Leishmania major promastigote entry of an autophagy-like compartment and amastigote escape from the parasitophorous vacuole

In this thesis, we investigated the interaction of the obligate intracellular parasite Leishmania (L.) major with two phenotypes of human monocyte derived macrophages (hMDMs). Thereby we focused on the development and maturation of the parasitophorous vacuole (PV) and could show that compartment development is dependent on the parasite stage.rnFocusing on the ultrastructure of PVs containing axenic amastigotes, we demonstrated that the parasites are partially located in damaged PVs or in the cytoplasm of the host. Moreover, we visualized multiple amastigotes in a common PV 144 h p.i. in pro-inflammatory hMDM I but not in anti-inflammatory hMDM II indicating different PV development. rnRegarding the promastigote form, we demonstrated a different uptake of viable and apoptotic L. major promastigotes by hMDMs. Viable promastigotes are predominantly taken up via the flagellum tip whereas apoptotic promastigotes enter the cells via the parasite body. Analyzing compartment maturation, we found that 20-30% of the PVs get positive for the early maturation markers PI3P and EEA1 independent of the viability of the parasites and unaffected by the human macrophage type. Subsequently, 25-40% of the parasites acquire the autophagy marker LC3 on their PV, what is independent of the viability of the parasites as well. We quantified this and in hMDM II less LC3-positive compartments formed compared to hMDM I. Analyzing the ultrastructure, we investigated that the compartments consist of a single-membrane PV characteristic for LC3-associated phagocytosis (LAP). Involvement of LAP was confirmed by demonstrating that the protein kinase ULK1 is dispensable for LC3-compartment formation around Leishmania PVs. Visualizing compartment dynamics in real time showed that apoptotic promastigotes are degraded in LC3-positve compartments, whereas viable promastigotes are able to get rid of LC3-protein on their PV suggesting an involvement in parasite development and survival. In this thesis, we established a lentiviral based fluorescent imaging technique that we combined with High-Pressure-Freezing (HPF) and high-resolution 3D electron microscopy. We visualized a promastigote in a LC3-compartment whose ultrastructure showed an opening of the PV to the outside. To identify new LAP markers involved in Leishmania infection, we established an immuno-magnetic isolation protocol for the purification of Leishmania containing compartments.rnIn conclusion, this study suggests that L. major compartment biogenesis and maturation in pro- and anti-inflammatory human macrophages is dependent on the parasite stage and is different between axenic amastigotes, viable promastigotes and apoptotic promastigotes. Understanding the development and maturation of Leishmania parasites in human host cells is important to control and combat the neglected disease leishmaniasis in the future.rn

Escape mechanisms after antiangiogenic treatment, or why are the tumors growing again?

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after cessation of treatment. In numerous preclinical studies, angiogenesis inhibitors were shown to be efficient in the treatment of many pathological conditions, including solid cancers. In most clinical trials, however, this approach turned out to have no significant effect, especially if applied as monotherapy. Recovery of tumors after therapy is a major problem in the management of cancer patients. The mechanisms underlying tumor recovery (or therapy resistance) have not yet been explicitly elucidated. This review deals with the transient switch from sprouting to intussusceptive angiogenesis, which may be an adaptive response of tumor vasculature to cancer therapy that allows the vasculature to maintain its functional properties. Potential candidates for molecular targeting of this angioadaptive mechanism are yet to be elucidated in order to improve the currently poor efficacy of contemporary antiangiogenic therapies.

Strain-specific spleen remodelling in Plasmodium yoelii infections in Balb/c mice facilitates adherence and spleen macrophage-clearance escape

Knowledge of the dynamic features of the processes driven by malaria parasites in the spleen is lacking. To gain insight into the function and structure of the spleen in malaria, we have implemented intravital microscopy and magnetic resonance imaging of the mouse spleen in experimental infections with non-lethal (17X) and lethal (17XL) Plasmodium yoelii strains. Noticeably, there was higher parasite accumulation, reduced motility, loss of directionality, increased residence time and altered magnetic resonance only in the spleens of mice infected with 17X. Moreover, these differences were associated with the formation of a strain-specific induced spleen tissue barrier of fibroblastic origin, with red pulp macrophage-clearance evasion and with adherence of infected red blood cells to this barrier. Our data suggest that in this reticulocyte-prone non-lethal rodent malaria model, passage through the spleen is different from what is known in other Plasmodium species and open new avenues for functional/structural studies of this lymphoid organ in malaria.

Panel 2: Rescue and Escape from the Holocaust

Panel 2: Rescue and Escape from the Holocaust Nina Paulovicova, University of Alberta, Canada: “The Silenced Phenomenon of Cross-National Rescue: 'Leaking Border' and Paid Smugglers” Download Paper (login required) Tomasz Frydel, University of Toronto: "Rescue or Denunciation of Jews? A Case Study of Southeastern Poland during German Occupation" Download Paper (login required) Tanja von Fransecky, Technical University, Berlin, Germany: "Escape and Attempted Escape of Jewish Deportees from Deportation Trains in France, Belgium and the Netherlands” Download Paper (login required) Chair: Adara Goldberg and Elizabeth Anthony, Clark UniversityComment: Deborah Dwork, Clark University

The Effect of Heat Treatment of Forages on Degradation Kinetics and Escape Protein Concentration

An in situ study was conducted to evaluate the effects of heat treatments on the degradation kinetics and escape protein concentrations of forages (alfalfa and berseem clover). Alfalfa collected at 4 and 7 weeks post-harvest and berseem clover collected at 5 and 7 weeks postharvest were freeze-dried and then heated to 100, 125, and 150o C for 2 hours. Heat treatment effects were determined by placing two bags of sample (for each treatment, maturity, and forage species for a given incubation times) into the rumen of one fistulated steer fed alfalfa hay. Bags were incubated for periods of 0 to 48 hours. Increasing levels of heat treatments of forages increased concentrations of neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent insoluble nitrogen (ADIN) and non-degradable protein (NDP), potentially degradable protein proportion (PDP), and protein escaping rumen degradation (PEP) while decreasing water soluble protein (WSP) and the rates of crude protein (CP), except immature berseem clover and cell wall (CW) degradation. PEP was greater and rate of CP degradation was lower at 100 and 150o C compared to 125o C in immature berseem clover.

The Effect of Maturity and Frost Killing of Forages on Degradation Kinetics and Escape Protein Concentration

Two consecutive in situ studies were conducted to determine the effects of maturity and frost killing of forages (alfalfa and berseem clover) on degradation kinetics and escape protein concentrations. Four maturities (3, 5, 7, and 9 weeks after second harvest) of forages collected from three locations were used to determine the effects of maturity. Four weeks after a killing frost (-2o C), berseem clover was harvested from the same locations previously sampled. To evaluate maturity, 336 DacronÒ bags containing all maturities of either alfalfa or berseem clover were placed into the rumen of two fistulated steers fed alfalfa-grass hay. Frost killing effects of berseem clover were compared with maturecut berseem clover by placing DacronÒ bags into the rumen of one fistulated steer fed alfalfa hay. Bags were incubated for periods of 0 to 48 hours. With increasing maturity, the proportion of non-degradable protein (NDP) and the rate of crude protein (CP) degradation increased in both forages. While the rate of neutral detergent fiber (NDF) degradation and potentially degradable protein proportion (PDP) increased with increasing maturity in alfalfa, the rate of NDF degradation and PDP proportion decreased and proportion of water soluble protein (WSP) increased in berseem clover. The proportion of protein escaping rumen degradation (PEP) was greater in berseem clover than alfalfa, but was not affected by maturity. Frost killing of mature berseem clover decreased WSP proportion and increased PDP proportion compared to mature berseem clover harvested live. Even though ADIN concentration was higher for frost-killed berseem clover, PEP and total escape protein concentration (CEP) was also higher for frostkilled berseem clover than mature berseem clover harvested live, due to decreases in the rate of ruminal N degradation with frost-killing.

The free escape continuum of diffuse ions upstream of the Earth's quasi-parallel bow shock

The Earth's bow shock is very efficient in accelerating ions out of the incident solar wind distribution to high energies (≈ 200 keV/e). Fluxes of energetic ions accelerated at the quasi-parallel bow shock, also known as diffuse ions, are best represented by exponential spectra in energy/charge, which require additional assumptions to be incorporated into these model spectra. One of these assumptions is a so-called "free escape boundary" along the interplanetary magnetic field into the upstream direction. Locations along the IBEX orbit are ideally suited for in situ measurements to investigate the existence of an upstream free escape boundary for bow shock accelerated ions. In this study we use 2 years of ion measurements from the background monitor on the IBEX spacecraft, supported by ACE solar wind observations. The IBEX Background Monitor is sensitive to protons > 14 keV, which includes the energy of the maximum flux for diffuse ions. With increasing distance from the bow shock along the interplanetary magnetic field, the count rates for diffuse ions stay constant for ions streaming away from the bow shock, while count rates for diffuse ions streaming toward the shock gradually decrease from a maximum value to ~1/e at distances of about 10 RE to 14 RE. These observations of a gradual decrease support the transition to a free escape continuum for ions of energy >14 keV at distances from 10 RE to 14 RE from the bow shock.

Seedling resistance, tolerance and escape from herbivores: insights from co-dominant canopy tree species in a resource-poor African rain forest

* Although plants can reduce the impacts of herbivory in multiple ways, these defensive traits are often studied in isolation and an understanding of the resulting strategies is incomplete. * In the study reported here, empirical evidence was simultaneously evaluated for the three main sets of traits available to plants: (i) resistance through constitutive leaf traits, (ii) tolerance to defoliation and (iii) escape in space, for three caesalpiniaceous tree species Microberlinia bisulcata, Tetraberlinia bifoliolata and T. korupensis, which co-dominate groves within the lowland primary rain forest of Korup National Park (Cameroon). * Mesh cages were placed around individual wild seedlings to exclude insect herbivores at 41 paired canopy gap and understorey locations. After following seedling growth and survival for c. 2 years, caged and control treatments were removed, leaves harvested to determine nutrient and phenolic concentrations, leaf mass per area estimated, and seedling performance in gaps followed for a further c. 2 years to quantify tolerance to the leaf harvesting. * The more nutrient-rich leaves of the weakly shade-tolerant M. bisulcata were damaged much more in gaps than the two strongly shade-tolerant Tetraberlinia species, which had higher leaf mass per area and concentrations of total phenols. Conversely, the faster-growing M. bisulcata was better able to tolerate defoliation in terms of height growth (reflushing capacity), but not at maintaining overall leaf numbers, than the other two species. * Across gaps, insect-mediated Janzen–Connell effects were most pronounced for M. bisulcata, less so for T. korupensis, and not detectable for T. bifoliolata. The three species differed distinctly in their secondary metabolic profiles. * Taken together, the results suggested a conceptual framework linking the three sets of traits, one in which the three co-dominant species adopt different strategies towards herbivore pressure depending on their different responses to light availability. This study is one of the first in a natural forest ecosystem to examine resistance to, tolerance of, and escape from herbivory among a group of co-occurring tropical tree species.

Viral escape in the CNS with multidrug-resistant HIV-1.

Introduction: HIV-1 viral escape in the cerebrospinal fluid (CSF) despite viral suppression in plasma is rare [1,2]. We describe the case of a 50-year-old HIV-1 infected patient who was diagnosed with HIV-1 in 1995. Antiretroviral therapy (ART) was started in 1998 with a CD4 T cell count of 71 cells/ìL and HIV-viremia of 46,000 copies/mL. ART with zidovudine (AZT), lamivudine (3TC) and efavirenz achieved full viral suppression. After the patient had interrupted ART for two years, treatment was re-introduced with tenofovir (TDF), emtricitabin (FTC) and ritonavir boosted atazanavir (ATVr). This regimen suppressed HIV-1 in plasma for nine years and CD4 cells stabilized around 600 cells/ìL. Since July 2013, the patient complained about severe gait ataxia and decreased concentration. Materials and Methods: Additionally to a neurological examination, two lumbar punctures, a cerebral MRI and a neuropsycological test were performed. HIV-1 viral load in plasma and in CSF was quantified using Cobas TaqMan HIV-1 version 2.0 (Cobas Ampliprep, Roche diagnostic, Basel, Switzerland) with a detection limit of 20 copies/mL. Drug resistance mutations in HIV-1 reverse transcriptase and protease were evaluated using bulk sequencing. Results: The CSF in January 2014 showed a pleocytosis with 75 cells/ìL (100% mononuclear) and 1,184 HIV-1 RNA copies/mL, while HIV-1 in plasma was below 20 copies/mL. The resistance testing of the CSF-HIV-1 RNA showed two NRTI resistance-associated mutations (M184V and K65R) and one NNRTI resistance-associated mutation (K103N). The cerebral MRI showed increased signal on T2-weighted images in the subcortical and periventricular white matter, in the basal ganglia and thalamus. Four months after ART intensification with AZT, 3TC, boosted darunavir and raltegravir, the pleocytosis in CSF cell count normalized to 1 cell/ìL and HIV viral load was suppressed. The neurological symptoms improved; however, equilibrium disturbances and impaired memory persisted. The neuro-psychological evaluation confirmed neurocognitive impairments in executive functions, attention, working and nonverbal memory, speed of information processing, visuospatial abilities and motor skills. Conclusions: HIV-1 infected patients with neurological complaints prompt further investigations of the CSF including measurement of HIV viral load and genotypic resistance testing since isolated replication of HIV with drug resistant variants can rarely occur despite viral suppression in plasma. Optimizing ART by using drugs with improved CNS penetration may achieve viral suppression in CSF with improvement of neurological symptoms.

Performance of HBsAg point-of-care tests for detection of diagnostic escape-variants in clinical samples

BACKGROUND Hepatitis B viruses (HBV) harboring mutations in the a-determinant of the Hepatitis B surface antigen (HBsAg) are associated with reduced reactivity of HBsAg assays. OBJECTIVES To evaluate the sensitivity and specificity of three HBsAg point-of-care tests for the detection of HBsAg of viruses harboring HBsAg mutations. STUDY DESIGN A selection of 50 clinical plasma samples containing HBV with HBsAg mutations was used to evaluate the performance of three HBsAg point-of-care tests (Vikia(®), bioMérieux, Marcy-L'Étoile, France. Alere Determine HBsAg™, Iverness Biomedical Innovations, Köln, Germany. Quick Profile™, LumiQuick Diagnostics, California, USA) and compared to the ARCHITECT HBsAg Qualitative(®) assay (Abbott Laboratories, Sligo, Ireland). RESULTS The sensitivity of the point-of-care tests ranged from 98% to 100%. The only false-negative result occurred using the Quick Profile™ assay with a virus harboring a D144A mutation. CONCLUSIONS The evaluated point-of-care tests revealed an excellent sensitivity in detecting HBV samples harboring HBsAg mutations.

Long-term live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms

Plasmodium parasites are transmitted by Anopheles mosquitoes to the mammalian host and actively infect hepatocytes after passive transport in the bloodstream to the liver. In their target host hepatocyte, parasites reside within a parasitophorous vacuole (PV). In the present study it was shown that the parasitophorous vacuole membrane (PVM) can be targeted by autophagy marker proteins LC3, ubiquitin, and SQSTM1/p62 as well as by lysosomes in a process resembling selective autophagy. The dynamics of autophagy marker proteins in individual Plasmodium berghei-infected hepatocytes were followed by live imaging throughout the entire development of the parasite in the liver. Although the host cell very efficiently recognized the invading parasite in its vacuole, the majority of parasites survived this initial attack. Successful parasite development correlated with the gradual loss of all analyzed autophagy marker proteins and associated lysosomes from the PVM. However, other autophagic events like nonselective canonical autophagy in the host cell continued. This was indicated as LC3, although not labeling the PVM anymore, still localized to autophagosomes in the infected host cell. It appears that growing parasites even benefit from this form of nonselective host cell autophagy as an additional source of nutrients, as in host cells deficient for autophagy, parasite growth was retarded and could partly be rescued by the supply of additional amino acid in the medium. Importantly, mouse infections with P. berghei sporozoites confirmed LC3 dynamics, the positive effect of autophagy activation on parasite growth, and negative effects upon autophagy inhibition.