Dissemination of Orientia tsutsugamushi and Inflammatory Responses in a Murine Model of Scrub Typhus

Central aspects in the pathogenesis of scrub typhus, an infection caused by Orientia (O.) tsutsugamushi, have remained obscure. Its organ and cellular tropism are poorly understood.The purpose of this study was to analyze the kinetics of bacterial dissemination and associated inflammatory responses in infected tissues in an experimental scrub typhus mouse model, following infection with the human pathogenic strain Karp. We provide a thorough analysis of O. tsutsugamushi infection in inbred Balb/c mice using footpad inoculation, which is close to the natural way of infection. By a novel, highly sensitive qPCR targeting the multi copy traD genes, we quantitatively monitored the spread of O. tsutsugamushi Karp from the skin inoculation site via the regional lymph node to the internal target organs. The highest bacterial loads were measured in the lung. Using confocal imaging, we also detected O. tsutsugamushi at the single cell level in the lung and found a predominant macrophage rather than endothelial localization. Immunohistochemical analysis of infiltrates in lung and brain revealed differently composed lesions with specific localizations: iNOS-expressing macrophages were frequent in infiltrative parenchymal noduli, but uncommon in perivascular lesions within these organs. Quantitative analysis of the macrophage response by immunohistochemistry in liver, heart, lung and brain demonstrated an early onset of macrophage activation in the liver. Serum levels of interferon (IFN)-γ were increased during the acute infection, and we showed that IFN-γ contributed to iNOS-dependent bacterial growth control.Our data show that upon inoculation to the skin, O. tsutsugamushi spreads systemically to a large number of organs and gives rise to organ-specific inflammation patterns. The findings suggest an essential role for the lung in the pathogenesis of scrub typhus. The model will allow detailed studies on host-pathogen interaction and provide further insight into the pathogenesis of O. tsutsugamushi infection.     

Response to “On the choice of methodology for evaluating dose-rate effects on radiation-related cancer risks” by Walsh et al.

Radiation and Environmental Biophysics -     

The zebrafish orphan nuclear receptor genes nr2e1 and nr2e3 are expressed in developing eye and forebrain

Mammalian Nr2e1 (Tailless, Mtll or Tlx) and Nr2e3 (photoreceptor-specific nuclear receptor, Pnr) are highly related orphan nuclear receptors, that are expressed in eye and forebrain-derived structures. In this study, we analyzed the developmental expression patterns of zebrafish nr2e1 and nr2e3. RT-PCR analysis showed that nr2e1 and nr2e3 are both expressed during embryonic and post-embryonic development. To examine the spatial distribution of nr2e1 and nr2e3 during development whole-mount in situ hybridization was performed. At tailbud stage, initial nr2e1 expression was localized to the rostral brain rudiment anterior to pax2.1 and eng2 expression at the prospective midbrain-hindbrain boundary. During subsequent stages, nr2e1 became widely expressed in fore- and midbrain primordia, eye and olfactory placodes. At 24hpf, strong nr2e1 expression was detected in telencephalon, hypothalamus, dorsal thalamus, pretectum, midbrain tectum, and retina. At 2dpf, the initially widespread nr2e1 expression became more restricted to distinct regions within the fore- and midbrain and to the retinal ciliary margin, the germinal zone which gives rise to retina and presumptive iris. Expression of nr2e3 was exclusively found in the developing retina and epiphysis. In both structures, nr2e3 expression was found in photoreceptor cells. The developmental expression profile of zebrafish nr2e1 and nr2e3 is consistent with evolutionary conserved functions in eye and rostral brain structures.     

Model-based monitoring of industrial reversed phase chromatography to predict insulin variants

Downstream processing in the manufacturing biopharmaceutical industry is a multistep process separating the desired product from process- and product-related impurities. However, removing product-related impurities, such as product variants, without compromising the product yield or prolonging the process time due to extensive quality control analytics, remains a major challenge. Here, we show how mechanistic model-based monitoring, based on analytical quality control data, can predict product variants by modeling their chromatographic separation during product polishing with reversed phase chromatography. The system was described by a kinetic dispersive model with a modified Langmuir isotherm. Solely quality control analytical data on product and product variant concentrations were used to calibrate the model. This model-based monitoring approach was developed for an insulin purification process. Industrial materials were used in the separation of insulin and two insulin variants, one eluting at the product peak front and one eluting at the product peak tail. The model, fitted to analytical data, used one component to simulate each protein, or two components when a peak displayed a shoulder. This monitoring approach allowed the prediction of the elution patterns of insulin and both insulin variants. The results indicate the potential of using model-based monitoring in downstream polishing at industrial scale to take pooling decisions.     

On the presence of serotonin in mammalian cardiomyocytes

Pleiotropic effects of serotonin (5-HT) in the cardiovascular system are well documented. However, it remains to be elucidated, whether 5-HT is present in adult mammalian cardiomyocytes. To address this issue, we investigated the levels of 5-HT in blood, plasma, platelets, cardiac tissue, and cardiomyocytes from adult mice and for comparison in human right atrial tissue. Immunohistochemically, 5-HT was hardly found in mouse cardiac tissue, but small amounts could be detected in renal preparations, whereas adrenal preparations revealed a strong positive immunoreaction for 5-HT. Using a sensitive HPLC detection system, 5-HT was also detectable in the mouse heart and human atrium. Furthermore, we could identify 5-HT in isolated cardiomyocytes from adult mice. These findings were supported by detection of the activity of 5-HT-forming enzymes-tryptophan hydroxylase and aromatic L-amino acid decarboxylase-in isolated cardiomyocytes from adult mice and by inhibition of these enzymes with p-chlorophenylalanine and 3-hydroxybenzyl hydrazine. Addition of the first intermediate of 5-HT generation, that is 5-hydroxytryptophan, enhanced the 5-HT level and inhibition of monoamine oxidase by tranylcypromine further increased the level of 5-HT. Our findings reveal the presence and synthesis of 5-HT in cardiomyocytes of the mammalian heart implying that 5-HT may play an autocrine and/or paracrine role in the heart.     

Site-specific cancer incidence and mortality after cerebral angiography with radioactive thorotrast

Few opportunities exist to evaluate the carcinogenic effects of long-term internal exposure to alpha-particle-emitting radionuclides. Patients injected with Thorotrast (thorium-232) during radiographic procedures, beginning in the 1930s, provide one such valuable opportunity. We evaluated site-specific cancer incidence and mortality among an international cohort of 3,042 patients injected during cerebral angiography with either Thorotrast (n = 1,650) or a nonradioactive agent (n = 1,392) and who survived 2 or more years. Standardized incidence ratios (SIR) for Thorotrast and comparison patients (Denmark and Sweden) were estimated and relative risks (RR), adjusted for population, age and sex, were generated with multivariate statistical modeling. For U.S. patients, comparable procedures were used to estimate standardized mortality ratios (SMR) and RR, representing the first evaluation of long-term, site-specific cancer mortality in this group. Compared with nonexposed patients, significantly increased risks in Thorotrast patients were observed for all incident cancers combined (RR = 3.4, 95% CI 2.9-4.1, n = 480, Denmark and Sweden) and for cancer mortality (RR = 4.0, 95% CI 2.5-6.7, n = 114, U.S.). Approximately 335 incident cancers were above expectation, with large excesses seen for cancers of the liver, bile ducts and gallbladder (55% or 185 excess cancers) and leukemias other than CLL (8% or 26 excess cancers). The RR of all incident cancers increased with time since angiography (P < 0.001) and was threefold at 40 or more years; significant excesses (SIR = 4.0) persisted for 50 years. Increasing cumulative dose of radiation was associated with an increasing risk of all incident cancers taken together and with cancers of the liver, gallbladder, and peritoneum and other digestive sites; similar findings were observed for U.S. cancer mortality. A marginally significant dose response was observed for the incidence of pancreas cancer (P = 0.05) but not for lung cancer. Our study confirms the relationship between Thorotrast and increased cancer incidence at sites of Thorotrast deposition and suggests a possible association with pancreas cancer. After injection with >20 ml Thorotrast, the cumulative excess risk of cancer incidence remained elevated for up to 50 years and approached 97%. Caution is needed in interpreting the excess risks observed for site-specific cancers, however, because of the potential bias associated with the selection of cohort participants, noncomparability with respect to the internal or external comparison groups, and confounding by indication. Nonetheless, the substantial risks associated with liver cancer and leukemia indicate that unique and prolonged exposure to alpha-particle-emitting Thorotrast increased carcinogenic risks.     

Helical growth of the Arabidopsis mutant tortifolia1 reveals a plant-specific microtubule-associated protein

Plants can grow straight or in the twisted fashion exhibited by the helical growth of some climbing plants. Analysis of helical-growth mutants from Arabidopsis has indicated that microtubules are involved in the expression of the helical phenotype. Arabidopsis mutants growing with a right-handed twist have been reported to have cortical microtubules that wind around the cell in left-handed helices and vice versa. Microtubular involvement is further suspected from the finding that some helical mutants are caused by single amino acid substitutions in alpha-tubulin and because of the sensitivity of the growth pattern to anti-microtubule drugs. Insight into the roles of microtubules in organ elongation is anticipated from analyses of genes defined by helical mutations. We investigated the helical growth of the Arabidopsis mutant tortifolia1/spiral2 (tor1/spr2), which twists in a right-handed manner, and found that this correlates with a complex reorientation of cortical microtubules. TOR1 was identified by a map-based approach; analysis of the TOR1 protein showed that it is a member of a novel family of plant-specific proteins containing N-terminal HEAT repeats. Recombinant TOR1 colocalizes with cortical microtubules in planta and binds directly to microtubules in vitro. This shows that TOR1 is a novel, plant-specific microtubule-associated protein (MAP) that regulates the orientation of cortical microtubules and the direction of organ growth.     

Proteomic and functional analyses reveal a mitochondrial dysfunction in P301L tau transgenic mice

Transgenic mice overexpressing the P301L mutant human tau protein exhibit an accumulation of hyperphosphorylated tau and develop neurofibrillary tangles. The consequences of tau pathology were investigated here by proteomics followed by functional analysis. Mainly metabolism-related proteins including mitochondrial respiratory chain complex components, antioxidant enzymes, and synaptic proteins were identified as modified in the proteome pattern of P301L tau mice. Significantly, the reduction in mitochondrial complex V levels in the P301L tau mice revealed using proteomics was also confirmed as decreased in human P301L FTDP-17 (frontotemporal dementia with parkinsonism linked to chromosome 17) brains. Functional analysis demonstrated a mitochondrial dysfunction in P301L tau mice together with reduced NADH-ubiquinone oxidoreductase activity and, with age, impaired mitochondrial respiration and ATP synthesis. Mitochondrial dys-function was associated with higher levels of reactive oxygen species in aged transgenic mice. Increased tau pathology as in aged homozygous P301L tau mice revealed modified lipid peroxidation levels and the up-regulation of antioxidant enzymes in response to oxidative stress. Furthermore, P301L tau mitochondria displayed increased vulnerability toward beta-amyloid (Abeta) peptide insult, suggesting a synergistic action of tau and Abeta pathology on the mitochondria. Taken together, we conclude that tau pathology involves a mitochondrial and oxidative stress disorder possibly distinct from that caused by Abeta.