Multiple Organ Dysfunction in Older Major Trauma Critical Care Patients: A Multicenter Prospective Observational Study.

The objective was to explore the characteristics and outcomes of multiple organ dysfunction syndrome (MODS) in older trauma patients. Background: Severely injured older people present an increasing challenge for trauma systems. Recovery for those who require critical care may be complicated by MODS. In older trauma patients, MODS may not be predictable based on chronological age alone and factors associated with its development and resolution are unclear. Methods: Consecutive adult patients (aged ≥16 years) admitted to 4 level 1 major trauma center critical care units were enrolled and reviewed daily until discharge or death. MODS was defined by a daily total sequential organ failure assessment score of >5. Results: One thousand three hundred sixteen patients were enrolled over 18 months and one-third (434) were aged ≥65 years. Incidence of MODS was high for both age groups (<65 years: 64%, ≥65 years: 70%). There were few differences in severity, patterns, and duration of MODS between cohorts, except for older traumatic brain injury (TBI) patients who experienced a prolonged course of MODS recovery (TBI: 9 days vs no TBI: 5 days, P < 0.01). Frailty rather than chronological age had a strong association with MODS development (odds ratio [OR], 6.9; 95% confidence intervals [CI], 3.0-12.4; P < 0.001) and MODS mortality (OR, 2.1; 95% CI, 1.31-3.38; P = 0.02). Critical care resource utilization was not increased in older patients, but MODS had a substantial impact on mortality (<65 years: 17%; ≥65 years: 28%). The majority of older patients who did not develop MODS survived and had favorable discharge outcomes (home discharge ≥65 years NoMODS: 50% vs MODS: 15%; P < 0.01). Conclusions: Frailty rather than chronological age appears to drive MODS development, recovery, and outcome in older cohorts. Early identification of frailty after trauma may help to predict MODS and plan care in older trauma.

Multicenter phase II trial (SWOG S1609, cohort 51) of ipilimumab and nivolumab in metastatic or unresectable angiosarcoma: a substudy of dual anti-CTLA-4 and anti-PD-1 blockade in rare tumors (DART).

PURPOSE: Angiosarcoma is a rare aggressive endothelial cell cancer with high mortality. Isolated reports suggest immune checkpoint inhibition efficacy in angiosarcoma, but no prospective studies have been published. We report results for angiosarcoma treated with ipilimumab and nivolumab as a cohort of an ongoing rare cancer study. METHODS: This is a prospective, open-label, multicenter phase II clinical trial of ipilimumab (1 mg/kg intravenously every 6 weeks) plus nivolumab (240 mg intravenously every 2 weeks) for metastatic or unresectable angiosarcoma. Primary endpoint was objective response rate (ORR) per RECIST 1.1. Secondary endpoints include progression-free (PFS) and overall survival, and toxicity. A two-stage design was used. RESULTS: Overall, there were 16 evaluable patients. Median age was 68 years (range, 25-81); median number of prior lines of therapy, 2. Nine patients had cutaneous and seven non-cutaneous primary tumors. ORR was 25% (4/16). Sixty per cent of patients (3/5) with primary cutaneous scalp or face tumors attained a confirmed response. Six-month PFS was 38%. Altogether, 75% of patients experienced an adverse event (AE) (at least possibly related to drug) (25% grade 3-4 AE); 68.8%, an immune-related AE (irAE) (2 (12.5%), grade 3 or 4 irAEs (alanine aminotransferase/aspartate aminotransferase increase and diarrhea)). There were no grade 5 toxicities. One of seven patients in whom tumor mutation burden (TMB) was assessed showed a high TMB (24 mutations/mb); that patient achieved a partial response (PR). Two of three patients with PDL1 immunohistochemistry assessed had high PDL1 expression; one achieved a PR. CONCLUSION: The combination of ipilimumab and nivolumab demonstrated an ORR of 25% in angiosarcoma, with three of five patients with cutaneous tumors of the scalp or face responding. Ipilimumab and nivolumab warrant further investigation in angiosarcoma. TRIAL REGISTRATION NUMBER: NCT02834013.

Facilitating diabetic retinopathy screening using automated retinal image analysis in underresourced settings.

AIM: To evaluate an automated retinal image analysis (ARIA) of indigenous retinal fundus images against a human grading comparator for the classification of diabetic retinopathy (DR) status. METHODS: Indigenous Australian adults with type 2 diabetes (n = 410) from three remote and very remote primary-care services in the Northern Territory, Australia, underwent teleretinal DR screening. A single, central retinal fundus photograph (opportunistic mydriasis) for each eye was later regraded using a single ARIA and a UK human grader and national DR classification system. The sensitivity and specificity of ARIA were assessed relative to the comparator. Proportionate agreement and a Kappa statistic were also computed. RESULTS: Retinal images from 391 and 393 participants were gradable for 'Any DR' by the human grader and ARIA grader, respectively. 'Any DR' was detected by the human grader in 185 (47.3%) participants and by ARIA in 202 (48.6%) participants (agreement =88.0%, Kappa = 0.76,), whereas proliferative DR was detected in 31 (7.9%) and 37 (9.4%) participants (agreement = 98.2%, Kappa = 0.89,), respectively. The ARIA software had 91.4 (95% CI, 86.3-95.0) sensitivity and 85.0 (95% CI, 79.3-89.5) specificity for detecting 'Any DR' and 96.8 (95% CI, 83.3-99.9) sensitivity and 98.3 (95% CI, 96.4-99.4) specificity for detecting proliferative DR. CONCLUSIONS: This ARIA software has high sensitivity for detecting 'Any DR', hence could be used as a triage tool for human graders. High sensitivity was also found for detection of proliferative DR by ARIA. Future versions of this ARIA should include maculopathy and referable DR (CSME and/or PDR). Such ARIA software may benefit diabetes care in less-resourced regions.

The impact of steeping, germination and hydrothermal processing of wheat (Triticum aestivum L.) grains on phytate hydrolysis and the distribution, speciation and bio-accessibility of iron and zinc elements.

Chelation of iron and zinc in wheat as phytates lowers their bio-accessibility. Steeping and germination (15 °C, 120 h) lowered phytate content from 0.96% to only 0.81% of initial dry matter. A multifactorial experiment in which (steeped/germinated) wheat was subjected to different time (2-24 h), temperature (20-80 °C) and pH (2.0-8.0) conditions showed that hydrothermal processing of germinated (15 °C, 120 h) wheat at 50 °C and pH 3.8 for 24 h reduced phytate content by 95%. X-ray absorption near-edge structure imaging showed that it indeed abolished chelation of iron to phytate. It also proved that iron was oxidized during steeping, germination and hydrothermal processing. It was further shown that zinc and iron bio-accessibility were respectively 3 and 5% in wheat and 27 and 37% in hydrothermally processed wheat. Thus, hydrothermal processing of (germinated) wheat paves the way for increasing elemental bio-accessibility in whole grain-based products.

Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice.

Human exposure to asbestos can cause a wide variety of lung diseases that are still a current major health concern, even if asbestos has been banned in many countries. It has been shown in many studies that asbestos fibers, ingested by alveolar macrophages, disrupt lung iron homeostasis by sequestering iron. Calcium can also be deposited on the fibers. The pathways along which iron and above all calcium interact with fibers are still unknown. Our aim was that of investigating if the iron accumulation induced by the inhaled asbestos fibers also involves calcium ions accumulation. Lung sections of asbestos-exposed mice were analyzed using an extremely sensitive procedure available at the synchrotron facilities, that provides morphological and chemical information based on X-ray fluorescence microspectroscopy (µ-XRF). In this study we show that (1) where conventional histochemical procedures revealed only weak deposits of iron and calcium, µ-XRF analysis is able to detect significant deposits of both iron and calcium on the inhaled asbestos fibers; (2) the extent of the deposition of these ions is proportionally directly related and (3) iron and calcium deposition on inhaled asbestos fibers is concomitant with the appearance of inflammatory and hyperplastic reactions.

The use of spectroscopic imaging and mapping techniques in the characterisation and study of DLD-1 cell spheroid tumour models.

Determining the chemical and biological compositions of the tumour models used in pharmacological studies is crucial for understanding the interactions between the drug molecules and the tumour micro-environment. Conventional techniques for spheroid characterisation require intensive chemical pre-treatments that result in the removal of unbound metabolites. In this study, the spectroscopic techniques, scanning transmission ion microscopy (STIM), proton-induced X-ray emission (PIXE) mapping, scanning X-ray fluorescence microscopy (SXFM), and Fourier transform infrared (FT-IR) imaging were employed to gain complementary information on the compositions of untreated DLD-l cancer cell spheroids. When used together, these techniques exhibited great potential for providing a comprehensive over-view of the density, biochemistry and elemental compositions within the different regions of the spheroids. STIM density and elemental maps correlated well with cellular density across the spheroid, and showed the accumulation of S, Cu and various lighter elements in the necrotic region. High levels of oxidative stress were evident in the hypoxic region, and different degrees of cellular necrosis as well as high levels of lactate and collagen within the necrotic region were suggested by FT-IR markers. FT-IR imaging was further employed to study the pharmacodynamics of known the cytotoxins, cisplatin and Pt1C3. Cisplatin was observed to induce minimal biochemical changes to the spheroids following 24 hour incubations, whereas Pt1C3 caused severe cellular damage to the spheroid periphery; consistent with their different modes of action.

In situ distribution and speciation of toxic copper, nickel, and zinc in hydrated roots of cowpea.

The phytotoxicity of trace metals is of global concern due to contamination of the landscape by human activities. Using synchrotron-based x-ray fluorescence microscopy and x-ray absorption spectroscopy, the distribution and speciation of copper (Cu), nickel (Ni), and zinc (Zn) was examined in situ using hydrated roots of cowpea (Vigna unguiculata) exposed to 1.5 µm Cu, 5 µm Ni, or 40 µm Zn for 1 to 24 h. After 24 h of exposure, most Cu was bound to polygalacturonic acid of the rhizodermis and outer cortex, suggesting that binding of Cu to walls of cells in the rhizodermis possibly contributes to the toxic effects of Cu. When exposed to Zn, cortical concentrations remained comparatively low with much of the Zn accumulating in the meristematic region and moving into the stele; approximately 60% to 85% of the total Zn stored as Zn phytate within 3 h of exposure. While Ni concentrations were high in both the cortex and meristem, concentrations in the stele were comparatively low. To our knowledge, this is the first report of the in situ distribution and speciation of Cu, Ni, and Zn in hydrated (and fresh) plant tissues, providing valuable information on the potential mechanisms by which they are toxic.

Tunable reagents for multi-functional bioconjugation: reversible or permanent chemical modification of proteins and peptides by control of maleimide hydrolysis.

Controlling maleimide hydrolysis allows the modular construction of bromomaleimide-mediated bioconjugates which are either stable or cleavable in an aqueous, thiol-mediated reducing environment. The application of this methodology to reversible protein biotinylation, the irreversible labeling of peptide disulfide bonds and the assembly of stable, fluorescein-labelled glycoprotein mimics is described.

In situ maleimide bridging of disulfides and a new approach to protein PEGylation.

The introduction of non-natural entities into proteins by chemical modification has numerous applications in fundamental biological science and for the development and manipulation of peptide and protein therapeutics. The reduction of native disulfide bonds provides a convenient method to access two nucleophilic cysteine residues that can serve as ideal attachment points for such chemical modification. The optimum bioconjugation strategy utilizing these cysteine residues should include the reconstruction of a bridge to mimic the role of the disulfide bond, maintaining structure and stability of the protein. Furthermore, the bridging chemical modification should be as rapid as possible to prevent problems associated with protein unfolding, aggregation, or disulfide scrambling. This study reports on an in situ disulfide reduction-bridging strategy that ensures rapid sequestration of the free cysteine residues in a bridge, using dithiomaleimides. This approach is then used to PEGylate the peptide hormone somatostatin and retention of biological activity is demonstrated.

Protein modification, bioconjugation, and disulfide bridging using bromomaleimides.

The maleimide motif is widely used for the selective chemical modification of cysteine residues in proteins. Despite widespread utilization, there are some potential limitations, including the irreversible nature of the reaction and, hence, the modification and the number of attachment positions. We conceived of a new class of maleimide which would address some of these limitations and provide new opportunities for protein modification. We report herein the use of mono- and dibromomaleimides for reversible cysteine modification and illustrate this on the SH2 domain of the Grb2 adaptor protein (L111C). After initial modification of a protein with a bromo- or dibromomaleimide, it is possible to add an equivalent of a second thiol to give further bioconjugation, demonstrating that bromomaleimides offer opportunities for up to three points of attachment. The resultant protein-maleimide products can be cleaved to regenerate the unmodified protein by addition of a phosphine or a large excess of a thiol. Furthermore, dibromomaleimide can insert into a disulfide bond, forming a maleimide bridge, and this is illustrated on the peptide hormone somatostatin. Fluorescein-labeled dibromomaleimide is synthesized and inserted into the disulfide to construct a fluorescent somatostatin analogue. These results highlight the significant potential for this new class of reagents in protein modification.

The application of synchrotron radiation induced X-ray emission in the measurement of zinc and lead in Wistar rat ameloblasts.

The development of analytical techniques for the measurement of trace elements in cellular compartments of developing teeth remains an important methodological issue in dental research. Recent advances in third generation synchrotron facilities have provided high brilliance X-ray sources that can be effectively used to study trace element distributions in small spatial regions with low detection limits. The present study describes for the first time the application of synchrotron radiation induced X-ray emission (SRIXE) in measuring the distribution of zinc and lead in the ameloblasts of developing Wistar rat teeth. Wistar rats were fed a standard rat diet, containing the normal dietary requirements of zinc, ad libitum and exposed to 100 ppm of lead in drinking water. Resin embedded sections of first mandibular molars were analysed using a 13.3 keV incident monochromatic X-ray beam focussed to a 0.2 microm spot. Characteristic X-rays arising from the entire thickness of the sample were measured using an energy dispersive detector for quantitative analysis of elemental concentrations. The results showed that intranuclear concentrations of zinc were greater than levels in the cytoplasm. Furthermore, nuclear and cytoplasmic concentrations of zinc in the maturation stage (742+/-27 and 424+/-25 ppm, respectively) were significantly higher than the zinc levels observed in the nucleus and cytoplasm of presecretory stage ameloblasts (132+/-10 and 109+/-10 ppm, respectively) (p<0.05). A clear lead signal above the background was not detected in the ameloblasts and lead concentrations could only be reliably measured in the developing enamel. Overall, SRIXE was an effective method of studying the spatial distribution of zinc in the cells of developing teeth and offered a unique combination of sub-micron spatial resolution and parts-per-million detection limits (0.8-1 and 0.6-1 ppm for zinc and lead, respectively).

Self-reported health: reliability and consequences for health inequality measurement.

Self-reported health (SRH) is one of the most frequently employed measures for assessing income-related health inequalities between counties. A previous study has shown that 28% of respondents changed their assessment of their health status when asked a SRH question on two occasions in the same survey (first as part of self-completed questionnaire and then in a personal interview). This study re-examines this issue using another survey where SRH was again asked twice of respondents, but this time the personal interview was first and self-completion second. We find the same variation in responses, but the predominant direction is away from the 'extreme' categories 'Excellent' and 'Poor' which is the opposite direction to the previous study. We therefore conclude that the most likely explanation is a mode of administration effect that makes people less likely to choose the extreme categories in a self-completion questionnaire, but not a personal interview. However, this effect has a relatively minor impact on measures of inequality. This is due to a large proportion of the movement (i.e. movement to the middle) not being related to income and hence does not systematically impact on the cumulative distribution of health across this measure of socio-economic status.

Spatial distribution of lead in enamel and coronal dentine of wistar rats.

Lead is one of the most hazardous environmental toxins known. The assessment of lead in dental hard tissues is important in the understanding of its toxic effects on oral tissues and in estimating exposure and body burden in individuals exposed to lead from the environment. However, current information on the uptake and distribution of lead in enamel and dentine is limited. The aim of this project was to study, at high resolution, the spatial distribution of lead in enamel and coronal dentine using an experimental rat model. A dose of 40 mg/L of lead nitrate was administered to pregnant female rats during the periods of gestation and lactation through drinking water. First mandibular molar teeth were removed from their 15-d-old pups and the distribution of lead was studied using a nuclear microprobe (NMP). The distribution of lead in enamel and coronal dentine showed four distinct zones with significantly different mean lead concentrations (p<0.05). High levels of lead were observed in the superficial regions of enamel and in the dentine directly adjacent to the pulp. Additionally, the results confirmed that the NMP is capable of mapping the distribution of lead in teeth at micron resolutions with a detection limit of approx 1 microg/g.

Effect of orthophosphate, nucleotide analogues, ADP, and phosphorylation on the cytoplasmic domains of Ca(2+)-ATPase from scallop sarcoplasmic reticulum.

The effects of orthophosphate, nucleotide analogues, ADP, and covalent phosphorylation on the tryptic fragmentation patterns of the E1 and E2 forms of scallop Ca-ATPase were examined. Sites preferentially cleaved by trypsin in the E1 form of the Ca-ATPase were detected in the nucleotide (N) and phosphorylation (P) domains, as well as the actuator (A) domain. These sites were occluded in the E2 (Ca(2+)-free) form of the enzyme, consistent with mutual protection of the A, N, and P domains through their association into a clustered structure. Similar protection of cytoplasmic Ca(2+)-dependent tryptic cleavage sites was observed when the catalytic binding site for substrate on the E1 form of scallop Ca-ATPase was occupied by Pi, AMP-PNP, AMP-PCP, or ADP despite the presence of saturating levels of Ca2+. These results suggest that occupation of the catalytic site on E1 can induce condensation of the cytoplasmic domains to yield a unique structural intermediate that may be related to the form of the enzyme in which the active site is prepared for phosphoryl transfer. The effect of Pi on the E2 form of the scallop Ca-ATPase was also investigated, when it was found that formation of E2-P led to extreme resistance toward secondary cleavage by trypsin and stabilization of enzymatic activity for long periods of time.