
Researchers from the Rowan University School of Osteopathic Medicine, have developed a test that could detect Alzheimer’s before symptoms start by identifying a series of obscure antibodies. Scientists narrowed down the auto-antibodies he was looking at from a sample of nearly 10,000 to just 10.

Role Found for Critical Gene in 95% of ALS
Cynthia Fox interviewed experts about a recent Science study that offered new insight surrounding a protein called TDP-43 in relation to amyotrophic lateral sclerosis.  The study found that in 95 percent of ALS cases the protein leaves its home – the nucleus of motor neuron cells – resulting in the creation of improper “cryptic” exons.

Like a bad teenager, in 95 percent of all amyotrophic lateral sclerosis (ALS) cases, a protein called TDP-43 leaves its home— the nucleus of motor neuron cells—to congregate, in suspect fashion, in the cytoplasm.

In a study published in Science this summer, the Johns Hopkins University (JHU) team of pathologist Phillip Wong, Ph.D., offered new insight into this molecular rebellion. It confirmed a function of normal TDP-43 in the nucleus: orchestrating proper RNA splicing and exon formation. It confirmed what lack of nuclear TDP-43 does: creates improper “cryptic” exons. And it identified proteins that mitigate effects of nuclear TDP-43 loss: potential drug leads.

“The recently published work in Science very clearly demonstrates that in the absence of TDP-43, RNA is misspliced, and in many cases targeted for degradation,” University of Michigan neurologist Sami Barmada, M.D., Ph.D., told Bioscience Technology. Barmada was uninvolved with the research. “A very real consequence of such dysfunctional RNA splicing and degradation is an inability to maintain cell health, ultimately resulting in neuron loss. The authors assembled an intriguing story that tells us quite a bit about how TDP-43 functions, and what happens to those functions in diseases such as ALS, and fronto-temporal dementia (FTD). If the mechanism identified in this manuscript does indeed underlie toxicity due to TDP-43 mislocalization, then it may very well contribute to neuron loss in the vast majority of ALS.”

High Stress Jobs May be Linked to Increased Stroke Risk
The final story in our round up this week takes a look at a new Neurology study, which found there may be a link between high stress jobs and an increased risk for stroke. After analyzing six studies, comprised of a total of 138,782 participants, researchers concluded that people with high stress jobs (such as waitresses and nursing aides) were 58 percent more likely to have an ischemic stroke than those in low stress jobs (such as natural scientists and architects).

New Scanner to Help Uncover Causes of Dementia

http://www.biosciencetechnology.com/news/2015/11/new-scanner-help-uncover-causes-dementia

The funding from the Medical Research Council will allow the SIGNA PET/MR scanner, made by GE Healthcare, to be installed in Central Manchester University Hospitals NHS Foundation Trust.  Currently there are only two of these scanners in the UK, but following the Manchester funding and money to other university centers in the UK, this number will increase to seven, from a number of manufacturers.

The new scanner will help scientists and clinicians understand the causes and progression of dementia, and provide ways to test the effects of new treatments.  Molecular changes in the brain are believed to be responsible for dementia and the scanners have the potential to link these with the brain changes that they cause – leading to new understanding and new treatments.

Professor Nigel Hooper is the University’s Director for Dementia Research.  He said: “Dementia is a condition that is poorly understood and difficult to treat effectively.  It’s going to become more of a problem in the coming decades, so our research response needs to pick up as well.

“This scanner and the wider network will give us that ability to understand dementia better and to develop more treatments.”

The scanner is expected to be operational from July 2016 and work is currently underway to refurbish a shelled space adjacent to the Nuclear Medicine Centre to house the scanner suite which will have three treatment rooms, a research office and a radiopharmacy room. CMFT was chosen as the ideal location for the scanner due to its central Manchester location and close proximity to the main University campus, the co-location of the suite adjacent to the clinical PET/CT scanner at CMFT and the opportunity to take research into clinical practice.

Christine Tonge, the Director of Medical Physics at Central Manchester said: “We are excited by this opportunity to contribute to this important area of research. This scanner will put Manchester at the forefront of dementia research and we look forward to collaborating not only with our colleagues from the University, but also  from other hospitals in Greater Manchester and beyond.”

The scanner is being funded as part of the Dementias Platform UK – a multi-million pound public-private partnership, developed by the Medical Research Council, to accelerate progress in dementias research. DPUK’s aims are early detection, improved treatment and, ultimately, prevention of dementias. It is the world’s largest study group for use in dementias research, pulling together two million well-characterized participants from over 30 national population studies.

The Manchester scanner will be supervised by Professor Alan Jackson, who is the director of the University’s Wolfson Molecular Imaging Centre, which hosts two PET scanners and one MR scanner and produces radiotracers for use in PET scanning.  He said: “Manchester now has a range of scanning facilities which mean that clinicians and scientists can produce high quality research across a range of conditions.

“With the growing urgency of developing treatments for dementia, this new equipment is vital in addressing a major growing health concern.

“Most importantly, being linked with the other four universities which are also purchasing PET-MR scanners will mean Manchester has the ability to become involved in multi-centre trials and research grants and contracts – increasing the effectiveness of the UK’s research in this area.”

Source: University of Manchester

 

Trends Mol Med. 2014 Feb;20(2):66-71. doi: 10.1016/j.molmed.2013.11.003. Epub 2013 Dec 16.
TDP-43-mediated neurodegeneration: towards a loss-of-function hypothesis?
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are clinically distinct fatal neurodegenerative disorders. Increasing molecular evidence indicates that both disorders are linked in a continuous spectrum (ALS-FTD spectrum). Neuronal cytoplasmic inclusions consisting of the nuclear TAR DNA-binding protein 43 (TDP-43) are found in the large majority of patients in the ALS-FTD spectrum and dominant mutations in the TDP-43 gene cause ALS. A major unresolved question is whether TDP-43-mediated neuronal loss is caused by toxic gain of function of cytoplasmic aggregates, or by a loss of its normal function in the nucleus. Here we argue that based on recent genetic studies in worms, flies, fish, and rodents, loss of function of TDP-43, rather than toxic aggregates, is the key factor in TDP-43-related proteinopathies.
Gains or losses: molecular mechanisms of TDP43-mediated neurodegeneration

Edward B. Lee, Virginia M.-Y. Lee & John Q. Trojanowski

Nature Reviews Neuroscience 13, 38-50 (January 2012) |   http://dx.doi.org:/10.1038/nrn3121

RNA-binding proteins, and in particular TAR DNA-binding protein 43 (TDP43), are central to the pathogenesis of motor neuron diseases and related neurodegenerative disorders. Studies on human tissue have implicated several possible mechanisms of disease and experimental studies are now attempting to determine whether TDP43-mediated neurodegeneration results from a gain or a loss of function of the protein. In addition, the distinct possibility of pleotropic or combined effects — in which gains of toxic properties and losses of normal TDP43 functions act together — needs to be considered.

 

Transposable Elements in TDP-43-Mediated Neurodegenerative Disorders

PLOS

Published: September 5, 2012   DOI: http://dx.doi.org:/10.1371/journal.pone.0044099

Elevated expression of specific transposable elements (TEs) has been observed in several neurodegenerative disorders. TEs also can be active during normal neurogenesis. By mining a series of deep sequencing datasets of protein-RNA interactions and of gene expression profiles, we uncovered extensive binding of TE transcripts to TDP-43, an RNA-binding protein central to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Second, we find that association between TDP-43 and many of its TE targets is reduced in FTLD patients. Third, we discovered that a large fraction of the TEs to which TDP-43 binds become de-repressed in mouse TDP-43 disease models. We propose the hypothesis that TE mis-regulation contributes to TDP-43 related neurodegenerative diseases.

Citation: Li W, Jin Y, Prazak L, Hammell M, Dubnau J (2012) Transposable Elements in TDP-43-Mediated Neurodegenerative Disorders. PLoS ONE 7(9): e44099. doi:10.1371/journal.pone.0044099

Editor: Koichi M. Iijima, Thomas Jefferson University, United States of America

 

Accumulation of TAR DNA-binding protein 43 (TDP-43) containing cytoplasmic inclusions is a shared pathological hallmark in a broad spectrum of neurodegenerative disorders, including ALS, FTLD and Alzheimer’s disease [1]. Mutations in this multifunctional RNA binding protein are also known to underlie some familial and sporadic cases of ALS [1]. Despite considerable progress, the mechanisms that link TDP-43 to neurodegeneration still are unclear. We conducted a meta-analysis of TDP-43 protein:RNA target binding datasets and of mRNA expression datasets. All previous analyses of such data focused on sequence reads that uniquely map to the reference genome, thereby excluding transcripts derived from transposable elements (TEs). In contrast, we included sequences that map to multiple locations and examined reads that align to TEs. Our analyses lead to the striking hypothesis that TE over-expression may contribute to TDP-43 mediated neurodegeneration.

Transposable elements (TEs) are highly abundant mobile genetic elements that constitute a large fraction of most eukaryotic genomes. Retrotransposons, which copy themselves through an RNA intermediate, represent approximately 40% of the human genome [2], [3]. Although the majority of TE copies are nonfunctional, a subset have retained the ability to mobilize and even the immobile copies can be expressed [4]. Because of their potential to copy themselves and insert into new genomic locations as well as to generate enormous levels of expression, transposable elements present a massive endogenous reservoir of genomic instability and cellular toxicity [3]. The impacts of these parasitic genetic elements normally are stifled by potent cellular mechanisms involving small interfering RNAs that act via the RNA induced silencing complex (RISC) to inhibit transposon expression ([5] for review). Although most investigations have naturally focused on the germline, where new insertions are heritable and thus favored by transposon evolution, somatic tissues also have an active transposon silencing mechanism whose functional significance is less understood. An emerging literature has established that certain TEs are normally active in brain [6], [7], [8], [9] and elevated expression of some LINE, SINE (which are non-LTR retrotransposons) and LTR elements have been correlated with several neurodegenerative disorders [10], [11], [12], [13], [14], [15], [16]. We therefore investigated whether the RNA targets of TDP-43 include transposon-derived transcripts.

Several recent studies used deep sequencing to profile the RNA targets that co-purify with immunoprecipitated mouse, rat or human TDP-43 and also to profile gene expression changes in mouse after knockdown or over-expression of TDP-43 [17], [18], [19], [20]. In each case, however, these studies analyzed annotated protein coding sequences and excluded TE-derived transcripts and other repetitive elements due to the difficulties inherent in working with ambiguously mapped reads from short read technologies [e.g. [21]]. Despite efforts to develop new algorithms for analyzing multiple alignments of short reads [22], these algorithms have not been applied systematically for analyzing TE-derived transcripts in any neurodegenerative disease. Because each of the above mentioned TDP-43 related studies provided public access to their raw data, we were able to use this resource to search for TDP-43 targets and for transcript mis-expression when we included sequence reads that map to multiple genomic locations, the majority of which are TE derived transcripts in these datasets. Our meta-analysis supports three main conclusions. First, TDP-43 broadly targets TE-derived transcripts, including many SINE, LINE and LTR classes as well as some DNA elements. Second, the association between TDP-43 and TE-derived RNA targets is reduced in FTLD patients relative to healthy subjects, consistent with the idea that loss of TE control might be part of the disease pathology. Third, we observe broad over-expression of TE derived transcripts in each of two different mouse models with TDP-43 dysfunction. Finally there is a striking overlap between the TE transcripts identified as targets and those that are over-expressed with TDP-43 misexpression.

 

Results

We first re-analyzed raw data from the rat TDP-43 RNA immunoprecipitation sequencing (RIP-seq) dataset [17] and the mouse and human TDP-43 in vivo crosslinking-immunoprecipitation sequencing (CLIP-seq) datasets [18], [19]. We tested three different analysis methods to examine effects on TEs (Fig. 1A–C; Methods and Figs. S1 and Tables S1, S2, S3). Because reads could potentially map to many regions, we first used an analysis in which each location was weighted based on the number of alignments (Figs. 1A,B) see methods). This analysis method (MULTI), which included both unique and multi mapped reads, assigns an enrichment level for each element, but does not distinguish contributions of individual instances of each element. Although this method can potentially include effects from TEs that are difficult to map with short read sequence, a disadvantage is that it does not distinguish which instances of a given TE are detected. In addition, because many TE copies are present within introns of genes, the MULTI method does not distinguish whether the TE sequences are co-expressed with genes or expressed from TEs per se. To address these issues, and to test the robustness of our observations, we also tested two additional mapping methods for the rat and human datasets (Figs. 1C and S1E,F; Methods). First, we examined only the subset of reads that map uniquely to the genome (UNIQ). This method does bias the results to the fraction of TEs that have diverged enough to have unique sequences, but provides confidence that signal derives from unique chromosomal locations. As a third mapping strategy (UNIQ+SameEle), we examined the effects of including both uniquely mapped sequences and those that map to multiple locations so long as they map to the same element (weighted for their contribution to each instance as above – see Methods).

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Figure 1. TDP-43 binds broadly to transposable element (TE)-derived transcripts.

Magnitude (log2-fold) of enrichments (up) or depletions (down) are shown (A, rat; B, mouse) for significantly bound repeat elements grouped by class. MULTI method (see text) was used for A and B. (C) The majority of rat TE targets identified with MULTI also are identified (Left Panel, Rat) when analysis is restricted to reads that map uniquely (UNIQ) or when both uniquely mapped and multi-mapped reads that map to the same TE were included (UNIQ+SameEle). These conclusions also hold for TE targets whose binding is reduced in FTLD samples from human tissue relative to healthy controls (Left panel, Human). Most rat TE targets and differentially bound human TE targets identified from uniquely mapped reads are intergenic (Right panel). (D) For TDP-43, peaks (UNIQ+SameEle) over TE targets are tall and sharp with mean peak height of 158 counts/peak. In contrast, peak heights are lower for FUS (mean peak height of 17).

doi:10.1371/journal.pone.0044099.g001

With all three mapping strategies we find a dramatic enrichment of sequences that derive from each major class of TE (Figs. 1A–C; S1; Table S3). With the MULTI method, we find 271 significantly enriched or depleted (most were enriched) repeat element sub-families in the rat TDP-43-IP samples versus control (Fig. 1A), of which 245 correspond to TEs. In the mouse dataset (Fig. 1B), MULTI detects significant enrichment of 352 repeat element sub-families of which 334 correspond to TEs (Table S3). These comprise all major classes of TEs, including LINE, SINE, LTR and some DNA elements [3]. For instance, 85 out of the 122 known mouse LINE elements and 6 out of the 7 known rat LINE elements are identified as TDP-43 targets. Similarly 26 out of 41 mouse SINE elements and 36 out of 37 rat SINE elements also were detected as TDP-43 targets. One caveat to the mouse clip-seq analysis was the lack of a control IP to use in estimating background counts for this single dataset, which could potentially lead to a larger false positive rate in the detected peaks (see Methods); however, the similarity in the results obtained for this dataset as compared to the well-controlled studies for rat (Fig. 1A) and human datasets (see below) argues for the inclusion of this dataset despite its caveats.

Overall, we detect the most extensive binding to TEs with the MULTI method, and these findings are not an artifact of the way we assigned weights with the MULTI method because even with the more restricted UNIQ analysis, we identify ∼80% of the rat elements that are differentially enriched when all mappable reads are included (Figs. 1C, S1F). Moreover, among the uniquely mapped subset of TE instances that we identify as TDP-43 targets, greater than 80% map to intergenic regions rather than to elements contained within genes (Fig. 1C). When we include both unique mappers and multi mappers from the same element (UNIQ+SameEle), we detect enrichment for 95% of the TE sub-families that were identified as TDP-43 targets with the MULTI method (Figs. 1C, S1F). The concordant results from these three different mapping strategies provide confidence that identification of TE derived transcripts as TDP-43 targets is a robust effect that is detected with a variety of methods for dealing with multi-copy elements.

As a test of the biological specificity of our finding that TDP-43 selectively binds to TE derived transcripts, we applied the UNIQ mapping method to a CLIP-seq dataset for an unrelated RNA binding protein. For this purpose we chose fused in sarcoma (FUS), which like TDP-43, is an hnRNP RNA binding protein that plays diverse roles in RNA biology, including splicing [23]. FUS is a relevant control for specificity because like TDP-43, it is implicated in neurodegenerative disorders including ALS [24]. The result with FUS is in stark contrast with TDP-43 (Fig. 1D). For TDP-43, peaks (defined within a 500 bp window) that map to TEs are relatively large, with a mean peak height of 158 counts. In contrast, with FUS we only see small peaks over TEs with a height of just a few counts (mean peak height of 17; Fig. 1D for distribution). Peaks that map over RefGene annotations, on the other hand, are similarly distributed for both FUS and TDP-43 (Mean height of 32 and 68 respectively, Fig. S1H). The distributions of mean peak heights (see histogram, Fig. 1D) shows a clear separation between TDP-43 peaks and those obtained with FUS and this separation between peak heights is statistically significant (Wilcoxon rank sum p-value<2.2e−16). Thus our findings show specificity for TDP-43 and are not a byproduct of inherent biases in library construction or analysis.

Because TDP-43 has a known binding motif among its mRNA targets, we used MEME ([25]and see Methods) to identify enriched motifs among both the RefGene and repetitive targets. We identify a UGUGU pentamer motif that is equivalently enriched in uniquely mapped and repetitive targets (Fig. S1C; Methods). This motif is consistent with the binding specificity of TDP-43 that has previously been observed for uniquely mapped sequences [17], [18], [19], [20]. Thus TDP-43 binds TE derived transcripts via a similar sequence motif as identified for RefGene targets.

Because the human dataset [18] includes samples from healthy and FTLD patients (which exhibit TDP-43 positive cytoplasmic inclusions), it also provided an opportunity to identify differences in the TDP-43 targets between FTLD and healthy controls. As in rat and mouse, we observe in human samples a dramatic and significant enrichment in target sequences that derive from many classes of TEs. As with the mouse and rat data, the distribution of peak heights for TE and RefGene targets of TDP-43 are similar (Fig. S1I), indicating that the targeting of TE transcripts is as robust as it is for RefGene targets. More striking, however, is the comparison between healthy subjects and FTLD patients. When we examine the relative enrichment for each repeat element within healthy vs. FTLD samples, we detect a dramatic difference in binding to TE derived RNAs (Fig. 1E–H). Overall, the association between TDP-43 and TE transcripts is significantly reduced in FTLD patients, which leads to a relative enrichment of 38 repeat elements in healthy versus FTLD, 28 of which correspond to transcripts derived from TEs (Fig. 2 and Table S3; See Methods for statistical analyses). We see reduced binding of TDP-43 to transcripts from all major classes of TE including SINE, LINE, LTR and a few DNA elements. Here too, we observe that the majority of the TE targets whose binding to TDP-43 was reduced in FTLD are consistently identified with all three methods (Fig. 1C). Most of the TE targets that show reduced binding to TDP-43 in FTLD samples are intergenic rather than contained within genes (Fig. 1C). Example peaks are shown for one RefGene control (Fig. 1F) as well as two differentially targeted TEs (Figs. 1G,H).

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Figure 2. TDP-43 binding to TEs is selectively lost in FTLD patients.

(A) In the human CLIP-seq data from FTLD versus healthy control, 38 repeat elements showed significant (p-value< = 1e-5 and fold changes> = 2) differential binding. Log2 fold binding differences are shown for significantly enriched/depleted elements. (B,C,D) Peaks are shown in genome browser for one RefGene control (B) and two differentially targeted TEs (C,D) in Healthy (top) versus FTLD (bottom). (E) Enrichment for the UGUGU motif relative to its prevalence in the genome is shown across a 51-nt window surrounding binding sites (−25 nt, 25 nt). Healthy samples (Blue) show similar enrichment for the UGUGU pentamer motif among RefGene (solid) and repeat (dashed) sequences (RefGene/repeat motif enrichment ratio ≈1.3). In contrast, motif enrichment in FTLD samples (Red) is significantly reduced among repeat (dashed) annotations relative to RefGene (solid; p-value< = 0.01; RefGene/repeat motif enrichment ratio ≈2.0).

doi:10.1371/journal.pone.0044099.g002

This reduced binding in FTLD patients of TDP-43 to TE-derived transcripts also is apparent when we examine over-all enrichment for the UGUGU pentamer motif (Figs. 2E and S1) relative to the genome. In the rat and mouse samples as well as in the dataset from healthy human brain samples, we observe equivalent enrichment of UGUGU binding motifs among uniquely mapped (RefGene) versus repetitively mapped (repeat) TDP-43 targets (RefGene/repeat enrichment ratio near 1.0; Fig. S1D; see Methods). In the FTLD-TDP-43-CLIP samples, we also see enrichment for the UGUGU motif among RefGene targets that is equivalent to that seen in healthy subjects (Fig. 2E), but the level of enrichment for this UGUGU motif is significantly lower among the sequences that map to repeat elements. In the FTLD samples, the RefGene/repeat enrichment ratio is increased to 2.0 (Fig. 2E; p-value< = 0.01, p-values were assigned with 100 iterations on randomly chosen sets containing 50% of original data; see Methods). In other words, FTLD samples exhibit a selective reduction of binding to TE transcripts and also exhibit reduced UGUGU motif enrichment among the remaining repetitive sequences that still co-purify with TDP-43. This difference in motif enrichment between FTLD and control samples is only manifested among repeat annotations.

The reduced binding of TE transcripts in FTLD patients suggested that TDP-43 pathology might include a loss of TE regulation. We investigated this possibility in two ways. First, we analyzed the repetitive sequence reads from two different mRNA-seq datasets from mouse models of TDP-43 pathology.

The first mRNA-seq study that we analyzed [20] used over-expression of human TDP-43 in transgenic mice. Overexpression of this aggregation prone protein is associated with toxic TDP-43 pathological effects and is thought to act as a dominant-negative, causing reduction in the normal functions of TDP-43. The second mRNA-seq study [19] used antisense oligonucleotide-mediated depletion of TDP-43 in mouse striatum to test the effects of TDP-43 loss of function. Both studies identified transcripts that are differentially expressed or spliced in response to these TDP-43 manipulations. To ask if the above TDP-43 depletion and over-expression/dominant-negative impacted TE derived transcripts, we again analyzed sequence reads including those that map to multiple locations. We found broad elevations of TE derived transcripts in both the over-expression transgenic mouse model and in the striatal depletion of TDP-43 (Figs. 3A,B). TDP-43 over-expression was associated with elevated expression of 86 repetitive elements (Fig. 3A), whereas TDP-43 depletion results in increased expression levels of 223 repetitive element species (Fig. 3B). In both cases, most of these correspond to LINE, SINE and LTR elements. Overall, the affected TE transcripts are expressed at comparable levels to those of the differentially expressed RefGene transcripts (Fig. S1J), suggesting that these are robust effects on transcripts whose expression levels are not at the limit of detection. More importantly, when TDP-43 function is compromised, we observe a striking degree of concordance between the TE transcripts that are elevated and the ones that we identified as RNA targets of TDP-43 in normal tissue (Red in Fig. 3; See Table S3). Indeed the majority of elevated TE transcripts in both mouse mRNA-seq datasets also were detected as TDP-43 targets in the iCLIP-seq binding dataset (Fig. 3; Table S3). This remarkable concordance between the transcripts that are targeted by TDP-43 and those that are elevated in response to TDP-43 misexpression is unique to the repetitive elements in the genome. In contrast, CLIP targets identified from the RefGene fraction of the transcriptome have little overlap with those that show over-expression when TDP-43 function is compromised suggesting that the coding gene expression increases are largely indirect effects [19]. RefGene transcripts whose expression is reduced show good concordance with direct target identification.

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Figure 3. Concordance between mis-regulated TE transcripts upon TDP-43 manipulation and TDP-43 bound TE transcripts.

(A,B) Over-expression [20] of TDP-43 in transgenic mice and depletion [19] of TDP-43 in mouse striatum each result in elevated expression of many TE derived transcripts. The majority of over-expressed TEs also were detected (Table S3) as binding targets by CLIP-seq (RED). A few showed elevated expression but were not detected as binding targets (BLUE).

doi:10.1371/journal.pone.0044099.g003

Discussion

TDP-43 aggregation and neuropathology plays a fundamental role in a broad spectrum of neurodegenerative disorders [1], [26], [27]. This hnRNP-like RNA binding protein already has been implicated in a remarkable number of cellular functions including repression of HIV-1, alternative splicing, regulation of mRNA stability and microRNA biogenesis [26], [27]. Importantly, a large number of cellular targets of TDP-43 have been characterized, leading to the hypothesis that one key role of this multi-functional protein is to regulate alternative splicing of mRNA targets with a preference for those with large UG rich introns [17], [18], [19], [26], [28]. Our findings support the novel hypothesis that TDP-43 also targets the mobile element derived transcriptome. This association is defective in FTLD patients and the TE transcriptome is broadly over-expressed in mouse models of TDP-43 pathology.

A large fraction of the genetic material of multicellular organisms is made up of mobile elements as well as inactivated TEs. A fraction of these TEs retain the capacity to copy themselves and insert at new genomic locations. During the co-evolution of TEs with their host genomes, organisms have evolved elaborate and efficient mechanisms to prevent or at least regulate such transposition events. As a result, even the potentially active TE copies rarely mobilize within the germline and are also largely constrained in somatic tissue. Several recent studies demonstrate, however, that LINE-1 elements are normally active and mobile during neurogenesis in both rodent and human tissue [7], [8], [9]. Somatic mobilization of Alu and SVA elements as well as LINEs also has recently been detected in several different human brain regions [6]. This raises the intriguing hypothesis that active mobilization of some TEs plays a role in normal brain development or physiology. On the other hand, there also is emerging evidence that unregulated activation of TEs is associated with neuropathology. TE activation in brain has been observed in macular degeneration [14], Rett syndrome [11], Prion diseases [13],[29], Fragile-X associated tremor/ataxia syndrome (FXTAS) [15] and ALS [12]. Moreover, for the cases of macular degeneration and FXTAS, there is evidence that activation of SINEs and an LTR-retrotransposon respectively may contribute to the observed pathology [14], [15].

Our findings support three conclusions. First, that TDP-43 broadly targets TE-derived transcripts, including many SINE, LINE and LTR classes as well as some DNA elements. This conclusion is replicated in three independent datasets from rat, mouse and human. Second, the association between TDP-43 and TE-derived RNA targets is reduced in FTLD patients relative to healthy subjects, consistent with the idea that loss of TE control might be part of the disease pathology. Third, we observe broad over-expression of TE derived transcripts in each of two different mouse models with TDP-43 dysfunction. And there is a striking overlap between the TE targets identified in the CLIP study and those that are over-expressed with TDP-43 misexpression. Taken together, our findings raise the hypothesis that TDP-43 normally functions to silence or regulate TE expression. When TDP-43 protein function is compromised, TEs become over-expressed. Unregulated TE expression can have a number of detrimental impacts including genome instability, activation of DNA-damage stress response or toxic effects from accumulation of TE-derived RNAs or proteins. Such toxicity from activation of mobile genetic elements may contribute to TDP-43-mediated neurodegenerative disorders.

 

Does a loss of TDP-43 function cause neurodegeneration?

Zuo-Shang Xu

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation St, 817 LRB, Worcester, MA, 01605, USA

Molecular Neurodegeneration 2012, 7:27  doi:10.1186/1750-1326-7-27     http://www.molecularneurodegeneration.com/content/7/1/27

In 2006, TAR-DNA binding protein 43 kDa (TDP-43) was discovered to be in the intracellular aggregates in the degenerating cells in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), two fatal neurodegenerative diseases [1,2]. ALS causes motor neuron degeneration leading to paralysis [3,4]. FTLD causes neuronal degeneration in the frontal and temporal cortices leading to personality changes and a loss of executive function [5]. The discovery triggered a flurry of research activity that led to the discovery of TDP-43 mutations in ALS patients and the widespread presence of TDP-43 aggregates in numerous neurodegenerative diseases. A key question regarding the role of TDP-43 is whether it causes neurotoxicity by a gain of function or a loss of function. The gain-of-function hypothesis has received much attention primarily based on the striking neurodegenerative phenotypes in numerous TDP-43-overexpression models. In this review, I will draw attention to the loss-of-function hypothesis, which postulates that mutant TDP-43 causes neurodegeneration by a loss of function, and in addition, by exerting a dominant-negative effect on the wild-type TDP-43 allele. Furthermore, I will discuss how a loss of function can cause neurodegeneration in patients where TDP-43 is not mutated, review the literature in model systems to discuss how the current data support the loss-of-function mechanism and highlight some key questions for testing this hypothesis in the future.

 

Amyotrophic lateral sclerosis (ALS) is a disorder where progressive degeneration of large motor neurons in the spinal cord and cerebral cortex leads to paralysis and death [3,4]. Frontotemporal lobar degeneration (FTLD) causes degeneration of neurons in frontal and temporal cortices, leading to deterioration of executive, cognitive and social functions, as well as loss of emotional control[5]. Although clinically distinct, a significant overlap exists between these two diseases in the patient population, resulting in a continuous spectrum ranging from patients with one disease at either end and patients with varying degrees of both diseases in the middle [6,7]. Recent genetic data has reaffirmed the connection between these two diseases. Some genetic mutations cause one disease but rarely the other, e.g. SOD1, FUS and TDP-43 for ALS, and tau, progranulin and CHMP2B for FTLD. Other mutations cause either or both diseases in the same patient or family, e.g. ubiquilin 2 and C9ORF72. In a significant population of patients (~95 % ALS and ~50 % FTLD), TDP-43 positive intracellular inclusions are present in the CNS even though the TDP-43 gene is not mutated [811], raising the question of how wild-type TDP-43 is involved in the pathogenesis of these cases.

TDP-43 is a RNA binding protein containing two RNA-recognition motifs (RRM), a nuclear localization signal (NLS) and a nuclear export signal (NES) [12]. The protein is normally concentrated in the nucleus but also shuttles back and forth between the nucleus and cytoplasm[13]. TDP-43 is a global regulator of gene expression and is involved in regulation of transcription and multiple aspects of RNA processing and functioning, including splicing, stability, transport, translation and microRNA maturation [1417]. TDP-43 interacts with many proteins and RNAs and functions in multi-protein/RNA complexes [1821]. TDP-43 maintains its protein expression at a constant level within a tight range by auto-feedback mechanisms, which involve TDP-43 binding to its own 3’ untranslated region [15,22]. Overexpression of TDP-43 leads to down-regulation of the endogenous TDP-43 [23,24], and blocking expression of one allele leads to a compensatory increase in the expression of the other allele [2527]. The tight regulation of TDP-43 levels is suggestive of its crucial role in the functioning of multi-protein/RNA complexes, where maintaining a certain stoichiometry between TDP-43 and the other components may be critical.

Because mutations in TDP-43 lead to ALS, a causal role of TDP-43 for neurodegeneration is firmly established [12,28,29]. Therefore, understanding how the mutants cause neurodegeneration offers a convenient entry point for exploring how TDP-43 plays this role. The first question is whether a gain, a loss of function or a dominant-negative effect mediates neurotoxicity. A resolution to this question is of critical importance because it sets the direction of further research on the disease mechanism and on the design of therapeutic strategies. To answer this question, model systems of both gain or loss of function must be employed (Table 1). Gain-of-function models are usually achieved by gene overexpression and loss-of-function models by gene knockout or knockdown. Based on the phenotypic readouts, the mechanism whereby the mutants cause neurodegeneration can be deduced (Table 1).

Table 1.Assay for disease mechanism using transgenic animals

A gain-of-function (Table 1, GF column) mechanism includes two scenarios: first, the mutant gene gains a novel toxic activity that is independent of the normal function of the gene, and second, the mutant becomes hyperactive in one of its normal functions leading to toxicity. In the first scenario, overexpression of the mutant gene, but not the wild type, will cause the disease phenotype. In the second scenario, overexpression of either the mutant or wild-type gene will cause the disease phenotype. In both gain-of-function scenarios, knockout or knockdown of the gene is not expected to cause the disease phenotype.

A loss of function (haploinsufficiency; Table 1, LF column) means that the mutant gene has no function or a reduced function but does not interfere with the function of the wild-type allele. In this scenario, neither overexpression of the mutant nor the wild type is expected to cause the disease phenotype. But knockout or knockdown reproduces the loss of function, and therefore, is expected to generate the disease phenotype.

A dominant-negative mechanism (Table 1, DN column) denotes the condition where the mutant allele is dysfunctional and inhibitory to the function of the wild-type allele. In this scenario, overexpression of the mutant gene is expected to cause the disease phenotype because it dominant-negatively inhibits the function of the endogenous wild-type protein. On the other hand, overexpression of the wild type is generally not expected to generate the disease phenotype because the wild-type gene can function normally and does not inhibit the function of the normal endogenous allele. However, there are exceptions under certain circumstances, for example, if the protein functions in a multi-protein complex (see details below). Knockout or knockdown of the gene is expected to reproduce the disease phenotype because this reduces the function of the wild-type gene. Thus, in model systems, the dominant-negative mechanism can display characteristics of both a gain and a loss of function—it is a loss of function in essence, yet its effect can dominate over the endogenous wild-type allele.

In the case of TDP-43, an abundance of gain-of-function models have been generated in various species, including worm, fly, fish and rodents [12]. In all models with rare exceptions, a consistent finding is that overexpression of both mutant and wild type TDP-43 can cause a neurodegenerative phenotype (Table 1, TDP-43 columns), thus supporting a gain-of-function mechanism and a potential overactivation of TDP-43 in the mutants [12]. Loss-of-function models have also been generated in non-mammalian species and all except the worm showed neurological and neurodegenerative phenotypes [3033,35,44]. The difference between worm and the other species may reflect some species difference, since TDP-43 is dispensable for survival in the worm but not so in other species. In general, the degenerative phenotypes in the loss-of-function models appear less overwhelming than the overexpression models and are often difficult to separate from the developmental effects stemming from a lack of TDP-43 function. Importantly, there is a lack of evidence in mammalian models that a loss of TDP-43 function causes neurodegeneration. This is largely due to the failure in generating such a model using a gene knockout approach [2527,36]. As a result, the current literature leans towards a gain-of-function mechanism as far as the role of TDP-43 in neurodegeneration is concerned.

Yet despite the preponderance of evidence for the gain-of-function mechanism, it has not been sufficient to rule out the loss-of-function mechanism, because the gain-of-function mechanism does not explain well a phenomenon that is consistently observed in numerous pathological studies, i.e. the nuclear clearance of TDP-43 that accompanies the presence of TDP-43 intracellular aggregates[1,2,45]. The question whether the depletion of TDP-43 in the nucleus is consequential in the pathogenesis remains unanswered. In addition, although the aggregates in the cytoplasm may generate gain-of-function type of toxicity, it is also conceivable that the aggregation of TDP-43 renders TDP-43 non-functional, and as such, causes TDP-43 dysfunction. In this review, I propose a model that is centered on the loss-of-function mechanism whereby TDP-43 plays its role in neurodegeneration. I will highlight the evidence in the current literature that is consistent with this model and the evidence that is still needed from future experiments to test this model.

A model for the loss of TDP-43 function as a central mechanism of pathogenesis in human disease

The TDP-43 protein is normally expressed through transcription and translation, and once produced, it regulates its own expression by a feedback mechanism, i.e., upregulating its own expression when the protein level is too low and inhibiting its expression when the protein level is too high [15,2227]. By this auto-regulatory mechanism, the intracellular level of TDP-43 is maintained within a narrow range (Figure 1, #1 normal). This tightly maintained TDP-43 level may be important because TDP-43 functions in multiprotein/RNA complexes [1821], where a proper structure and function of the complex requires a certain stoichiometric ratio between TDP-43 and its protein and RNA partners (Figure 1, #1 normal). Such a requirement is not unique to TDP-43 complexes as it has been demonstrated in other protein-RNA or protein complexes. For example, in the primary micro RNA (pri-miRNA) processing Drosha complex, overexpression of one subunit DGCR8 leads to an inhibition in the processing activity [46]. As another example, in the kinesin-2 heterotrimeric complex that drives the antegrade transport of late endosomes and lysosomes, overexpression of one subunit KAP3 inhibited the transport similar to the KAP3 knockdown [47].

Figure 1 .
Mechanisms that can cause TDP-43 dysfunction in ALS, FTLD and other neurodegenerative conditions. AD means Alzheimer’s disease, PD Parkinson’s disease, HD Huntington’s disease, LBD Lewy body dementia, DS Down syndrome, HSD hippocampal sclerosis dementia, FBD familial British dementia, and SCA spinal cerebellar ataxia. See the section subtitled “A model for the loss of TDP-43 function as a central mechanism of pathogenesis in human disease” for a detailed description of this diagram.

Xu Molecular Neurodegeneration 2012 7:27   doi:10.1186/1750-1326-7-27   Download authors’ original image

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In the disease situation, conditions in patients’ cells become conducive for TDP-43 aggregation. For example, TDP-43 mutants and its C-terminal fragments associated with ALS and FTLD have enhanced aggregation propensity [4851], and therefore, can drive TDP-43 aggregation. The aggregation can lead to a reduction in the pool of TDP-43 that can be incorporated into the TDP-43 protein/RNA complexes (Figure 1, #2 aggregation), thereby reducing the complex function and leading to neurodegeneration.

In model systems where TDP-43 is overexpressed (Figure 1, #3), the function of TDP-43 can be inhibited because an oversupply of exogenous TDP-43 mismatches with a limited supply of its endogenous interacting protein/RNA partners, resulting in the formation of incomplete and dysfunctional complexes. Below I highlight the evidence in the current literature that is consistent with this model and the future experiments that are need to test this model.

TDP-43 performs functions of vital importance, but the consequence of its dysfunction in neurodegeneration remains unclear

A crucial piece of evidence for a loss-of-function mechanism would be demonstration that a loss of TDP-43 function can cause neurodegeneration. This has not yet been experimentally achieved in a convincing manner, particularly in mammalian species. Knockouts in rodents cause early embryonic lethality [2527,36]. Inducible knockout in adult mice causes a rapid loss of fat tissue and lethality [36]. These results have not been informative as to the consequences of TDP-43 dysfunction in the nervous system. Nevertheless, the severity of the phenotype in the knockout models suggests a critical functional importance of TDP-43 in the health and survival of mammalian cells. Indeed, the conditional knockout of TDP-43 in mouse embryonic stem cells causes cell death [36]. Therefore, it is conceivable that TDP-43 function may also be vital for the survival and function of neurons. Supporting this notion are the experiments where TDP-43 knockdown causes morphological abnormalities and cell death in cultured neurons [50,52,53] and a large change in gene expression in cells of the CNS [15,16].

Experimental data from non-mammalian species have also been consistent with the critical functional importance of TDP-43. In C. Elegans, TDP-43 deletion mutants are viable, but show low fertility, slow growth and locomotor defects [44]. In Drosophila, TDP-43 knockout causes abortive embryonic development and lethality [30,31]. Although some escape the lethality and develop to adults, they display severe locomotor defects, premature death and abnormal neuronal morphology [30,31]. Evidence for progressive axonal degeneration and locomotor defects has also been reported in adult TDP-43 knockdown flies [32]. In zebrafish, TDP-43 knockdown during embryonic development causes selective defects in motor axonal growth and results in motor behavioral abnormalities [35]. These results do not conclusively demonstrate a role of TDP-43 dysfunction in neurodegeneration in ALS and FTLD, but do indicate that TDP-43 is important in the development and functioning of the nervous system, thus leaving open the possibility that TDP-43 dysfunction could play a role in neurodegeneration.

How a loss of TDP-43 function explains the pathogenic mechanism of TDP-43 mutants

Mutations in TDP-43 cause motor neuron degeneration and ALS [28,29]. The overwhelming majority of the mutations are located in the C-terminal glycine-rich domain [12], which is unstructured and responsible for interactions with other proteins [17,21,54]. How mutant TDP-43 causes neurodegeneration is not known. Overexpression models support a gain of function, but the reliance of overexpression to elicit neurodegenerative phenotypes risks over-interpretation. A lack of convincing evidence that TDP-43 levels are elevated in human disease leaves open the question of whether the results from the overexpression models are relevant for the human disease.

While there is room for doubt for the gain-of-function mechanism, evidence for the loss-of-function mechanism is also weak, primarily because few experiments have generated data directly relevant to this question, especially in mammalian systems. Nevertheless, reasonable scenarios for this mechanism can be formulated based on the current, albeit fragmented and incomplete, experimental literature. First, wild-type TDP-43 is an aggregation-prone protein and mutant TDP-43 is even more so [48,51,55]. Therefore, TDP-43 mutants can initiate and drive protein aggregation, leading to TDP-43 depletion from the cell nucleus, as has been observed in patients [1,2,56]. In addition, mutant TDP-43 may have an enhanced susceptibility for polypeptide fragmentation, which generates the patient-specific 25-kDa fragments [29,57]. These fragments have a high propensity for aggregation [50,55,58] and can coaggregate with wild-type TDP-43, thereby sequestering wild-type TDP-43 into the aggregates and depleting TDP-43 from the nucleus [50].

Second, the mutant may be functionally less active or inactive but may still retain its autoregulation capability. As a result, the overall TDP-43 level would be maintained but the function of TDP-43 would be reduced because the protein expressed from the mutant allele is dysfunctional. Some experimental data support this scenario. In mice, overexpression of mutant TDP-43 inhibited the expression of the endogenous TDP-43 to the same extent as wild type overexpression [23,37,38], suggesting that the disease-causing mutants retain their autoregulatory function. In Drosophila, wild-type TDP-43 is capable of promoting growth of dendrites and increasing the size of synaptic terminals at the neuromuscular junction. However, these activities are lost in the ALS-causing mutants [31,34], suggesting that the mutants have lost some of the wild-type functions.

Third, mutant TDP-43 may form defective TDP-43 protein/RNA complexes, thereby poisoning the function of the complex. In this capacity, the mutant TDP-43 can act dominant-negatively to inhibit the function of the wild-type allele. There is evidence that TDP-43 forms a homodimer [59] and that multiple TDP-43 molecules are incorporated into each complex [19]. Therefore, if a mutant TDP-43 molecule were capable of rendering dysfunction to the whole complex that contains both mutant and wild-type TDP-43 molecules, then the function of the wild-type allele would be inhibited.

These scenarios are consistent with a model where TDP-43 mutants cause a loss of TDP-43 function by a dominant negative mechanism. Notably, while the first scenario requires the formation of aggregates for cellular toxicity, the second and third scenarios make such a requirement unnecessary. Indeed, in both cellular and animal models, toxicity induced by mutant TDP-43 does not require its aggregation [33,37,39,60].

How TDP-43 dysfunction could contribute to neurotoxicity from overexpression of either mutant or wild-type TDP-43 in model systems

The prevailing interpretation for the observation that overexpression of mutant TDP-43 causes neurodegeneration is that mutant TDP-43 exert its toxicity by a gain of function. However, these results are also consistent with a dominant-negative mechanism, as discussed above (also see Table 1). The dominant-negative model predicts that overexpression of the mutant in sufficient quantities will inhibit the function of the two endogenous wild-type alleles in the model systems.

A puzzling observation is that overexpression of wild-type TDP-43 causes similar neurotoxic phenotypes in model systems [23,33,35,37,38,4043,60,61]. Because of the autoregulatory mechanism, overexpression of human wild-type TDP-43 leads to a suppression of the endogenous TDP-43 [23,24]. This has led to a proposal that a loss of the endogenous TDP-43 caused neurotoxicity [24]. While this proposal can reasonably explain the toxicity of the mutants on the premise that they are dysfunctional, the toxicity from the wild-type TDP-43 poses a problem because several studies have shown that the human wild-type TDP-43 gene can substitute the function of its homologue in species as distant as Drosophila and C. Elegans[30,44]. A more plausible explanation can be derived from the fact that TDP-43 functions in multiprotein/RNA complexes, whose function may depend on a certain stoichiometric composition of the different protein/RNA components. Overexpression of wild-type TDP-43 provides an amount of TDP-43 in excess of the other components that form the complexes, thereby sequestering those components into incomplete and dysfunctional complexes (Figure 1, #3 overexpression). Therefore, both overexpression of the mutants and the wild-type TDP-43 can cause neurodegeneration by dominant-negatively inhibiting the normal function of TDP-43 complexes so long as it interacts with two or more components in the complexes simultaneously and with near equal binding affinities.

While the above interpretation of the literature remains to be confirmed by further experimentation, some of the predictions from this loss-of-function/dominant-negative hypothesis are supported by observations in the current literature. First, overexpression of mutant should be more potent in causing neurodegeneration than overexpression of the wild type, which has been the case in several overexpression models [35,40,60,61]. Although this finding is not inconsistent with the gain-of-function mechanism, the result can also be explained readily by the dominant-negative mechanism outlined above. Overexpression of mutants can inhibit normal TDP-43 function by three mechanisms: (1) displacing the endogenous TDP-43 through the autoregulation mechanism, (2) inserting itself into the TDP-43 complexes in the place of the wild-type protein, and (3) forming dysfunctional complexes by disruption of the stoichiometry between TDP-43 and other protein/RNA components. In contrast, overexpression of the wild-type TDP-43 can inhibit TDP-43 function only through the third mechanism because unlike the mutant protein, it has full function. Therefore, to inhibit TDP-43 function to the same degree, a higher level of expression will be required for the wild-type TDP-43 than the mutant.

Second, if the dominant-negative hypothesis is correct, overexpression and knockout or knockdown of the gene can cause similar phenotypes. Currently, data from mammalian species is lacking to address this point. However, evidence can be drawn from other species. For example, overexpression of either mutant or the wild-type TDP-43 in Drosophila motor neurons causes progressive locomotor defects and a shortening of lifespan [33]. These phenotypes are similar to those caused by TDP-43 knockdown [33]. As another example, expression of human TDP-43 mutants but not the wild type in zebrafish embryos compromised motor axonal growth and caused locomotor defects. Similarly as in flies, knocking down the endogenous TDP-43 caused the same phenotypes [35]. Importantly, the phenotypes in the knockdown fish are rescued by the expression of human wild-type TDP-43 but not the mutants. These results are consistent with the view that the ALS-relevant TDP-43 mutants are dysfunctional and are capable of inhibiting TDP-43 function in a dominant negative manner.

Third, the loss-of-function/dominant-negative hypothesis predicts that ALS-causing mutants should be loss-of-function alleles. As discussed above, the observations that the mutants lost their ability to stimulate the growth of dendrites and axons in flies [31,34,35] and their inability to rescue phenotypes from TDP-43 knockdown in zebrafish [35] supports the loss-of-function proposition. However, key evidence from mammalian species remains to be produced.

While the case for a loss of function by a dominant-negative mechanism can be argued for, it may be overly simplistic to argue that a gain of function does not contribute to the phenotypes caused by TDP-43 overexpression in the model systems. Some evidence indicate that TDP-43 is capable of causing cellular toxicity by a gain of function under ectopic and overexpressed conditions. For example, TDP-43 causes toxicity in yeast, which does not possess an endogenous TDP-43 homologue [62]. Similarly, TDP-43 is not essential in C. Elegans, yet overexpression of human TDP-43 can still cause toxicity that is not observed in knockouts [44,61,63,64]. Therefore, in model systems where TDP-43 performs vital functions, phenotypes caused by TDP-43 overexpression are likely derived from both an interference of endogenous TDP-43 function and a gain of function. Given the complexity in the protein/RNA interaction networks of TDP-43, perhaps this would not be surprising. Overexpression is likely to generate new aberrant interactions as well as to disrupt the authentic interactions that are vital for the cell. Therefore, disentangling these effects will be complex in the overexpression models.

What is the role of wild type TDP-43 in human neurodegeneration

While the case for a loss of function in the TDP-43 mutants and in the overexpression model systems can be made, can the loss-of-function mechanism play a role in patients where TDP-43 is not mutated and not overexpressed? This is an important question because the vast majority of patients with ALS and FTLD-TDP do not have TDP-43 mutations. The answer to this question is yes because even though the primary trigger of the degenerative process lies not in TDP-43 but elsewhere, the same kind of TDP-43 aggregation and nuclear clearance is observed in the CNS of these patients [1,2,45] (Figure 1). The loss-of-function/dominant-negative model will predict that the nuclear clearance and the cytoplasmic aggregation of TDP-43 are probably a significant contributor to neurodegeneration by causing a loss of TDP-43 function. However, the experimental data for testing this prediction is scarce. In Drosophila and zebrafish, knockout or knockdown of TDP-43 produced similar neurodegenerative phenotypes [33,35]. However, further analysis is needed to differentiate the effects of TDP-43 dysfunction on neurodegeneration from those on neurodevelopment, and the relevance of these observations to human neurodegeneration remains to be established. A mammalian model with TDP-43 dysfunction in the mature CNS is urgently needed to understand the effects from a loss of TDP-43-function.

Based on the loss-of-function/dominant-negative hypothesis outlined above, what triggers TDP-43 aggregation will be one of the most intriguing and important questions in understanding the pathogenic mechanisms in ALS and FTLD. Recent investigations have shown that multiple causes can trigger secondary TDP-43 aggregation and nuclear clearance. These causes can be classified into several categories: (1) Gene mutations that enhance the mutant protein aggregation propensity and cause ALS-FTLD with TDP-43 aggregation. Examples in this category include VCP, optineurin, dynactin, ataxin 2 and ubiquilin 2. All the mutant proteins form aggregates and some form coaggregates with wild-type TDP-43 [9,6569]. The mechanism whereby these mutants cause TDP-43 aggregation is not understood. One possibility is that the aggregation of these proteins weakens the capacity of cellular proteostasis [70], which creates an environment conducive for aggregation-prone proteins such as TDP-43 to aggregate. Some of the proteins such as VCP and ubiquilin may be involved in TDP-43 degradation [71,72]. Therefore, mutations in these proteins may directly alter the TDP-43 economy and cause TDP-43 aggregation. (2) Gene mutations that cause ALS and FTLD with TDP-43 aggregation, but the mutant proteins are not involved in protein aggregation themselves. Examples in this category include progranulin, angiogenin and C9ORF72[1,11,73,74]. At present, it is not known how these mutations lead to TDP-43 aggregation. (3) Traumatic brain injury that lead to ALS-FTLD without gene mutations. Repetitive traumatic brain injury has been shown to be associated with ALS and FTLD with intracellular TDP-43 aggregation[75,76]. (4) Other neurodegenerative diseases that are not ALS-FTLD but trigger secondary TDP-43 aggregation. Examples of this category include some of the most common neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease and numerous others [8,77,78] (Figure1). Aggregation of TDP-43 in these cases may also be attributed to a disruption of proteostasis environment due to the aggregation of other proteins, although direct experimental evidence for this hypothesis is not yet in existence. (5) Unknown causes in sporadic ALS and FTLD cases. Some of the speculated causes include genetic predisposition in combination with environmental stress, e.g. environmental toxins, trauma and high physical activity [7982].

Recent studies have suggested that a redistribution of TDP-43 to the cytoplasm may be a precursor to TDP-43 aggregation. In ALS and FTLD patients, some neurons show an increase in cytoplasmic TDP-43 immunoreactivity with diffused or granular appearance, which may represent an early stage of TDP-43 aggregation [8386]. The cause for the cytoplasmic redistribution is not clear. However, a recent study demonstrate that a single traumatic brain injury can be followed by a persistent increase in the cytoplasmic levels of TDP-43 [87], suggesting that injuries to the CNS can be an initial trigger for increased levels of cytoplasmic TDP-43. In model systems, the redistribution of TDP-43 can be triggered by various stresses, including neuronal injury [8890], overexpression of disease-associated mutant TDP-43 and VCP [9193], oxidative stress [93,94] and proteasome inhibition [53]. The functional consequence of the cytoplasmic localization of TDP-43 will require further characterization. Nevertheless, some studies suggest that the cytoplasm-localized TDP-43 is recruited to stress granules before being transformed into aggregates that can persist independent of stress granules [9395]. Another study demonstrated that a modest knockdown of TDP-43 exacerbated, rather than alleviated, cell death that is induced by proteasome inhibition and associated with TDP-43 cytoplasmic translocation [53], suggesting that any toxicity that might be associated with TDP-43 cytoplasmic translocation is derived from a loss of TDP-43 function. These data are consistent with the hypothesis that an increased cytoplasmic level of TDP-43, which follows the initial cellular stress, can lead to TDP-43 aggregation and nuclear depletion.

Therapeutic implications from the dominant-negative model

Discussion on therapeutic implication based on the loss-of-function hypothesis may be premature since the hypothesis remains to be tested. However, such an exercise may be helpful for illustration of the critical importance for a resolution of this question. In the case of a gain of function, strategies that reduce the function should be effective. This may be achieved by lowering the protein levels through an inhibition of its synthesis or a stimulation of its degradation. If the toxic activity is known, strategies that inhibit the specific toxic activity may also be effective. In the case of a loss of function, on the other hand, strategies that increase the function should be effective. This may be achieved by increasing expression and stability of the protein, or stimulating its activity.

The therapeutic strategy for the dominant negative mechanism differs from both purely gain- or loss-of-function mechanisms and will be most challenging. We cannot simply increase the level of TDP-43 because uncontrolled increase of TDP-43 may inhibit the function of TDP-43 rather than improving it. High levels of TDP-43 could also further accelerate its aggregation and produce aberrant interactions with other proteins and RNA. Moreover, we do not understand why TDP-43 stays in the cytoplasm and becomes depleted from the nucleus in the disease. Therefore, it is not clear whether a simple increase of TDP-43 will replenish its level in the nucleus. In the case of mutant TDP-43, allele-specific inhibition of the mutant TDP-43 may be helpful but may not be sufficient to compensate for the lost function of the mutant allele. If the hypothesis that TDP-43 aggregation drives nuclear depletion of the TDP-43 is correct, preventing or reversing the aggregation may be a rational and safe approach to mitigate the loss of TDP-43 function. To achieve this, we need to understand how TDP-43 aggregation is triggered and propagated. We also need to understand the TDP-43 aggregation process at molecular and structural levels. Alternatively, strategies that enhance the function of TDP-43 without resorting to increase the protein level, or retain TDP-43 in the nucleus may also be effective.

Conclusions

TDP-43 aggregation and nuclear depletion have been observed widely in neurodegenerative diseases. The role of TDP-43 in neurodegeneration remains to be defined. Chief among the questions is whether a gain of function, a loss of function or a dominant-negative mechanism is responsible for neurotoxicity. The answer to this question is of critical importance because it guides the future direction of research and sets the foundation for therapeutic strategies. Current experimental data from model systems has been predominantly invoked to support the gain-of-function mechanism. However, a careful review of the data suggests that a loss of TDP-43 function caused by its mutations, its aggregation and nuclear depletion, and the inhibition of TDP-43 function by a dominant-negative mechanism in the overexpression models, are at least as plausible as the gain-of-function theory, if not more so. Therefore, in our future research, we need to gain a more detailed understanding of the normal function of TDP-43, particularly in the cells of the CNS. We need models of loss of TDP-43 function in the CNS, particularly in mammalian species, to understand the consequence of TDP-43 dysfunction. In such a pursuit, models with a partial loss of TDP-43 function may be especially desirable because in humans, it is unlikely that the TDP-43 function is totally lost. We need evidence from human diseases to determine whether the conditions are more in tune with a gain or a loss of TDP-43 function. Lastly, we need to design strategies to address the difficult problem of how to restore the normal levels of TDP-43 function as a therapy.

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Nanotechnology therapy for non-cancerous diseases

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Nanotechnology in respiratory medicine

Albert Joachim Omlor1, Juliane Nguyen2, Robert Bals3 and Quoc Thai Dinh13

Respiratory Research 2015, 16:64  http://dx.doi.org:/10.1186/s12931-015-0223-5

http://respiratory-research.com/content/16/1/64

Like two sides of the same coin, nanotechnology can be both boon and bane for respiratory medicine. Nanomaterials open new ways in diagnostics and treatment of lung diseases. Nanoparticle based drug delivery systems can help against diseases such as lung cancer, tuberculosis, and pulmonary fibrosis. Moreover, nanoparticles can be loaded with DNA and act as vectors for gene therapy in diseases like cystic fibrosis. Even lung diagnostics with computer tomography (CT) or magnetic resonance imaging (MRI) profits from new nanoparticle based contrast agents. However, the risks of nanotechnology also have to be taken into consideration as engineered nanomaterials resemble natural fine dusts and fibers, which are known to be harmful for the respiratory system in many cases. Recent studies have shown that nanoparticles in the respiratory tract can influence the immune system, can create oxidative stress and even cause genotoxicity. Another important aspect to assess the safety of nanotechnology based products is the absorption of nanoparticles. It was demonstrated that the amount of pulmonary nanoparticle uptake not only depends on physical and chemical nanoparticle characteristics but also on the health status of the organism. The huge diversity in nanotechnology could revolutionize medicine but makes safety assessment a challenging task.

Keywords: Nanoparticles; Lung; Airways; Nanotoxicology; Biodistribution; Nanomedicine

Over the past years nanomaterials have found their way into more and more areas of life. Examples are new coatings and pigments, electronic devices as well as cosmetic products like sunscreens and toothpastes. On top of that, much effort is done to adopt nanotechnology for the treatment of human diseases. The term “Nano” refers to structures in the range of 1 to 100 nm. In contrast to nanoparticles, which have to measure between 1 and 100 nm in all dimensions, nanomaterials may consist of elements bigger than 100 nm but need to be structured in the nanoscale and exhibit characteristic features associated with their nanostructure [1]. In this context, the International Organization for Standardization defined the term nano-object as a material with one, two or three external dimensions in the nanoscale [2] (Fig. 1). Nanomaterials have an extremely high surface area to volume ratio. Therefore, some of them are very reactive or catalytically active. Moreover, in the nanoworld quantum effects become visible and lead to some of the unique properties of nanoparticles. Like viruses and cellular structures, some nanoparticles are able to self-assemble to more complex structures [3]. This makes them interesting candidates for novel drugs. On the other hand it is necessary to redefine toxicology because of nanotechnology. Unlike classical toxicology, where dose and composition matter, in nanotoxicology the focus has to be set on properties like morphology, size, size distribution, surface charge, and agglomeration state as well. Nanotechnology is important for respiratory medicine for several reasons. Firstly, it offers new approaches to treat diseases of the respiratory tract. However, as nanotechnology usage in consumer products, cosmetics, and medicine is continuously increasing, it is also pivotal to understand potentially adverse effects of nanomaterials on the respiratory system. Additionally, studying respiratory effects of manufactured nanomaterials helps to understand the impact of combustion exhaust and ultra-fine dusts on human health. On top of that, the lung is probably the most important gateway of nanoparticles to the human organism. For the assessment of safety in nanotechnology it is therefore also important to elucidate which nanoparticle properties determine pulmonary resorption and biodistribution (Fig. 2).

Fig. 1. Nano-Objects can be divided into nanoparticles, nanofibres and nanoplates depending on the number of external dimensions in the nanoscale

Nano-Objects can be divided into nanoparticles, nanofibres and nanoplates

Nano-Objects can be divided into nanoparticles, nanofibres and nanoplates

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Fig. 2. The increasing use of nanotechnology affects respiratory medicine in three main areas. Firstly, nanotechnology enables more sophisticated options in therapy and diagnostics. Secondly, the use of nanomaterials can cause toxic effects in the respiratory system. Health risks associated with the use of nanomaterials are not fully understood and merit further investigation. Moreover, it will be essential to understand the effects of inhaled nanoparticles on extrapulmonary organs

nanotechnology affects respiratory medicine in three main areas

nanotechnology affects respiratory medicine in three main areas

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Applications of nanotechnology in therapeutics and diagnostics

Although clinical application of nanotechnology in therapeutics and diagnostics is still rare, there are multiple promising candidates for future use in the field of respiratory medicine.

Drug delivery

Nanoparticles can act as vessels for drugs because they are small enough to reach almost any region of the human organism. Drugs can be bound chemically to the nanoparticles by a multitude of different linker molecules or by encapsulation. This allows better control of toxicokinetics. However, the main advantage is the capability of targeted drug delivery. The targeting can be active or passive. In case of tumor diseases, the leaky and immature vasculature of fast growing tumors can be taken advantage of in order to achieve passive targeting of chemotherapeutic loaded nanoparticles. This is called the enhanced permeability and retention (EPR) effect [4]. The first generation nano drug delivery systems rely entirely on the EPR effect. One example is Genoxol-PM, a polymeric paclitaxel loaded poly(lactic acid)-block-poly(ethylene glycol) micelle-formulation [5]. This nanocarrier has recently been tested in a phase II trial in patients with advanced non-small cell lung cancer (NSCLC). 43 patients were treated with four 3-week cycles of Genexol-PM at 230 mg/m2 on day 1 combined with gemcitabine 1000 mg/m2 on day 1 and day 8. With a response rate of 46.5 %, the therapy showed favorable antitumor activity. Moreover, emetogenicity was low. However, frequent grade 3/4 adverse events like neutropenia and pneumonia were observed [6]. The second generation nanoparticle drug delivery systems possess targeting ligands. These can be antibodies, aptamers, small molecules and proteins (Fig. 3). The attached ligands actively guide the nanoparticles and therefore the drugs to the tumor cells. Tumor specific monoclonal antibodies are already widely used in cancer therapy. Those antibodies can be attached to nanoparticles for active targeting. In a recent study polyglycolic acid nanoparticles, that were conjugated with cetuximab antibodies for targeting and loaded with the drug paclitaxel palmitate, were administered intravenously to mice with A549-luc-C8 lung tumors. The survival rate of these mice increased significantly compared to the control group [7]. Another approach involves aptamers as targeting agents. Aptamers are synthetic oligonucleotides that are capable of binding specific target structures. Their small size, their simple synthesis, and their lack of immunogenicity make them promising ligands for nanoparticles. Moreover, small molecules such as folate can be used for targeting tumor cells that express a high density of folate receptors. In addition, tumors often overexpress receptors for several proteins. Proteins like transferrin therefore are common targeting ligands [8]. These second generation nanocarriers are already used clinically against lung cancer with substances like Aurimmune Cyt-6091 and Bind-014. Aurimmune Cyt-6091 is a drug delivery system based on gold nanoparticles functionalized with polyethylene glycol (PEG) and tumor necrosis factor alpha (TNF-α). It has been used against adenocarcinoma of the lung in a phase I clinical trial. The TNF-α serves both as targeting and therapeutic agent in this case [9]. A phase II clinical trial for non-small cell lung cancer patients has been planned [10]. The nano drug delivery system Bind-014 is currently tested in a phase II clinical trial as second-line therapy for patients with non-small cell lung cancer [11]. Bind-014 nanoparticles consist of a polylactic acid (PLA) core, in which the anti-tumor drug docetaxel is physically entrapped. The particles are surface-decorated with PEG to reduce elimination from the immune system and contain ligands against prostate-specific membrane antigen (PSMA) for targeting. PSMA is expressed in prostate cancer cells and in the neovasculature of nonprostate solid tumors, such as NSCLC [12]. Preliminary data demonstrates, that Bind-014 is clinically active and well tolerated. It also showed promising effects on patients with KRAS mutations, where ordinary anti-tumor agents usually fail. Additionally, adverse effects like anemia, neutropenia and neuropathy were significantly reduced compared to solvent based docetaxel [13].

Four different strategies for active targeting of nanoparticle based drug delivery systems

Four different strategies for active targeting of nanoparticle based drug delivery systems

Fig. 3. Four different strategies for active targeting of nanoparticle based drug delivery systems are shown. The nanoparticles can be conjugated with tumor specific antibodies or aptamers. Additionally, small molecules, such as folate, as well as proteins, such as transferrin, can be used for targeting receptors that are overexpressed on tumors

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Nanoparticle based drug delivery also offers potential in other fields of respiratory medicine. In experiments with tuberculosis infected guinea pigs, it was demonstrated that inhaled alginate nanoparticles encapsulating isoniazid, rifampicin, and pyrazinamide showed better bioavailability and higher efficiency than oral drug medication [14]. Similar results were presented by Pandey et al. with the three antitubercular drugs encapsulated in poly (DL-lactide-co-glycolide) nanoparticles[15]. Moreover, another study demonstrated that pirfenidone loaded nanoparticles have higher anti-fibrotic efficacy in the treatment of mice with bleomycin-induced pulmonary fibrosis than dissolved pirfenidone [16].

Hyperthermia

Nanoparticle induced hyperthermia can be used to locally destroy tumor cells. Heat generation is usually achieved by two approaches, magnetic and photothermal hyperthermia. In magnetic hyperthermia, an extracorporeal coil creates an alternating magnetic field that heats magnetic nanoparticles inside a tumor. This increases the temperature in the tumor without affecting healthy tissue. A recent study assessed the effect of inhalable superparamagnetic iron oxide nanoparticles in a mouse model of NSCLC. Compared to the non-targeted nanoparticles, the epidermal growth factor receptor (EGFR) targeted nanoparticles showed significantly more effective tumor shrinkage after magnetic hyperthermia treatment [17]. The other approach, photothermal therapy uses laser radiation in the visible or near infrared spectrum and photosensitizing nanoparticles such as gold or graphene. A commercial product called auroshell is available for tumor therapy. Auroshell nanoparticles consist of a silica core surrounded by a thin layer of gold. The gold nanoshells are administered intravenously and accumulate in the tumor due to the EPR effect. Upon exposure of the tumor to a near infrared laser, the laser energy is efficiently converted to heat by the gold nanoshells [18]. This therapy, which is called AuroLase, is currently undergoing clinical trial in patients with primary and/or metastatic lung tumors [19] (Fig. 4).

Two different approaches of nanoparticle based hyperthermia therapy

Two different approaches of nanoparticle based hyperthermia therapy

Fig. 4. Two different approaches of nanoparticle based hyperthermia therapy are shown. a In magnetic hyperthermia, magnetic nanoparticles (MNP) are applied intravenously and accumulate inside the tumor. When an oscillating magnetic field is created by an extracorporeal coil the magnetic nanoparticles produce heat inside the tumor. b In photothermal hyperthermia, gold nanoshells (GNS) or similar photosensitizing nanoparticles are applied intravenously and accumulate inside the tumor. Upon exposure of the tumor to near infrared (NIR) laser radiation, the gold nanoshells convert the laser light into heat

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Gene therapy

Like viruses, nanoparticles can be used as vectors for genes. But in contrast to viruses, they are less immunogenic and have higher DNA transport capacity. In a study, DNA loaded polyethylenimine nanoparticles were used in order to treat lipopolysaccharide induced acute lung injury in mice. After intravenous injection of the nanoparticles, the beta2-Adrenic Receptor genes in the nanoparticles led to a short lived transgene expression in alveolar epithelia cells. As a result the 5-day survival rate improved from 28 % to 64 %. The severity of the symptoms measured by alveolar fluid clearance, lung water content, histopathology, bronchioalveolar lavage cellularity, protein concentration, and inflammatory cytokines was also significantly attenuated [20]. DNA loaded nanoparticles are also promising candidates in the treatment of cystic fibrosis. It was shown in a clinical trial that nasal application of DNA nanoparticles is safe and evidently leads to vector gene transfer [21]. One major problem in this context is to overcome the mucus barrier. In a recent study, it was demonstrated that densely PEG-coated DNA nanoparticles can rapidly penetrate extracorporeal human cystic fibrosis and extracorporeal mouse airway mucus. In addition, those particles exhibited better gene transfer after intranasal administration to mice than conventional carriers [22].

Diagnostics

Nanoparticles have the potential to improve pulmonary x-ray diagnostics. Folic acid-modified dendrimer-entrapped gold nanoparticles were utilized as imaging probes for targeted CT imaging. In in-vitro and in-vivo tests, the nanoparticles were trapped in the lysosomes of folic acid receptor expressing lung adenocarcinoma cells (SPC-A1). It was possible to detect the tumor cells by micro-CT imaging after nanoparticle uptake. In addition, it was also shown that the particles possess good biocompatibility, with no impact on cell morphology, viability, cell cycle, and apoptosis [23]. Nanoparticles can also be used to enhance MR diagnostics of lung tissue. In experiments with intratracheal administration of Gadolinium-DOTA nanoparticles in mice, signal enhancements in several organs including the lung were measured with ultrashort-echo-time-proton-MRI. The signal change over time in the different organs demonstrated the passage of the nanoparticles from the lung to the blood, then to the kidneys, and finally to the bladder [24].

Toxicological aspects of nanomaterials

Toxic effects of nanoparticles are a major concern in pulmonary medicine. Especially ultrafine particles of low soluble, low toxic materials like titanium dioxide, carbon black, and polystyrene are overall more toxic and inflammatory than fine particles of the same material. This applies to both synthesized nanoparticles and natural dusts [25]. For nano related toxicity multiple mechanisms seem to be important. In the following, the interaction with the immune system, the creation of oxidative stress, and toxic effects on the genome are taken a closer look at. In order to correlate toxic effects with nanoparticle properties, it is necessary to thoroughly characterize the selected nanoparticles prior to administration.

Nanoparticle characterization

The most commonly used methods to characterize nanoparticles for toxicology studies are transmission electron microscopy (TEM) for size, morphology, and agglomeration, dynamic light scattering (DLS) for the size distribution of the particles, zeta potential measurement for nanoparticle surface charge, and x-ray diffraction (XRD) for the particles’ crystal structure. In some cases such as gold and silver nanoparticles, UV-vis spectroscopy can be used to determine size and size distribution due to a special size dependent optical activity [26]. Ideally, nanoparticle characterization is repeated after administration as changes of the nanoparticles during the application process are possible. In in-vitro experiments, nanoparticles are usually applied by mixing with cell culture medium. The dissolved components of the medium, especially the ions, lead to agglomeration and precipitation of many nanoparticles, causing significant changes in their physicochemical properties. Similar effects are to be expected when nanoparticles come into contact with surfactant or other biological fluids. It was shown, that some nanoparticles tend to form protein coronae in biological systems [27].

Effects on immune system and inflammation

Many nanoparticles possess properties that give them the potential to influence the immune system. In this context, nanoparticles’ ability to penetrate cellular boundaries, to escape phagocytation by macrophages, to act as haptens, and even to disturb the Th1/Th2 balance might be essential [28]. For carbon black nanoparticles, a recent study investigated the effects of inhalative exposure on mice with bleomycin-induced pulmonary fibrosis. The analysis of histology as well as cytokine expression suggested that the nanoparticles triggered an inhalation exacerbated lung inflammation. The author concluded that especially for people with pulmonary preconditions inhalation of nanoparticles can lead to serious health problems [29]. In this context, another study found out that PEGylated cationic shell-cross-linked knedel-like (cSCK) nanoparticles produced significantly less airway inflammation than non-PEGylated ones. This was explained by a change in endocytosis. In contrast to the clathrin-dependent endocytosis of non-PEGylated particles, the PEGylated cSCK nanoparticles showed a clathrin-independent route [30]. On the other hand, some nanomaterials exhibit impressive immune modulating activity. As an example, [Gd@C82(OH)22]n, a fullerene derivate with a gadolinium atom inside showed anticancer activity without being cytotoxic (Fig. 5). In vitro studies demonstrated that [Gd@C82(OH)22]n activated dendritic cells (DCs) and even induced phenotypic maturation of those cells. Moreover, the [Gd@C82(OH)22]n treated DCs also stimulated allogenic T cells in a Th1 characteristic. The effect of [Gd@C82(OH)22]n was comparable, probably even stronger than the effect of lipopolysaccharide (LPS) on DCs. The study also verified that the nanoparticles were free of LPS contamination. In-vivo experiments on ovalbumin (OVA) immunized mice showed enhanced immune responses comparable to the adjuvant effect of Alum on OVA mice. However, whereas Alum lead to a Th2 response pattern with IL-4, IL-5 and IL-10 upregulation, [Gd@C82(OH)22]n caused a Th1 pattern with upregulation of IFNγ [31]. Similar results were demonstrated in another study using a murine asthma model. OVA sensitized mice that were additionally treated with the nanomaterial graphene oxide during allergen sensitization had stronger airway remodeling and hyperresponsiveness than mice that have only been treated with OVA. The graphene oxide lead to a downregulation of Th2 dependent markers such as IL-4, IL-5, IL-13 IgE and IgG1 but increased Th1-associated IgG2a. Moreover, the graphene oxide increased the macrophage production of mammalian chinitases, chitinase-3-like protein 1 (CHI3L1), and AMCase, which could be the reason for the overall augmentation in airway remodeling and hyperresponsiveness [32]. However, this kind of immune modulation can also be utilized for therapeutic purposes. In a recent study a nanoparticle-based vaccine has been used to treat dust mite allergies in mice. The immune-modulating carriers were generated by loading dust mite allergen Der p2 and the potent Th1 adjuvant unmethylated cytosine-phosphate-guanine (CpG) into biodegradable poly(lactic-co-glycolic acid) (PLGA) polymer particles. Mice treated with those nanoparticles showed significantly lower airway hyperresponsiveness as well as lower IgE antibody levels after a 10 day intranasal Der p2 instillation compared to the control group. The authors conclude that this biodegradable nanoparticle-based vaccination strategy has significant potential for treating HDM allergies [33].

Gd@C82(OH)22 consists of a gadolinium atom (green) inside a 2 nm cage of carbon atoms (grey)

Gd@C82(OH)22 consists of a gadolinium atom (green) inside a 2 nm cage of carbon atoms (grey)

Fig. 5. Gd@C82(OH)22 consists of a gadolinium atom (green) inside a 2 nm cage of carbon atoms (grey). The hydroxyl groups (red) outside the cage are responsible for water solubility. In water the molecule forms aggregates [Gd@C82(OH)22]n with average size of 25 nm

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Oxidative stress and catalysis

Oxidative stress is often brought in context with nanotoxicology. It can be measured directly with dichlorofluorecein or indirectly by the upregulation of reactive oxygen species (ROS) eliminating enzymes like superoxide dismutase [34]. Another approach involves tests whether the nanoparticle dependent toxicity can be reduced by the application of an antioxidant. Widely used semiconductor materials such as lead sulfide nanoparticles may have the potential to generate oxidative stress in the lung. A recent study tested the toxicity of intratracheally applied 30 nm and 60 nm lead sulfide nanoparticles on rats. Oxidative damage was evaluated based on superoxide dismutase, total antioxidant capacity, and concentration of malondialdehyde. In addition to inflammatory responses, both 30 nm and 60 nm groups showed increased oxidative damage compared to control groups. The effect was significantly stronger for the 30 nm lead sulfide compared to the 60 nm nanoparticles [35]. Another nanomaterial which is associated with oxidative stress is nanosized titanium dioxide. Li et al. induced pulmonary injury in mice by daily intranasal instillation of suspended 294 nm TiO2 nanoparticles for 90 days, demonstrating that the rate of reactive oxygen species (ROS) generation increased with increasing TiO2 doses. Moreover lipid, protein and DNA peroxidation products were identified in elevated doses, which suggests that ROS dependent lung damage was significant in the nanoparticle treated animals [36]. Furthermore, in vitro tests on BEAS-2B and A549 lung cell lines demonstrated that the commonly used nanoparticles ZnO and Fe2O3 are very different in terms of creating oxidative stress. The Fe2O3nanoparticles with an average diameter of 39 nm were distributed in the cytoplasm, whereas the 63 nm ZnO nanoparticles were trapped in organelles such as the endosome. In contrast to the Fe2O3 nanoparticles the ZnO nanoparticles caused reactive oxygen species production as well as cell cycle arrest, cell apoptosis, mitochondrial dysfunction and glucose metabolism perturbation[37] (Table 1).

Table 1. Oxidative stress induction in respiratory tissue by different nanoparticles

Genotoxicity

Another important type of toxicity caused by nanoparticles is genotoxicity. A common method to quantify genotoxicity is the comet assay, which uses electrophoresis to detect DNA strand breaks. This assay was used in a recent study to check whether intratracheal instilled fullerene C60nanoparticles induced DNA damage in male rats. However, despite inflammatory responses and hemorrhages in the alveoli of the C60 treated rats, there was no significant increase in fractured DNA in their lung cells. Therefore, it was concluded that even at inflammation inducing doses, fullerene C60 nanoparticles have no potential for DNA damage in the lung cells of rats [38]. Similarly, another study demonstrated that intratracheal instillation of anatase TiO2 nanoparticles on rats did not result in genotoxicity. None of the TiO2 groups showed an increase in fractured DNA while the positive control with ethyl methanesulfonate exhibited significant increases [39]. In contrast to those results, Kyjovska ZO et al. found that even in low doses, where no inflammation occurs, Printex 90 carbon black nanoparticles induce genotoxicity in mice. There was no inflammation, cell damage and acute phase response, which means that the increased DNA strand breaks are related to direct DNA damage caused by the nanoparticles [40]. On the other hand, a recent study suggests that CeO2 nanoparticles may be even used as antioxidant and anti-genotoxic agents in the lung. After treatment with the oxidative stress-inducing agent KBrO3, BEAS-2B cells pretreated with the CeO2 nanoparticles showed significantly less intracellular ROS as well as a reduction in DNA damage compared to non-pretreated cells [41].

Biodistribution

Nanoparticle detection

Research on the biodistribution of nanoparticles requires tracking of the applied nanoparticles in the test animal. Conventional light microscopy is not able to detect nanoparticles because of Abbe’s law. Therefore, electron microscopic imaging is often required. However, light microscopy can be used to describe the nanoparticle induced changes in the cell morphology without being able to see the nanoparticles themselves. Additionally, nanoparticles can be indirectly made detectable in light microscopy by a method called autometallography. This is a silver staining that can be used to increase the size of several types of nanoparticles like gold, silver, and some metal sulfides and selenides in the histological section [42]. This technique was used to detect silver nanoparticles in the olfactory bulb and lateral brain ventricles of mice that had been intranasally treated with 25 nm silver nanoparticles [43].

Particle deposition and resorption in the respiratory tract

Most research about biodistribution of nanoparticles in organisms focuses on intravenous injection. However, nanoparticles were shown to be able to pass the blood air barrier of the lung. Whether or not nanoparticles can travel through the lung into the body seems to be size dependent. This was evaluated by injecting neutron activated radioactive gold nanoparticles of 1.4 nm and 18 nm intratracheally to rats. The bigger nanoparticles almost completely retained in the lung while significant amounts of the smaller 1.4 nm particles were found in blood, liver, skin and carcass 24 h after instillation [44]. Choi H. S. et al. applied nanoparticles of different size and charge to mice. The nanoparticles were tracked in different organs through fluorescence labeling. It was demonstrated that nanoparticles rapidly translocated to the mediastinal lymph nodes if they possess a hydrodynamic diameter of 34 nm or less and a neutral or anionic surface. Bigger and positively charged nanoparticles exhibited no significant uptake [45] (Fig. 6). In addition to physical parameters of the applied nanoparticles the health status of the exposed organism also seems to play an important role. A recent study showed that the distribution of oropharyngeal instilled 40 nm gold nanoparticles is influenced by additional LPS treatment. The gold content of organs was measured with inductively coupled plasma mass spectroscopy. BALB/C mice that had been oropharyngeal treated with LPS 24 h prior to the nanoparticle administration exhibited less gold content in their lungs than untreated mice. In both groups gold was detected in different organs. High concentrations were found in heart and thymus in the non LPS group, while the LPS treated mice accumulated most of the gold in the spleen. The author concluded that nanoparticle uptake may depend on medical preconditions [46].

Fig. 6. Pulmonary uptake of nanoparticles depends on size and surface charge. Positively charged nanoparticles and nanoparticles that are bigger than 34 nm cannot pass the epithelial barrier of the lung. Only small and not positively charged nanoparticles can translocate from the lung over blood and lymph system to the organism

Pulmonary uptake of nanoparticles depends on size and surface charge

Pulmonary uptake of nanoparticles depends on size and surface charge

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Conclusions

Over the last decade, major breakthroughs in nanotechnology have been achieved. It is only a matter of time before new nano based drugs reach respiratory medicine. Especially the fields of targeted drug delivery, gene therapy, and hyperthermia offer great potential for modern drugs. On the other hand the increased use of nanomaterials in all fields of life also bears the risk of exposure through inhalation. It is therefore essential to understand pulmonary toxicology of nanomaterials in all its facets. However, it is still very unclear why the toxic effects of nanoparticles in the respiratory tract are so inhomogeneous and not well predictable. In this context, not only local reactions of lung and airways but also nanoparticle uptake and distribution in the organism are important factors and therefore fields of current research. As only few nanoparticle compositions have been tested, it is questionable whether those results can be easily adapted to other nanoparticles. Because of the continuously increasing diversity of engineered nanoparticles, toxicology can hardly keep pace with the safety assessment of future products. Therefore, more attention should be set on this wide field of research.

Abbreviations

CHI3L1: Chitinase-3-like protein 1

cSCK: Cationic shell-cross-linked knedel-like

CT: Computer tompgraphy

DC: Dendritic cell

DLS: Dynamic light scattering

EGFR: Epidermal growth factor receptor

EPR: Enhanced permeability and retention

LPS: Lipopolysaccharide

MRI: Magnetic resonance imaging

NSCLC: Non-small-cell lung carcinoma

OVA: Ovalbumin

PEG: Polyethylene glycol

PLA: Polylactic acid

PLGA: Poly(lactic-co-glycolic acid)

PSMA: Prostate-specific membrane antigen

ROS: Reactive oxygen species

TEM: Transmission electron microscopy

TNF-α: Tumor necrosis factor alpha

XRD: X-ray diffraction

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Pulmonary applications and toxicity of engineered nanoparticles.

Because of their unique physicochemical properties, engineered nanoparticles have the potential to significantly impact respiratory research and medicine by means of improving imaging capability and drug delivery, among other applications. These same properties, however, present potential safety concerns, and there is accumulating evidence to suggest that nanoparticles may exert adverse effects on pulmonary structure and function. The respiratory system is susceptible to injury resulting from inhalation of gases, aerosols, and particles, and also from systemic delivery of drugs, chemicals, and other compounds to the lungs via direct cardiac output to the pulmonary arteries. As such, it is a prime target for the possible toxic effects of engineered nanoparticles. The purpose of this article is to provide an overview of the potential usefulness of nanoparticles and nanotechnology in respiratory research and medicine and to highlight important issues and recent data pertaining to nanoparticle-related pulmonary toxicity.

PMID:
18641236
[PubMed – indexed for MEDLINE]
PMCID:
PMC2536798

Free PMC Article

The possibility of nanotechnology dramatically improving the health and quality of life of people throughout the world holds great promise. Predictions of beneficial effects of nanotechnology in numerous industrial, consumer, and medical applications have been promising. By no means an exhaustive list, these applications include those that may lead to more efficient water purification, stronger and lighter building materials, increased computing power and speed, improved generation and conservation of energy, and new tools for the diagnosis and treatment of disease. The optimistic outlook for a future improved by nanotechnology must be tempered, however, by the realization that relatively little is known about the potential adverse effects of nanomaterials on human health and the environment.

The definition of a nanoparticle is generally considered to be a particle with at least one dimension of 100 nm or less. As a result of their small size and unique physicochemical properties, the toxicological profiles of nanoparticles may differ considerably from those of larger particles composed of the same materials (15, 98). Furthermore, nanoparticles of different materials (e.g., gold, silica, titanium, carbon nanotubes, quantum dots) are not expected to interact with and affect biological systems in a similar fashion. As a result, it seems unlikely that the toxic potential and/or mechanisms of nanoparticles can be predicted or explained by any single unifying concept.

The respiratory system represents a unique target for the potential toxicity of nanoparticles due to the fact that in addition to being the portal of entry for inhaled particles, it also receives the entire cardiac output. As such, there is potential for exposure of the lungs to nanoparticles that are introduced to the body via the act of breathing and by any other exposure route that may result in systemic distribution, including dermal and gastrointestinal absorption and direct injection. Interest in the respiratory system as a target for the potential effects, both beneficial and adverse, of nanoparticles is reflected by the steady increase in the number of scientific publications on these subjects during the past decade (Fig. 1).

publications related to the pulmonary toxicity and applications of engineered nanoparticles

publications related to the pulmonary toxicity and applications of engineered nanoparticles

Scientific publications related to the pulmonary toxicity and applications of engineered nanoparticles. The number of articles published in each of the past 10 years was identified by searching the PubMed database

he purpose of this article is to complement and expand on previous reviews of the pulmonary effects of nanoparticles (11, 14, 34, 35) by providing an overview of potential applications of nanotechnology in pulmonary research and in diagnosis and treatment of disease. In addition, recent advances regarding the potential pulmonary toxicity of nanoparticles as assessed in human, experimental animal, and in vitro studies are discussed. For the purposes of this article, only intentionally engineered nanoparticles are considered; unintentionally generated (e.g., via combustion engines, grilling, welding) and naturally occurring nanoparticles (e.g., via forest fires or volcanic eruptions) are not included in this discussion.

NANOPARTICLES AND THE LUNG

There are myriad nanoparticles to which the respiratory system may be exposed.

There is the potential for the respiratory system to be exposed to a seemingly countless number of unique nanoparticles, essentially none of which has been sufficiently examined for potential toxicity at this time. A substantial number of nanoparticles are already present in the marketplace in consumer products such as sunscreens, cosmetics, and car wax, and many more are sure to follow (a comprehensive list is maintained and updated by the Project on Emerging Nanotechnologies at the Woodrow Wilson International Center for Scholars: http://www.wilsoncenter.org/nano). Although the toxicity of the majority of nanoparticles may prove to be minimal, the fact that there is any potential for adverse effects to result from exposure suggests that prudence is warranted.

Various types of nanoparticles exist including those that are carbon-based (e.g., nanotubes, nanowires, fullerenes) and metal-based (e.g., gold, silver, quantum dots, metal oxides such as titanium dioxide and zinc oxide) and those that are arguably more biological in nature (e.g., liposomes and viruses designed for gene or drug delivery). To demonstrate the complexity of the situation, it is worthwhile to consider the case of carbon nanotubes as an example. Carbon nanotubes can be: 1) produced and/or cleaned using one of several different methods; 2) produced using one of several different metal catalysts; 3) single- or multi-walled; 4) of various lengths; and 5) subjected to numerous surface modifications. The result of these permutations is that a vast number of unique carbon nanotubes can be derived, all of which fall under one broad category, namely the carbon nanotube. Dividing these into single-walled and multi-walled forms reduces the ambiguity only so much, and we are still left with potentially thousands of each type. Furthermore, as has been demonstrated in recent in vitro experiments (37), the potential for nanotube agglomeration or for adhesion of nanotubes to biological molecules and the resultant alteration of their reactivity must be considered. Needless to say, the variations in nanoparticle form and functionality, not only for carbon nanotubes but also for nanoparticles in general, present significant challenges in the assessment of their potential usefulness and toxicity.

Nanoparticle accumulation within the lung.

Nanoparticles may reach the lung via inhalation or systemic delivery and do so by incidental/accidental or intentional means. Intentional pulmonary administration is being examined as a means of nanoparticle delivery for imaging and therapeutic purposes and is discussed separately below. Incidental or accidental inhalation exposure to nanoparticles can be envisioned most likely to occur as a result of exposure to occupational aerosols during the production or packaging of nanoparticles or nanostructured materials (89). In addition to pulmonary effects resulting from such exposures, translocation and subsequent systemic exposure and accumulation are also possible and are being investigated. It should be noted that nanoparticles naturally tend to agglomerate into larger particles that can be microns in size, thereby reducing the likelihood of free nanoparticles being respired. However, surface modifications designed to limit particle-particle interactions and protein binding may reduce the tendency for nanoparticle agglomeration and increase the potential for inhalation and deposition within the lungs (131).

Incidental pulmonary exposure as a result of systemic delivery is likely inherent for any nanoparticle that is injected or that might be absorbed following dermal application or ingestion. Although no published human data pertaining to pulmonary accumulation of nanoparticles following systemic exposure were identified, several animal studies have demonstrated pulmonary accumulation of nanoparticles (or of drug-conjugated nanoparticles) by means of determining their quantity in total lung homogenate preparations following their ingestion or intravenous or subcutaneous injection (43, 71, 109, 138, 142, 155). None of these studies investigated whether systemically administered nanoparticles traversed the blood-air barrier to gain access to the interstitium or lung epithelium; however, this is not necessarily a requirement for beneficial (or detrimental) effects to ensue. Although the levels and duration of accumulation appear to vary for the different nanoparticles examined, these data highlight the potential for exposure of the lungs to nanoparticles via the systemic route.

PULMONARY APPLICATIONS OF NANOPARTICLES

Imaging and diagnostic applications.

Many improvements in imaging capabilities that will benefit basic and clinical pulmonary research and disease diagnosis can be envisioned through the application of nanotechnology. Advances that include the delivery of nanoparticle imaging agents to specific cells or tissues of interest, the development of nanoprobes for molecular imaging of disease pathways, and the development of better contrast agents are forthcoming (21, 22, 115). Quantum dots are one type of nanoparticle that is proving to be particularly useful for imaging and diagnostic purposes. These semiconductor nanocrystals have broad absorption spectra and narrow emission spectra, and as their fluorescence is dependent on their chemical composition and size, multiple quantum dots (each with a unique color emission) can be detected simultaneously. Moreover, their relatively large surface area provides the opportunity for attachment of peptides or antibodies that precisely target cell types or tissues for imaging, thereby increasing specificity and decreasing background. In this regard, Akerman et al. (2) demonstrated that quantum dots coated with a peptide that binds to membrane dipeptidase on pulmonary endothelial cells were detected in the lung but not in brain or kidney 5 min after intravenous administration in BALB/c mice. Furthermore, in a study using quantum dots conjugated to monoclonal antibodies, rapid and specific detection of respiratory syncytial virus infection was demonstrated in vitro and in the lungs of BALB/c mice in vivo (137). Quantum dots have also been used to study tumor cell extravasation into lung tissue in C57BL/6 mice (140), highlighting the utility of these nanoparticles in the study of tumor metastasis.

Other nanoprobes for pulmonary imaging and diagnostics are also being examined experimentally. A recent study by le Masne de Chermont et al. (78) demonstrated that inorganic luminescent nanoparticles can be optically excited before injection into mice to provide long-lasting imaging of the lung. This was particularly evident for the positively charged nanoparticles that were studied, as noninvasive external detection revealed significant pulmonary accumulation of these nanoparticles up to 1 h following intravenous injection (78).

Therapeutic applications.

The potential therapeutic applications of nanoparticles in respiratory and systemic diseases are numerous (20, 21, 112,115, 133). A considerable thrust of recent research has been focused on determining the suitability of nanoparticles of various types to serve as vectors for the pulmonary delivery of drugs or genes via inhalation or systemic administration, whereas other efforts have been directed toward developing and delivering nano-sized drug particles to the lung (Table 1). The majority of the studies reported to date have focused on the utility of these strategies for the treatment of pulmonary infection. As an example, gene transfer using intranasal administration of chitosan-DNA nanospheres was shown to prophylactically inhibit respiratory syncytial virus infection and to reduce allergic airway inflammation in mice when given prophylactically or therapeutically (74, 75). Moreover, nanoparticle-mediated intranasal delivery of short interfering RNA (siRNA) targeted against a specific viral gene, NS1, has also been shown to inhibit respiratory syncytial virus infection in mice and rats (72, 161).

Table 1.

logo-ajplung

The usefulness of nano-sized drug particles as treatment modalities in models of pulmonary infection has also been investigated. Inhalation of aerosolized nano-sized itraconazole resulted in significantly higher lung concentrations in mice than did oral administration (138) and was found to prophylactically inhibit invasive pulmonary aspergillosis and reduce infection-related deaths in mice, whereas oral drug administration did not (4, 59). In addition, Pandey et al. (110) demonstrated that a single inhalation of aerosolized poly (DL-lactide-co-glycolide) nanoparticles loaded with antitubercular drugs (isoniazid, rifampicin, or pyrazinamide) resulted in therapeutic plasma drug levels for up to 6 days in guinea pigs and found that repeated inhalations were as effective as more frequent oral administrations of free drug in treating experimental tuberculosis. A subsequent study revealed that a single subcutaneous injection of these antitubercular drug-containing nanoparticles in mice resulted in therapeutic plasma drug levels for up to 32 days and was more effective at reducing bacterial counts in the lungs and spleen than was daily oral administration of free drug (109). Finally, Zahoor et al. (158) reported that the same antitubercular drugs were more effective than free oral drugs when they were encapsulated in alginate nanoparticles and administered via inhalation to guinea pigs.

Other studies relevant to the potential utility of nano-sized drugs in disease treatment have examined siRNA-mediated suppression of target mRNA levels following intranasal administration of chitosan-based nanoparticles in mice (61) and the pharmacokinetics of lipid-coated nanoparticles of 5-fluorouracil in hamsters (58). Moreover, allergic airway inflammation in mice has been shown to be reduced by intravenous administration of polymer nanoparticles coated with a P-selectin inhibitor (67) and by intranasal administration of chitosan nanoparticles carrying theophylline (79). Importantly, Dames et al. (30) recently reported on the ability to externally direct inhaled magnetically charged iron oxide nanoparticles to specific areas of the lungs of mice without adversely affecting respiratory mechanics, demonstrating for the first time that targeted aerosol delivery to the lungs is achievable. Such an approach could prove to be beneficial in the treatment of localized lung infections or tumors.

Although the majority of the toxicity studies that are discussed below focused on nonbiodegradable nanoparticles such as metals and carbon nanotubes, nanoparticles designed for clinical pulmonary drug delivery will likely be biodegradable (133). In this regard, Dailey et al. (29) reported that intratracheal administration of biodegradable polymeric nanoparticles to BALB/c mice did not induce pulmonary inflammation (measured as bronchoalveolar lavage fluid neutrophil influx, protein content, and lactate dehydrogenase activity), whereas nonbiodegradable polystyrene nanoparticles did. In addition to the treatment of lung diseases, the inhalation route is being explored for the systemic delivery of drugs to treat a variety of nonpulmonary ailments. This is due in part to the large surface area of the lungs and the relatively high bioavailability of many small molecules when administered by this route (113). As discussed below, human studies have not demonstrated systemic translocation of nanoparticles following inhalation, although some animal studies suggest that it is possible. Indeed, experimental animal data demonstrating achievement of therapeutic plasma drug levels following inhalation of nanoparticle-encapsulated antitubercular drugs (109, 110, 158) indicate that this approach may be feasible. Efforts to develop safe and effective nanoparticles for aerosol delivery are ongoing (33, 41, 52, 53, 124, 130) and will undoubtedly lead to significant advances in the treatment of respiratory and systemic diseases.

A simplified depiction of potential factors that may influence the effects of engineered nanoparticles on the respiratory system.

A simplified depiction of potential factors that may influence the effects of engineered nanoparticles on the respiratory system.

Fig. 2. From: Pulmonary applications and toxicity of engineered nanoparticles.

A simplified depiction of potential factors that may influence the effects of engineered nanoparticles on the respiratory system.
Jeffrey W. Card, et al. Am J Physiol Lung Cell Mol Physiol. 2008 September;295(3):L400-L411.
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Studies in humans.

As summarized elsewhere (7, 107), inhaled particles of different sizes exhibit different fractional depositions within the human respiratory tract. Although inhaled ultrafine particles (<100 nm) deposit in all regions, tracheobronchial deposition is highest for particles <10 nm in size, whereas alveolar deposition is highest for particles approximately 10–20 nm in size (7, 107). Particles <20 nm in size also efficiently deposit in the nasopharyngeal-laryngeal region. Human studies of potential adverse pulmonary effects resulting from exposure to engineered nanoparticles appear to be limited, although a number of investigations into pulmonary deposition patterns of inhaled nanoparticles in the healthy and diseased lung have been conducted (5, 24, 28, 93). Computational models predict increased deposition of inhaled nanoparticles in diseased or constricted airways (44), and, consistent with this prediction, obstructive lung disease and asthma have both been demonstrated to increase their pulmonary retention (5, 24). Nonetheless, Pietropaoli et al. (114) did not observe differences between healthy and asthmatic subjects in respiratory parameters assessed up to 45 h after a 2-h inhalation of ultrafine carbon particles (up to 25 μg/m2), nor was airway inflammation observed in either group (measured as exhaled nitric oxide). Moreover, the same study reported that exposure of healthy subjects to a higher concentration of ultrafine carbon particles (50 μg/m2 for 2 h) resulted in decreased midexpiratory flow rate and carbon monoxide diffusing capacity 21 h after exposure, albeit still in the absence of airway inflammation (114). Thus nanoparticles may influence respiratory function and gas exchange without a concomitant induction of inflammation.

Several studies have also examined the potential for inhaled manufactured ultrafine particles (i.e., 99mtechnetium-labeled carbon nanoparticles) to translocate from the lungs to the systemic circulation in humans. This is an important issue to consider as inhaled engineered nanoparticles may exert adverse cardiovascular effects, similar to the proposed mechanism for the nanoparticulate fraction of urban air pollution (15, 40). All but one of the studies reported to date indicate that inhaled 99mtechnetium-labeled carbon nanoparticles are not detected outside of the lungs in appreciable quantities after inhalation (17, 91, 93, 100, 150, 151). However, as alluded to by Mills et al. (91), these findings do not indicate that other nanoparticles will behave in the same manner, nor do they rule out the possibility that nanoparticles may interact with and influence the vasculature. Moreover, the studies conducted to date have used a single inhalation exposure protocol, and it is possible that repeated exposures may result in greater pulmonary accumulation and translocation of significant quantities of nanoparticles to the circulation.

Studies in experimental animals.

Pulmonary effects resulting from airway administration of nanoparticles have been examined in a number of experimental animal studies, a summary of which is presented in Table 2. Although the primary outcomes of interest in the majority of these studies have been pulmonary inflammation and fibrosis, several have investigated distribution patterns within the lung and the potential translocation and systemic distribution of nanoparticles following pulmonary administration; these are summarized in Table 3. In addition to the endpoints listed in Tables 2 and and3,3, carcinogenic effects of inhaled nanoparticles (ultrafine particles) have, in some instances, been found to be more severe than those of larger size analogs. This is thought to result primarily from lung particle overload due to the inability of alveolar macrophages to recognize and/or clear particles of this size, leading to particle build up, chronic inflammation, fibrosis, and tumorigenesis. These effects are discussed in detail elsewhere (14, 101) and will not be covered here.

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Improvements in the diagnosis and treatment of respiratory diseases as a result of the application of nanotechnology are anticipated, and experimental evidence indicates that engineered nanoparticles have unique properties that may render them beneficial in visualizing disease processes earlier and in delivering therapeutics to the lung, possibly even to specific areas within the lung. Using the lungs as a portal of entry for nanoparticles in the treatment of systemic diseases is also being explored and holds tremendous promise. However, nanotechnology is not without its limitations, and of foremost concern is the current lack of knowledge regarding the potential toxicity of engineered nanoparticles. As has been summarized here, a considerable amount of data from in vitro and in vivo studies indicates that nanoparticles have the capacity to exert adverse pulmonary effects, although not all nanoparticles are equivalent in this regard. In addition, in vitro toxicities are not always predictive of in vivo effects or potencies and vice versa, underscoring the need for the continued development and refinement of a suitable testing strategy for assessing the pulmonary effects of nanoparticles. It is anticipated that continued investigation into the mechanisms underlying the adverse in vitro and in vivo effects summarized in this review and their relevance to human lung physiology and disease will lead to a better understanding of the potential hazards associated with nanoparticle exposure and to the development of safe and effective respiratory medical applications and therapeutics based on nanotechnology.

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