Optimizing reduction of western blotting analytical variations: Use of replicate test samples, multiple normalization methods, and sample loading positions.

Western blot (WB) analysis is widely used, but obtaining consistent results can be problematic, especially when using multiple gels. This study examines WB performance by explicitly applying a method commonly used to test analytical instrumentation. Test samples were lysates from RAW 264.7 murine macrophages treated with LPS to activate MAPK and NF-kB signaling targets. Samples from the pooled cell lysates placed in every lane on multiple gels were analyzed by WBs for levels of p-ERK, ERK, IkBß and non-target protein. Different normalization methods and sample groupings were applied to the density values and the resulting coefficients of variation (CV) and ratios of maximal to minimal values (Max/Min) were compared. Ideally with identical sample replicates the CVs would be 0 and the Max/Min 1; deviation indicating introduction of variability by the WB process. Common normalizations to reduce analytical variance, total lane protein, % Control, and p-ERK/ERK ratios, did not have the lowest CVs or Max/Min values. Normalization using the sum of target protein values combined with analytical replication most effectively reduced variability, resulting CV and Max/Min values as low as 5-10% and 1.1. These methods should allow reliable interpretation of complex experiments that require samples to be placed on multiple gels.

Celebrating 60 Years of Accomplishments of the Armed Forces Radiobiology Research Institute1.

Chartered by the U.S. Congress in 1961, the Armed Forces Radiobiology Research Institute (AFRRI) is a Joint Department of Defense (DoD) entity with the mission of carrying out the Medical Radiological Defense Research Program in support of our military forces around the globe. In the last 60 years, the investigators at AFRRI have conducted exploratory and developmental research with broad application to the field of radiation sciences. As the only DoD facility dedicated to radiation research, AFRRI's Medical Radiobiology Advisory Team provides deployable medical and radiobiological subject matter expertise, advising commanders in the response to a U.S. nuclear weapon incident and other nuclear or radiological material incidents. AFRRI received the DoD Joint Meritorious Unit Award on February 17, 2004, for its exceptionally meritorious achievements from September 11, 2001 to June 20, 2003, in response to acts of terrorism and nuclear/radiological threats at home and abroad. In August 2009, the American Nuclear Society designated the institute a nuclear historic landmark as the U.S.'s primary source of medical nuclear and radiological research, preparedness and training. Since then, research has continued, and core areas of study include prevention, assessment and treatment of radiological injuries that may occur from exposure to a wide range of doses (low to high). AFRRI collaborates with other government entities, academic institutions, civilian laboratories and other countries to research the biological effects of ionizing radiation. Notable early research contributions were the establishment of dose limits for major acute radiation syndromes in primates, applicable to human exposures, followed by the subsequent evolution of radiobiology concepts, particularly the importance of immune collapse and combined injury. In this century, the program has been essential in the development and validation of prophylactic and therapeutic drugs, such as Amifostine, Neupogen®, Neulasta®, Nplate® and Leukine®, all of which are used to prevent and treat radiation injuries. Moreover, AFRRI has helped develop rapid, high-precision, biodosimetry tools ranging from novel assays to software decision support. New drug candidates and biological dose assessment technologies are currently being developed. Such efforts are supported by unique and unmatched radiation sources and generators that allow for comprehensive analyses across the various types and qualities of radiation. These include but are not limited to both 60Co facilities, a TRIGA® reactor providing variable mixed neutron and γ-ray fields, a clinical linear accelerator, and a small animal radiation research platform with low-energy photons. There are five major research areas at AFRRI that encompass the prevention, assessment and treatment of injuries resulting from the effects of ionizing radiation: 1. biodosimetry; 2. low-level and low-dose-rate radiation; 3. internal contamination and metal toxicity; 4. radiation combined injury; and 5. radiation medical countermeasures. These research areas are bolstered by an educational component to broadcast and increase awareness of the medical effects of ionizing radiation, in the mass-casualty scenario after a nuclear detonation or radiological accidents. This work provides a description of the military medical operations as well as the radiation facilities and capabilities present at AFRRI, followed by a review and discussion of each of the research areas.

Measuring type I interferon using reporter gene assays based on readily available cell lines.

Cell lines stably transfected with genes responding to Type I interferons (IFN) are potentially a useful alternative to enzyme linked immuo-assays (ELISAs) or assays based on resistance of a test cell line to virus infection using cell death or infection endpoints. Increasingly available are a variety of commercial cell lines developed for reporter gene assays (RGAs) which are responsive to IFN exposure. These cells produce a soluble gene product which can be readily quantified using multiwell plate spectrophotometers or luminometers. We have investigated RAW-Blue ISG™ and B16-Blue IFNα/ß™ cells (InvivoGen) which produce secreted embryonic alkaline phosphatase (SEAP) as a RGA to measure Interferon alpha (IFNα) and beta (IFNß). These cells showed a log-linear response over 4 logs of IFN concentration between 10 and 100,000 Units/ml (U/ml). Concentration dependent responses could be observed as early as 6 h but greater sensitivity was obtained at 24 h. Neutralizing antibodies to IFNα and IFNß reduced the response to baseline. As proof of principle supernatants from RAW 264.7 (murine macrophage; parental cell line) infected with 1 multiplicity of infection (moi) of influenza A virus (X31/H3N2) were used as test samples. Pre-treatment of the supernatant with anti-IFNα failed to reduce the cell response but it was reduced to background by anti-IFNß. The high level of IFNß but very low level of IFNα was confirmed by ELISA. Availability, ease of use and maintenance, and possible cost savings make application of this reporter gene cell approach a valuable alternative to other methods for measuring Type I interferon.

Early activation of MAP kinases by influenza A virus X-31 in murine macrophage cell lines.

Early molecular responses to Influenza A (FLUA) virus strain A/X-31 H3N2 in macrophages were explored using J774.A1 and RAW 264.7 murine cell lines. NF-kappa B (NFκB) was reported to be central to FLUA host-response in other cell types. Our data showed that FLUA activation of the classical NFκB dependent pathway in these macrophages was minimal. Regulator proteins, IkappaB-alpha and -beta (IκBα, IκBß), showed limited degradation peaking at 2 h post FLUA exposure and p65 was not observed to translocate from the cytoplasm to the nucleus. Additionally, the non-canonical NFκB pathway was not activated in response to FLUA. The cells did display early increases in TNFα and other inflammatory cytokine and chemokine production. Mitogen activated phosphokinase (MAPK) signaling pathways are also reported to control production of inflammatory cytokines in response to FLUA. The activation of the MAPKs, cJun kinases 1 and 2 (JNK 1/2), extracellular regulated kinases 1 and 2 (ERK 1/2), and p38 were investigated in both cell lines between 0.25 and 3 h post-infection. Each of these kinases showed increased phosphorylation post FLUA exposure. JNK phosphorylation occurred early while p38 phosphorylation appeared later. Phosphorylation of ERK 1/2 occurred earlier in J774.A1 cells compared to RAW 264.7 cells. Inhibition of MAPK activation resulted in decreased production of most FLUA responsive cytokines and chemokines in these cells. The results suggest that in these monocytic cells the MAPK pathways are important in the early response to FLUA.

A multidisciplinary approach to teach responses to weapons of mass destruction and terrorism using combined simulation modalities.

STUDY OBJECTIVE: To reinforce concepts presented in the lectures; understand the complexity and speed of casualty and information generation during a Weapons of Mass Destruction and Terrorism (WMD/T) event; experience the novelty of combined weapons' effects; recognize the time course of the various chemical, biological, and radiation agents; and make challenging decisions with incomplete and conflicting information. SETTINGS: Two environments simulated simultaneously: one a major trauma center emergency room (ER) with two patient simulators and several human actors; the other an Emergency Operations Command Center (EOC). TARGET AUDIENCE: Students for this course included: clinicians, scientists, military and intelligence officers, lawyers, administrators, and logistic personnel whose jobs involve planning and executing emergency response plans to WMD/T. SIMULATION SCRIPT: A WMD/T attack in Washington, D.C., has occurred. Clinical students performed in their real life roles in the simulated ER, while nonclinical students did the same in the simulated EOC. Six ER casualties with combined WMD/T injuries were presented and treated over 40 minutes. In the EOC, each person was given his or her role title with identification tag. The EOC scenario took cues from the action in the ER via two television (TV) news feeds and telephone calls from other Emergency Operations Assets. PERFORMANCE EXPECTATIONS: Students were expected to actively engage in their roles. Student performances were self-evaluated during the debriefing. DEBRIEFING: The two groups were reunited and debriefed utilizing disaster crisis resource management tools. ASSESSMENT OF EFFECTIVENESS: Students answered an 18-point questionnaire to help evaluate the usefulness and acceptance of multimodality patient simulation. LESSONS LEARNED: Large-scale multimodality patient simulation can be used to train both clinicians and nonclinicians for future events of WMD/T. Students accepted the simulation experience and thought that scenario was appropriately realistic, complex, and overwhelming. Difficulties include the extensive man-hours involved in designing and presenting the live simulations. EOC-only sessions could be staged with only a few video cassette recorders, TVs, telephones, and callers.

A Multidisciplinary Approach to Teach Responses to Weapons of Mass Destruction and Terrorism Using Combined Simulation Modalities.

STUDY OBJECTIVE: To reinforce concepts presented in the lectures; understand the complexity and speed of casualty and information generation during a Weapons of Mass Destruction and Terrorism (WMD/T) event; experience the novelty of combined weapons' effects; recognize the time course of the various chemical, biological, and radiation agents; and make challenging decisions with incomplete and conflicting information. SETTINGS: Two environments simulated simultaneously: one a major trauma center emergency room (ER) with two patient simulators and several human actors; the other an Emergency Operations Command Center (EOC). TARGET AUDIENCE: Students for this course included: clinicians, scientists, military and intelligence officers, lawyers, administrators, and logistic personnel whose jobs involve planning and executing emergency response plans to WMD/T. SIMULATION SCRIPT: A WMD/T attack in Washington, D.C., has occurred. Clinical students performed in their real life roles in the simulated ER, while nonclinical students did the same in the simulated EOC. Six ER casualties with combined WMD/T injuries were presented and treated over 40 minutes. In the EOC, each person was given his or her role title with identification tag. The EOC scenario took cues from the action in the ER via two television (TV) news feeds and telephone calls from other Emergency Operations Assets. PERFORMANCE EXPECTATIONS: Students were expected to actively engage in their roles. Student performances were self-evaluated during the debriefing. DEBRIEFING: The two groups were reunited and debriefed utilizing disaster crisis resource management tools. ASSESSMENT OF EFFECTIVENESS: Students answered an 18-point questionnaire to help evaluate the usefulness and acceptance of multimodality patient simulation. LESSONS LEARNED: Large-scale multimodality patient simulation can be used to train both clinicians and nonclinicians for future events of WMD/T. Students accepted the simulation experience and thought that scenario was appropriately realistic, complex, and overwhelming. Difficulties include the extensive man-hours involved in designing and presenting the live simulations. EOC-only sessions could be staged with only a few video cassette recorders, TVs, telephones, and callers.

Active and inactive influenza virus induction of tumor necrosis factor-alpha and nitric oxide in J774.1 murine macrophages: modulation by interferon-gamma and failure to induce apoptosis.

Infection of J774.1 murine macrophages by influenza A virus (IAV) induces two major responses, production of host defense molecules and death by apoptosis. We investigated whether induction of two cytotoxic compounds, tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO), directly caused IAV-induced apoptosis, and whether induction could be modulated by interferon-gamma (IFN-gamma) or the replication competence of the virus. Live IAV potently induced production of both TNF-alpha and NO, but UV inactivated virus was a poor inducer of both molecules. When cells were pre-treated with IFN-gamma, inactive IAV became as effective an inducer of NO, but not TNF-alpha, as live IAV. Amantadine, which antagonizes viral entry and replication, partly inhibited TNF-alpha and NO production in unprimed cells, but did not inhibit NO in IFN-gamma primed cells. IAV-induced cytotoxicity was not due to the induction of TNF-alpha or NO. Cells were insensitive to either TNF-alpha-containing supernatants or to recombinant TNF-alpha. Anti-TNF-alpha antibody did not protect cells from IAV-induced cell death, and anti-oxidants that inhibited TNF-alpha production also failed to increase cell survival. Inhibitors of NO production did not protect from IAV-induced cell death, either alone or in combination with superoxide dismutase (SOD). We conclude that, even though IAV was a potent inducer of TNF-alpha and NO in macrophages, IAV-induced apoptosis was not mediated directly by them. Importantly, viral replication was not required for the induction of TNF-alpha or NO, and the action of inactive IAV could be potentiated by IFN-gamma.

Influenza virus induction of apoptosis by intrinsic and extrinsic mechanisms.

It is now firmly established that apoptosis is an important mechanism of influenza virus-induced cell death both in vivo and in vitro. Data are predominantly from experiments with influenza A virus and in vitro experimental systems. Multiple influenza virus factors have been identified that can activate intrinsic or extrinsic apoptotic induction pathways. Currently there is no evidence for influenza virus directly accessing the apoptosis execution factors. The best-studied influenza virus inducers of apoptosis are dsRNA, NS1, NA, and a newly described gene product PB1-F2. PB1-F2 is the only influenza virus factor to date identified to act intrinsically by localization and interaction with the mitochondrial-dependent apoptotic pathway. Both dsRNA and NA have been shown to act via an extrinsic mechanism involving proapoptotic host-defense molecules: PKR by induction of Fas-Fas ligand and NA by activation of TGF-beta. PKR is capable of controlling several important cell-signaling pathways and therefore may have multiple effects; a predominant one is increased interferon (IFN) production and activity. NS1 has been shown to be both proapoptotic and antiapoptotic. Use of influenza virus NS1 deletion mutants has provided evidence for NS1 interference with apoptosis, IFN induction, and related cell-signaling pathways. Influenza virus also has important exocrine paracrine effects, which are likely mediated via TNF family ligands and oxygen, free radicals capable of inducing apoptosis. Little is known about activation of inhibitors of apoptosis such as inhibitory apoptotic proteins. Whether all these factors always have a role in influenza virus-induced apoptosis is unknown. The kinetics of synthesis of influenza virus factors affecting apoptosis during the replication cycle may be an important aspect of apoptosis induction.