Label-free nonlinear optical signatures of extracellular vesicles in liquid and tissue biopsies of human breast cancer.

Extracellular vesicles (EVs) have been implicated in metastasis and proposed as cancer biomarkers. However, heterogeneity and small size makes assessments of EVs challenging. Often, EVs are isolated from biofluids, losing spatial and temporal context and thus lacking the ability to access EVs in situ in their native microenvironment. This work examines the capabilities of label-free nonlinear optical microscopy to extract biochemical optical metrics of EVs in ex vivo tissue and EVs isolated from biofluids in cases of human breast cancer, comparing these metrics within and between EV sources. Before surgery, fresh urine and blood serum samples were obtained from human participants scheduled for breast tumor surgery (24 malignant, 6 benign) or healthy participants scheduled for breast reduction surgery (4 control). EVs were directly imaged both in intact ex vivo tissue that was removed during surgery and in samples isolated from biofluids by differential ultracentrifugation. Isolated EVs and freshly excised ex vivo breast tissue samples were imaged with custom nonlinear optical microscopes to extract single-EV optical metabolic signatures of NAD(P)H and FAD autofluorescence. Optical metrics were significantly altered in cases of malignant breast cancer in biofluid-derived EVs and intact tissue EVs compared to control samples. Specifically, urinary isolated EVs showed elevated NAD(P)H fluorescence lifetime in cases of malignant cancer, serum-derived isolated EVs showed decreased optical redox ratio in stage II cancer, but not earlier stages, and ex vivo breast tissue showed an elevated number of EVs in cases of malignant cancer. Results further indicated significant differences in the measured optical metabolic signature based on EV source (urine, serum and tissue) within individuals.

Surveying the Landscape: A Review of Longitudinal Traumatic Brain Injury Studies in Service Member and Veteran Populations.

Traumatic brain injury (TBI) is known to be a signature wound of the post-9/11 conflicts. In response, the U.S. Department of Defense (DOD) and other federal organizations have directed significant investments toward TBI research on characterizing injury populations and understanding long-term outcomes. To address legislative requirements and research gaps, several observational, longitudinal TBI studies were initiated as an effective means of investigating TBI clinical management, outcomes, and recovery. This review synthesizes the landscape (i.e., requirements and gaps, infrastructure, geography, timelines, TBI severity definitions, military and injury populations of interest, and measures) of DOD-funded longitudinal TBI studies being conducted in service member and veteran (SMV) populations. Based on the landscape described here, we present recommended actions and solutions that would allow a consolidated and cooperative future state of longitudinal TBI research, optimized continued investments, and advances in the state of the science without redundancy.

Computational Photon Counting Using Multithreshold Peak Detection for Fast Fluorescence Lifetime Imaging Microscopy.

Time-resolved photon counting methods have a finite bandwidth that restricts the acquisition speed of techniques like fluorescence lifetime imaging microscopy (FLIM). To enable faster imaging, computational methods can be employed to count photons when the output of a detector is directly digitized at a high sampling rate. Here, we present computational photon counting using a hybrid photodetector in conjunction with multithreshold peak detection to count instances where one or more photons arrive at the detector within the detector response time. This method can be used to distinguish up to five photon counts per digitized point, whereas previous demonstrations of computational photon counting on data acquired with photomultiplier tubes have only counted one photon at a time. We demonstrate in both freely moving C. elegans and a human breast cancer cell line undergoing apoptosis that this novel multithreshold peak detection method can accurately characterize the intensity and fluorescence lifetime of samples producing photon rates up to 223%, higher than previously demonstrated photon counting FLIM systems.

Military Traumatic Brain Injury: The History, Impact, and Future.

This review examines how lessons learned from United States military conflicts, beginning with the United States Civil War through the engagements in Iraq and Afghanistan, have shaped current traumatic brain injury (TBI) care in the United States military, influenced congressional mandates and directives, and led to best practices in caring for the warfighter. Prior to the most recent war, emphasis was placed on improving the surgical and medical care of service members (SM) with severe and especially penetrating brain injuries. However, during the Iraq and Afghanistan conflicts, also known as the Global War on Terrorism (GWOT), blast injury from improvised explosive devices most often caused mild TBI (mTBI), an injury that was not always recognized and was labelled the "signature wound" of the GWOT. This has led to extensive research on objective diagnostic technologies for mTBI, the association of mTBI with post-traumatic stress disorder (PTSD), and the long term consequences of mTBI. Here we summarize the key findings and most important advances from those efforts, and discuss the way forward regarding future military conflicts.

Label-free characterization of single extracellular vesicles using two-photon fluorescence lifetime imaging microscopy of NAD(P)H.

The heterogeneous nature of extracellular vesicles (EVs) creates the need for single EV characterization techniques. However, many common biochemical and functional EV analysis techniques lack single EV resolution. Two-photon fluorescence lifetime imaging microscopy (FLIM) is widely used to functionally characterize the reduced form of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H) in cells and tissues. Here, we demonstrate that FLIM can also be used to image and characterize NAD(P)H in single isolated EVs. EVs were isolated using standard differential ultracentrifugation techniques from multiple cell lines and imaged using a custom two-photon FLIM system. The presented data show that the NAD(P)H fluorescence lifetimes in isolated cell-derived EVs follow a wide Gaussian distribution, indicating the presence of a range of different protein-bound and free NAD(P)H species. EV NAD(P)H fluorescence lifetime distribution has a larger standard deviation than that of cells and a significantly different fluorescence lifetime distribution than the nuclei, mitochondria, and cytosol of cells. Additionally, changes in the metabolic conditions of cells were reflected in changes in the mean fluorescence lifetime of NAD(P)H in the produced EVs. These data suggest that FLIM of NAD(P)H could be a valuable tool for EV research.

Military TBI-What civilian primary care providers should know.

In June 2019, the Department of Veterans Affairs (VA) launched the VA Mission Act, which expanded veterans' health-care access to the private sector. Since civilian primary care providers may see more veterans in their practice, it will be important to understand the unique experiences, comorbidities, and culture of this population in order to provide optimal care. Military service members (SMs) are at an increased risk for traumatic brain injury (TBI), and comorbidities, such as post traumatic stress disorder (PTSD), increasing the likelihood of prolonged symptoms. Military training and repetitive low-level blast exposure may cause symptoms similar to TBI or increase long-term negative effects in SMs. Military culture often has a strong influence in this population. Those who serve in the military identify with military values and have a strong team mentality, which places emphasis on the mission above all else, not accepting defeat, and not ever leaving a fellow SM behind. These values can impact the way a SM/veteran seeks care and/or communicates with his or her health-care provider. Taking a detailed history to understand how these factors apply, as well as screening for mental health comorbidities, are recommended. Understanding the military cultural influences can assist in promoting a stronger therapeutic alliance and encourage more open communication. Ultimately, it is the trusting and respectful relationship between the SM/veteran and the provider that will determine the most effective treatment and result in the most effective resolution of TBI and comorbid symptoms.

Mild TBI/Concussion Clinical Tools for Providers Used Within the Department of Defense and Defense Health Agency.

BACKGROUND: Military personnel are at greater risk for sustaining mild traumatic brain injury (mTBI), or concussion, whether they are in a combat or garrison setting. Consequently, mTBI is a major health concern for health practitioners to understand, in order to provide timely assessment and treatment to service members (SM) who are suspected to have mTBI. OBSERVATIONS: Providing early diagnosis and effective management of symptoms can optimize recovery and promote healthy outcomes. Understanding what resources and guidelines are available is important for those evaluating active duty SMs within the Military Health System. CONCLUSIONS: This article showcases clinical tools for screening, evaluating, and diagnosing concussion used within the US Department of Defense, and provides resources for practitioners to find these clinical tools online.

Mortality Following Hospital Admission for US Active Duty Service Members Diagnosed With Penetrating Traumatic Brain Injury, 2004-2014.

OBJECTIVE: To examine mortality among active duty US military service members (SMs) with the diagnosis of penetrating traumatic brain injury (PTBI) and a hospital admission between 2004 and 2014. DESIGN: Data on SMs with PTBI and an admission to a military or civilian hospital were obtained from the 2004 to 2014 Military Health System data repository. After applying exclusion criteria, data on 1226 SMs were analyzed. MAIN MEASURES: The number of observed deaths per 100 identified patients with PTBI and time to death from admission were used as main measures. RESULTS: Approximately 25% of the 1226 patients with PTBI included in this study died following admission, with 44.6% of the all deaths occurring within a day following hospital admission and 75% occurring within the first week. Severe comorbid conditions and intentionally self-inflicted injuries are associated with higher mortality rate. SMs' gender, age, year of hospital admission, and service were significantly associated with likelihood of death following PTBI hospitalization. Males had a higher likelihood of dying following hospital admission compared with females (odds ratio = 2.7, confidence interval = 1.03-7.9). SMs in the 35- to 44-year-old and 45- to 64-year-old groups had up to a 2.6 times higher odds of death following their admission compared with the 25- to 34-year-old group. Age, admission year, service, and rank were significantly associated with SMs' time to death from hospitalization. Patients between the ages of 45 and 64 years were significantly more likely to die earlier than other age groups. Furthermore, cases in the Navy Afloat group had a higher fatality rate and were more likely to die earlier than patients in other services. PTBI comorbid conditions and injury type did not significantly affect time to death. CONCLUSION: This study quantifies case fatality rate among hospitalized US SMs with the diagnosis of PTBI. We report a 23.1% crude case fatality rate among the current cohort. Early intensive care for these patients may be the key to improving survival rates.

A Needs Review of Caregivers for Adults With Traumatic Brain Injury.

A literature review of the stresses of caregivers of active-duty service members and veterans with a traumatic brain injury provides clinicians with the information and resources they can use in caring for this patient population.

TNFR2 maintains adequate IL-12 production by dendritic cells in inflammatory responses by regulating endogenous TNF levels.

Sepsis-induced immune reactions are reduced in TNF receptor 2 (TNFR2)-deficient mice as previously shown. In order to elucidate the underlying mechanisms, the functional integrity of myeloid cells of TNFR2-deficient mice was analyzed and compared to wild type (WT) mice. The capacity of dendritic cells to produce IL-12 was strongly impaired in TNF-deficient mice, mirroring impaired production of IL-12 by WT dendritic cells in sepsis or after LPS or TNF pre-treatment. In addition, TNFR2-deficient mice were refractory to LPS pre-treatment and also to hyper-sensitization by inactivated Propionibacterium acnes, indicating habituation to inflammatory stimuli by the immune response when TNFR2 is lacking. Constitutive expression of TNF mRNA in kidney, liver, spleen, colon and lung tissue, and the presence of soluble TNFR2 in urine of healthy WT mice supported the conclusion that TNF is continuously present in naïve mice and controlled by soluble TNFR2. In TNFR2-deficient mice endogenous TNF levels cannot be balanced and the continuous exposure to enhanced TNF levels impairs dendritic cell function. In conclusion, TNF pre-exposure suppresses secondary inflammatory reactions of myeloid cells; therefore, continuous control of endogenous TNF by soluble TNFR2 seems to be essential for the maintenance of adequate sensitivity to inflammatory stimuli.

Sepsis leads to a reduced antigen-specific primary antibody response.

Immunosuppression, impaired cytokine production and high susceptibility to secondary infections are characteristic for septic patients, and for mice after induction of polymicrobial septic peritonitis by sublethal cecal ligation and puncture (CLP). Here, we demonstrate that CLP markedly altered subsequent B-cell responses. Total IgG and IgM levels, as well as the memory B-cell response, were increased in septic mice, but antigen-specific primary antibody production was strongly impaired. We found that two days after CLP, CD11b(+) splenocytes were activated as demonstrated by the increased expression of activation markers, expression of arginase and production of NO by immature myeloid cells. The in vivo clearance of a bacterial infection was not impaired. DCs demonstrated reduced IL-12 production and altered antigen presentation, resulting in decreased proliferation but enhanced IFN-γ production by CD4(+) cells. CD4(+) T cells from mice immunized on day 2 after CLP showed reduced Th1 and Th2 cytokine production. In addition, there was an increase in Treg cells. Interestingly, levels of immature B cells decreased but levels of mature B cells increased two days after CLP. However, adoptive transfer of naïve CD4(+) T cells, naïve B cells, or naïve DCs did not rescue the antigen-specific antibody response.