Virtual autopsy with multiphase postmortem computed tomographic angiography versus traditional medical autopsy to investigate unexpected deaths of hospitalized patients: a cohort study.

BACKGROUND: "Virtual" autopsy by postmortem computed tomography (PMCT) can replace medical autopsy to a certain extent but has limitations for cardiovascular diseases. These limitations might be overcome by adding multiphase PMCT angiography. OBJECTIVE: To compare virtual autopsy by multiphase PMCT angiography with medical autopsy. DESIGN: Prospective cohort study. (ClinicalTrials.gov: NCT01541995) SETTING: Single-center study at the University Medical Center Hamburg-Eppendorf, Hamburg, Germany, between 1 April 2012 and 31 March 2013. PATIENTS: Hospitalized patients who died unexpectedly or within 48 hours of an event necessitating cardiopulmonary resuscitation. MEASUREMENTS: Diagnoses from clinical records were compared with findings from both types of autopsy. New diagnoses identified by autopsy were classified as major or minor, depending on whether they would have altered clinical management. RESULTS: Of 143 eligible patients, 50 (35%) had virtual and medical autopsy. Virtual autopsy confirmed 93% of all 336 diagnoses identified from antemortem medical records, and medical autopsy confirmed 80%. In addition, virtual and medical autopsy identified 16 new major and 238 new minor diagnoses. Seventy-three of the virtual autopsy diagnoses, including 32 cases of coronary artery stenosis, were identified solely by multiphase PMCT angiography. Of the 114 clinical diagnoses classified as cardiovascular, 110 were confirmed by virtual autopsy and 107 by medical autopsy. In 11 cases, multiphase PMCT angiography showed "unspecific filling defects," which were not reported by medical autopsy. LIMITATION: These results come from a single center with concerted interest and expertise in postmortem imaging; further studies are thus needed for generalization. CONCLUSION: In cases of unexpected death, the addition of multiphase PMCT angiography increases the value of virtual autopsy, making it a feasible alternative for quality control and identification of diagnoses traditionally made by medical autopsy. PRIMARY FUNDING SOURCE: University Medical Center Hamburg-Eppendorf.

Isolation and characterization of major histocompatibility complex (MHC) class II B genes in the Barn owl (Aves: Tyto alba)

We isolated major histocompatibility complex class II B (MHCIIB) genes in the Barn owl (Tyto alba). A PCR-based approach combined with primer walking on genomic and complementary DNA as well as Southern blot analyses revealed the presence of two MHCIIB genes, both being expressed in spleen, liver, and blood. Characteristic structural features of MHCIIB genes as well as their expression and high non-synonymous substitution rates in the region involved in antigen binding suggest that both genes are functional. MHC organization in the Barn owl is simple compared to passerine species that show multiple duplications, and resembles the minimal essential MHC of chicken.

Molecular and therapeutic characterization of anti-ectodysplasin A receptor (EDAR) agonist monoclonal antibodies.

The TNF family ligand ectodysplasin A (EDA) and its receptor EDAR are required for proper development of skin appendages such as hair, teeth, and eccrine sweat glands. Loss of function mutations in the Eda gene cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition that can be ameliorated in mice and dogs by timely administration of recombinant EDA. In this study, several agonist anti-EDAR monoclonal antibodies were generated that cross-react with the extracellular domains of human, dog, rat, mouse, and chicken EDAR. Their half-life in adult mice was about 11 days. They induced tail hair and sweat gland formation when administered to newborn EDA-deficient Tabby mice, with an EC(50) of 0.1 to 0.7 mg/kg. Divalency was necessary and sufficient for this therapeutic activity. Only some antibodies were also agonists in an in vitro surrogate activity assay based on the activation of the apoptotic Fas pathway. Activity in this assay correlated with small dissociation constants. When administered in utero in mice or at birth in dogs, agonist antibodies reverted several ectodermal dysplasia features, including tooth morphology. These antibodies are therefore predicted to efficiently trigger EDAR signaling in many vertebrate species and will be particularly suited for long term treatments.

Peripheral projections of calretinin-immunoreactive primary sensory neurons in chick hindlimbs.

In chicken dorsal root ganglia, calretinin immunoreactivity is expressed by a subpopulation of large A-neurons, most of which co-express calbindin D-28k. The myelinated axons of these neurons selectively innervate all muscle spindles and most Herbst corpuscles associated to feathers in hindlimbs. It is suggested that the presence of calretinin in primary afferents may be correlated with the electrophysiological properties of rapidly adapting mechanoreceptors.

Expression of calbindin immunoreactivity by subpopulations of primary sensory neurons in chick embryo dorsal root ganglion cells grown in coculture or conditioned medium.

Primary sensory neurons which innervate neuromuscular spindles in the chicken are calbindin-immunoreactive. The influence exerted by developing skeletal muscle on the expression of calbindin immunoreactivity by subpopulations of dorsal root ganglion (DRG) cells in the chick embryo was tested in vitro in coculture with myoblasts, in conditioned medium (CM) prepared from myoblasts and in control cultures of DRG cells alone. Control cultures of DRG cells grown at the 6th embryonic day (E6) did not show any calbindin-immunostained ganglion cell. In coculture of myoblasts previously grown for 14 days, about 3% of calbindin-immunoreactive ganglion cells were detected while about 1% were observed in some cultures grown in CM. Fibroblasts from various sources were devoid of effect. Skin or kidney cells were more active than myoblasts to initiate calbindin expression by subpopulations of DRG cells in coculture or, to a lesser degree, in CM. The results suggest that cellular factors would rather induce calbindin expression in certain sensory neurons than ensure a selective neuronal survival.

Sequence homologies in the region preceding the transcription initiation site of the liver estrogen-responsive vitellogenin and apo-VLDLII genes.

In the liver of oviparous vertebrates vitellogenin gene expression is controlled by estrogen. The nucleotide sequence of the 5' flanking region of the Xenopus laevis vitellogenin genes A1, A2, B1 and B2 has been determined. These sequences have been compared to each other and to the equivalent region of the chicken vitellogenin II and apo-VLDLII genes which are also expressed in the liver in response to estrogen. The homology between the 5' flanking region of the Xenopus genes B1 and B2 is higher than between the corresponding regions of the other closely related genes A1 and A2. Four short blocks of sequence homology which are present at equivalent positions in the vitellogenin genes of both Xenopus laevis and chicken are characterized. A short sequence with two-fold rotational symmetry (GGTCANNNTGACC) was found at similar positions upstream of the five vitellogenin genes and is also present in two copies close to the 5' end of the chicken apo-VLDLII gene. The possible functional significance of this sequence, common to liver estrogen-responsive genes, is discussed.

Exposure to bioaerosols in poultry houses at different stages of fattening; use of real-time PCR for airborne bacterial quantification

Previous studies have demonstrated that poultry house workers are exposed to very high levels of organic dust and consequently have an increased prevalence of adverse respiratory symptoms. However, the influence of the age of broilers on bioaerosol concentrations has not been investigated. To evaluate the evolution of bioaerosol concentration during the fattening period, bioaerosol parameters (inhalable dust, endotoxin and bacteria) were measured in 12 poultry confinement buildings in Switzerland, at three different stages of the birds' growth; samples of air taken from within the breathing zones of individual poultry house employees as they caught the chickens ready to be transported for slaughter were also analysed. Quantitative polymerase chain reaction (Q-PCR) was used to assess the quantity of total airborne bacteria and total airborne Staphylococcus species. Bioaerosol levels increased significantly during the fattening period of the chickens. During the task of catching mature birds, the mean inhalable dust concentration for a worker was 26 +/- 1.9 mg m(-3) and endotoxin concentration was 6198 +/- 2.3 EU m(-3) air, >6-fold higher than the Swiss occupational recommended value (1000 EU m(-3)). The mean exposure level of bird catchers to total bacteria and Staphylococcus species measured by Q-PCR is also very high, respectively, reaching values of 53 (+/-2.6) x 10(7) cells m(-3) air and 62 (+/-1.9) x 10(6) m(-3) air. It was concluded that in the absence of wearing protective breathing apparatus, chicken catchers in Switzerland risk exposure beyond recommended limits for all measured bioaerosol parameters. Moreover, the use of Q-PCR to estimate total and specific numbers of airborne bacteria is a promising tool for evaluating any modifications intended to improve the safety of current working practices

Occupational exposure to solar ultraviolet radiation of outdoor workers in France

Question: Outdoor occupational exposure could be associated with important cumulative and intense exposure to ultraviolet (UV) solar radiation. Such exposure would increase risk of skin cancer. However, little information exists on jobs associated with intense UV exposure. The objective of this study was to characterise occupational UV exposure in a representative sample in France. Methods: A population-based survey was conducted in May-June 2012 through computer-assisted telephonic interviews in population 25 to 69 years of age. Individual UV irradiation was computed with declared time and place of residence matched to UV records from satellite measurement (Eurosun project). We analysed factors influencing exposure to UV (annual average and seasonal peak). Results: A total of 1442 individuals declared having an occupational exposure to UV which represents 18% of population aged 25 to 69 years. Outdoor workers were more frequently men (58%), aged 40-54 (43%), with a phototype III or IV (69%). Occupations associated with highest UV exposure were: construction workers (annual daily average 62.8 Joules/m2), gardeners (62.6), farmers (52.8), culture/art/social sciences workers (52.0) and transport workers/mail carriers (49.5). The maximum of UVA exposure was found for occupation with a strong seasonality of exposure: culture, art or social sciences works (98.1 Joules/m2), construction works (97.2), gardening (96.7) and farming (95.0). Significant factors associated with high occupational UV exposure were gender (men vs. women: 53.6 vs. 42.6), phototype (IV vs. I: 51.9 vs. 45.5) and taking lunch outdoors (always vs. never: 59.8 vs. 48.6). Conclusion: Our study showed that some occupations were associated with particularly intense UV exposure such as farmers, gardeners, construction workers. Other unexpected occupations were also associated with high UV exposure such as transport workers, mail carriers and culture/art/social sciences workers.

Risk factors of sunburns from occupational sun exposure in outdoor workers

Question: Outdoor workers can be exposed to intense ultraviolet (UV) solar radiation likely to results to sunburns. As sunburn is an important risk factor for skin cancer, in particular melanoma, we investigated the causes of occupational sunburns (OS) in French outdoor workers. Methods: A population-based survey was conducted in May-June 2012 through computer-assisted telephonic interviews in population 25 to 69 years of age. History of sunburn from occupational exposure within the year preceding interview was collected. We analysed the risk of OS in multivariate logistic regression. Results: Out of 1442 individuals who declared having an occupational exposure to solar UV radiation, 403 (27.9%) reported a sunburn from occupational exposure in the year preceding the interview. Sunburns were more frequent in women (30% vs. 26.4% in men although not significant p = 0.14), in younger workers (p = 0.0099), in sensitive phototype (40% in phototype I/II vs. 23% in phototype III/IV, p < 0.001) and in workers taking lunch outdoor (p = 0.0355). Some occupations were more associated with OS (more than 30%): health occupations, managing, research/engineering, construction workers and culture/art/social sciences workers. In multivariate analysis, risk factors for OS are phototype (I vs. IV, OR = 4.30 95% CI [2.65-6.98]), sunburn during leisure time (OR = 3.46 95% CI [2.62-4.59]), seasonality of exposure (seasonal vs. constant exposure OR = 1.36 95% CI [1.02-1.81] and annual UVA exposure (OR for 10J/m² daily average increment 1.08 95% CI [1.02-1.14]). In multivariate analysis the type of occupation was not associated with increased OS. Conclusion: Sunburns from occupation was also observed in non sensitive population, phototype IV, which shows that outdoor workers are potentially exposed to intense UV radiations. This study suggests that prevention should target UV sensitive outdoor workers as well as those cumulating intense UV exposure.

Differential expression of 240 kDa ConA-binding glycoprotein in vitro and in vivo detected by immunochemical methods.

The expression of the 240 ConA-binding glycoprotein (240 kDa), a marker of synaptic junctions isolated from the rat cerebellum, was studied by immunocytochemical techniques in forebrain and cerebellum from rat and chicken, and in chick dorsal root ganglia. Parallel studies were carried out either on tissue sections or in dissociated cell cultures. In all cases non neuronal cells were not immunostained. The tissue sections of cerebellum from rat and chick exhibited 240 kDa glycoprotein immunoreactivity, especially in the molecular layer, while the forebrain sections from rat and chick did not show any significant immunostaining. In contrast, in dissociated forebrain cell cultures, all neuronal cells expressed 240 kDa glycoprotein immunoreactivity, while glial cells remained totally unlabelled. In tissue sections of dorsal root ganglion (DRG), sensory neurons expressed the 240 kDa only after the embryonic day (E 10). A large number of small neurons in the dorsomedial part of DRG were immunostained with 240 kDa glycoprotein antiserum, whereas only a small number of neurons in the ventrolateral part of the ganglia displayed 240 kDa immunoreactivity. In dissociated DRG cells cultures (mixed or neuron-enriched DRG cell cultures) all the neuronal perikarya but not their processes were stained. These studies indicate that 240 kDa glycoprotein expression is completely modified in cultures of neurons of CNS or PNS since the antigen becomes synthetized in high amount by all cells independent of synapse formation. This demonstrates that the expression of 240 kDa is controlled by the cell environment.

Development of stable cell lines for production or regulated expression using matrix attachment regions.

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection procedure. Given the variation in the expression levels of the same construct in individual clones, hundreds of clones must be isolated and tested to identify one or more with the desired characteristics. Various boundary elements (BEs), matrix attachment regions, and locus control regions (LCRs) were screened for their ability to augment the expression of heterologous genes in Chinese hamster ovary (CHO) cells. Of the chromatin elements assayed, the chicken lysozyme matrix-attachment region (MAR) was the only element to significantly increase stable reporter expression. We found that the use of the MAR increases the proportion of high-producing clones, thus reducing the number of clones that need to be screened. These benefits are observed both for constructs with MARs flanking the transgene expression cassette, as well as when constructs are co-transfected with the MAR on a separate plasmid. Moreover, the MAR was co-transfected with a multicomponent regulatable beta-galactosidase expression system in C2C12 cells and several clones exhibiting regulated expression were identified. Hence, MARs are useful in the development of stable cell lines for production or regulated expression.

Angiostatic kinase inhibitors to sustain photodynamic angio-occlusion.

Targeted angiostatic therapy receives major attention for the treatment of cancer and exudative age-related macular degeneration (AMD). Photodynamic therapy (PDT) has been used as an effective clinical approach for these diseases. As PDT can cause an angiogenic response in the treated tissue, combination of PDT with anti-angiogenic compounds should lead to improved therapy. This study was undertaken to test the clinically used small molecule kinase inhibitors Nexavar® (sorafenib), Tarceva® (erlotinib) and Sutent® (sunitinib) for this purpose, and to compare the results to the combination of Visudyne®-PDT with Avastin® (bevacizumab) treatment. When topically applied to the chicken chorioallantoic membrane at embryo development day (EDD) 7, a clear inhibition of blood vessel development was observed, with sorafenib being most efficient. To investigate the combination with phototherapy, Visudyne®-PDT was first applied on EDD11 to close all <100 μm vessels. Application of angiostatics after PDT resulted in a significant decrease in vessel regrowth in terms of reduced vessel density and number of branching points/mm(2) . As the 50% effective dose (ED50) for all compounds was approximately 10-fold lower, Sorafenib outperformed the other compounds. In vitro, all kinase inhibitors decreased the viability of human umbilical vein endothelial cells. Sunitinib convincingly inhibited the in vitro migration of endothelial cells. These results suggest the therapeutic potential of these compounds for application in combination with PDT in anti-cancer approaches, and possibly also in the treatment of other diseases where angiogenesis plays an important role.

Deletion of the Vaccinia Virus Gene A46R, Encoding for an Inhibitor of TLR Signalling, Is an Effective Approach to Enhance the Immunogenicity in Mice of the HIV/AIDS Vaccine Candidate NYVAC-C.

Viruses have developed strategies to counteract signalling through Toll-like receptors (TLRs) that are involved in the detection of viruses and induction of proinflammatory cytokines and IFNs. Vaccinia virus (VACV) encodes A46 protein which disrupts TLR signalling by interfering with TLR: adaptor interactions. Since the innate immune response to viruses is critical to induce protective immunity, we studied whether deletion of A46R gene in a NYVAC vector expressing HIV-1 Env, Gag, Pol and Nef antigens (NYVAC-C) improves immune responses against HIV-1 antigens. This question was examined in human macrophages and in mice infected with a single A46R deletion mutant of the vaccine candidate NYVAC-C (NYVAC-C-ΔA46R). The viral gene A46R is not required for virus replication in primary chicken embryo fibroblast (CEF) cells and its deletion in NYVAC-C markedly increases TNF, IL-6 and IL-8 secretion by human macrophages. Analysis of the immune responses elicited in BALB/c mice after DNA prime/NYVAC boost immunization shows that deletion of A46R improves the magnitude of the HIV-1-specific CD4 and CD8 T cell immune responses during adaptive and memory phases, maintains the functional profile observed with the parental NYVAC-C and enhances anti-gp120 humoral response during the memory phase. These findings establish the immunological role of VACV A46R on innate immune responses of macrophages in vitro and antigen-specific T and B cell immune responses in vivo and suggest that deletion of viral inhibitors of TLR signalling is a useful approach for the improvement of poxvirus-based vaccine candidates.