Ultrasensitive Label-Free Detection of Protein-Membrane Interaction Exemplified by Toxin-Liposome Insertion.

Measuring the high-affinity binding of proteins to liposome membranes remains a challenge. Here, we show an ultrasensitive and direct detection of protein binding to liposome membranes using high throughput second harmonic scattering (SHS). Perfringolysin O (PFO), a pore-forming toxin, with a highly membrane selective insertion into cholesterol-rich membranes is used. PFO inserts only into liposomes with a cholesterol concentration >30%. Twenty mole-percent cholesterol results in neither SHS-signal deviation nor pore formation as seen by cryo-electron microscopy of PFO and liposomes. PFO inserts into cholesterol-rich membranes of large unilamellar vesicles in an aqueous solution with Kd = (1.5 ± 0.2) × 10-12 M. Our results demonstrate a promising approach to probe protein-membrane interactions below sub-picomolar concentrations in a label-free and noninvasive manner on 3D systems. More importantly, the volume of protein sample is ultrasmall (<10 µL). These findings enable the detection of low-abundance proteins and their interaction with membranes.

Primordial GATA6 macrophages function as extravascular platelets in sterile injury.

Most multicellular organisms have a major body cavity that harbors immune cells. In primordial species such as purple sea urchins, these cells perform phagocytic functions but are also crucial in repairing injuries. In mammals, the peritoneal cavity contains large numbers of resident GATA6+ macrophages, which may function similarly. However, it is unclear how cavity macrophages suspended in the fluid phase (peritoneal fluid) identify and migrate toward injuries. In this study, we used intravital microscopy to show that cavity macrophages in fluid rapidly form thrombus-like structures in response to injury by means of primordial scavenger receptor cysteine-rich domains. Aggregates of cavity macrophages physically sealed injuries and promoted rapid repair of focal lesions. In iatrogenic surgical situations, these cavity macrophages formed extensive aggregates that promoted the growth of intra-abdominal scar tissue known as peritoneal adhesions.

Impact of intensive care unit relocation and role of tap water on an outbreak of Pseudomonas aeruginosa expressing OprD-mediated resistance to imipenem.

BACKGROUND: To assess the impact of the incidental relocation of an intensive care unit (ICU) on the risk of colonizations/infections with Pseudomonas aeruginosa exhibiting OprD-mediated resistance to imipenem (PA-OprD). AIM: The primary aim was to compare the proportion of PA-OprD among P. aeruginosa samples before and after an incidental relocation of the ICU. The role of tap water as a route of contamination for colonization/infection of patients with PA-OprD was assessed as a secondary aim. METHODS: A single-centre, observational, before/after comparison study was conducted from October 2013 to October 2015. The ICU was relocated at the end of October 2014. All P. aeruginosa-positive samples isolated from patients hospitalized ≥48 h in the ICU were included. Tap water specimens were collected every three months in the ICU. PA-OprD strains isolated from patients and tap water were genotyped using pulse-field gel electrophoresis. FINDINGS: A total of 139 clinical specimens of P. aeruginosa and 19 tap water samples were analysed. The proportion of PA-OprD strains decreased significantly from 31% to 7.7% after the relocation of the ICU (P = 0.004). All PA-OprD clinical specimens had a distinct genotype. Surprisingly, tap water was colonized with a single PA-OprD strain during both periods, but this single clone has never been isolated from clinical specimens. CONCLUSION: Relocation of the ICU was associated with a marked decrease in P. aeruginosa strains resistant to imipenem. The polyclonal character of PA-OprD strains isolated from patients and the absence of tap-water-to-patient contamination highlight the complexity of the environmental impact on the endogenous colonization/infection with P. aeruginosa.

Cell-free reconstitution reveals centriole cartwheel assembly mechanisms.

How cellular organelles assemble is a fundamental question in biology. The centriole organelle organizes around a nine-fold symmetrical cartwheel structure typically ∼100 nm high comprising a stack of rings that each accommodates nine homodimers of SAS-6 proteins. Whether nine-fold symmetrical ring-like assemblies of SAS-6 proteins harbour more peripheral cartwheel elements is unclear. Furthermore, the mechanisms governing ring stacking are not known. Here we develop a cell-free reconstitution system for core cartwheel assembly. Using cryo-electron tomography, we uncover that the Chlamydomonas reinhardtii proteins CrSAS-6 and Bld10p together drive assembly of the core cartwheel. Moreover, we discover that CrSAS-6 possesses autonomous properties that ensure self-organized ring stacking. Mathematical fitting of reconstituted cartwheel height distribution suggests a mechanism whereby preferential addition of pairs of SAS-6 rings governs cartwheel growth. In conclusion, we have developed a cell-free reconstitution system that reveals fundamental assembly principles at the root of centriole biogenesis.

Immediate postoperative extubation in bilateral lung transplantation: predictive factors and outcomes.

BACKGROUND: We reviewed our experience with tracheal extubation in the operating room (E-OR) among cystic fibrosis patients requiring bilateral lung transplantation to evaluate safety and determine predictive factors of E-OR. METHODS: The charts of 89 recipients (from May 2007 to June 2013) were analysed. Patients were divided into E-OR and E-ICU (intensive care unit extubation) groups. Data are expressed as numbers (percentages) or medians [25th-75th percentiles]. RESULTS: There were 41 patients in the E-OR group (46%). Donor and recipient characteristics were similar between groups. Intraoperative complications occurred less frequently in the E-OR group, and fluid and transfusion requirements were lower. Postoperative courses were different in the E-OR group, including a lower rate of grade 3 primary graft dysfunction (0 compared with 19 patients, P<0.0001) and shorter ICU (5.0 [3.7-7.2] compared with 11.5 [7.0-15.5] days) and hospital stays (22.0 [18.0-25.5] compared with 33.0 [25.0-56.5] days, respectively; P<0.0001 for both). The 1 yr survival rates were similar: 95% in the E-OR group and 98% in the E-ICU group. A statistical model built on a development cohort of 60 randomly selected patients predicted 95% of E-OR instances in this cohort and 82% of E-OR instances in the validation cohort (28 patients). Predictive factors were complications during single-lung ventilation (second graft implantation), complications during bipulmonary ventilation (end of surgery), and the ratio of arterial partial pressure of oxygen to fractional inspired oxygen (end of surgery). CONCLUSIONS: Our protocol allowed for extubation of 46% of bilateral lung transplant patients without increased postoperative risks.

Safe high-pressure freezing of infectious micro-organisms.

We describe how high-pressure freezing of infectious biological material can safely be accomplished with the help of membrane carriers. The method described is easy to perform; however, careful manipulations are required. Existing safety regulations must still be followed. However, the procedure reduces the risk of dissemination of infectious material.

Necrotizing fasciitis and septic shock related to the uncommon gram-negative pathogen Sphingobacterium multivorum.

We report the first case of necrotizing fasciitis due to the uncommon Gram-negative pathogen Sphingobacterium multivorum in an immunocompromised patient, who presented with septic shock. This case adds necrotizing fasciitis to the spectrum of S. multivorum-related infections and highlights the emergence of Gram-negative bacteria in severe soft tissue infections.

[Management of acute and severe complications in adults with cystic fibrosis]. / Prise en charge des complications aiguës sévères chez l'adulte mucoviscidosique.

The natural history of cystic fibrosis (CF) may be associated both with acute respiratory complications (respiratory exacerbations, haemoptysis, pneumothorax) and with non-respiratory complications (distal intestinal obstruction syndrome, dehydration) that may result in hospitalizations. The aim of this article is to describe the main therapeutic approaches that are adopted in the management of acute complications occurring in CF adults, and to discuss indications for admission of these patients to intensive care units. Adult CF patients admitted to intensive care unit often benefit from antibiotic courses adapted to their chronic bronchial infection, especially when the hospitalization is related to respiratory disease (including haemoptysis and pneumothorax). Nutritional support, including hypercaloric diet, control of hyperglycemia and pancreatic enzyme supplementation is warranted. The recommended therapy for major haemoptysis is bronchial artery embolization. Patient with significant pneumothorax should have a chest tube inserted, while the treatment of distal intestinal obstruction syndrome will most often be medical. In case of respiratory failure, non-invasive ventilation is the preferred mode of ventilatory support because invasive ventilation is associated with poor outcomes. Therapeutic options should always have been discussed between the patient, family members and the CF medical team to allow for informed decision making.

Comparison of different methods for thin section EM analysis of Mycobacterium smegmatis.

Bacteria are generally difficult specimens to prepare for conventional resin section electron microscopy and mycobacteria, with their thick and complex cell envelope layers being especially prone to artefacts. Here we made a systematic comparison of different methods for preparing Mycobacterium smegmatis for thin section electron microscopy analysis. These methods were: (1) conventional preparation by fixatives and epoxy resins at ambient temperature. (2) Tokuyasu cryo-section of chemically fixed bacteria. (3) rapid freezing followed by freeze substitution and embedding in epoxy resin at room temperature or (4) combined with Lowicryl HM20 embedding and ultraviolet (UV) polymerization at low temperature and (5) CEMOVIS, or cryo electron microscopy of vitreous sections. The best preservation of bacteria was obtained with the cryo electron microscopy of vitreous sections method, as expected, especially with respect to the preservation of the cell envelope and lipid bodies. By comparison with cryo electron microscopy of vitreous sections both the conventional and Tokuyasu methods produced different, undesirable artefacts. The two different types of freeze-substitution protocols showed variable preservation of the cell envelope but gave acceptable preservation of the cytoplasm, but not lipid bodies, and bacterial DNA. In conclusion although cryo electron microscopy of vitreous sections must be considered the 'gold standard' among sectioning methods for electron microscopy, because it avoids solvents and stains, the use of optimally prepared freeze substitution also offers some advantages for ultrastructural analysis of bacteria.

Multifocal Epstein-Barr virus-associated smooth muscle tumor in adults with AIDS: case report and review of the literature.

OBJECTIVES: The aim of this study was to provide a systematic review of Epstein-Barr virus-associated smooth muscle tumors (EBV-SMT) in human immunodeficiency virus (HIV)-infected adults, focusing on clinical and histopathologic features and outcome. METHODS: A literature search was performed using Medline, Embase and the Cochrane Library. RESULTS: We reviewed 35 cases including our case of a patient with a progressive multifocal EBV-SMT. Patients were mainly men (n = 24) with a mean age of 35.5 years. Median CD4 count was 21/mm(3). Main locations were brain (n = 12), liver (n = 8), spinal cord (n = 7) and adrenal gland (n = 6). The tumors were multifocal in 34% of cases, whereas analysis of clonality showed different clones in tumors from different sites. Treatment included removal surgery in 17 cases and/or radiotherapy in 9 and therapeutic abstention in 4. Mean follow-up after diagnosis was 12.3 months. Nine patients died during this period essentially from opportunistic infection and only 2 from the disease. CONCLUSION: EBV-SMT should be added to the list of virally induced tumors in severely immunocompromised HIV-infected adults. Multifocality of independent tumor clones, especially in liver, brain, spinal cord and adrenal gland, and a slow disease progression seem to be the key features of these tumors, the treatment of which remains poorly defined.

Compression and crevasses in vitreous sections under different cutting conditions.

Compression and crevasses are common cutting artefacts in cryo-ultramicrotomy of vitreous sections. They can be reduced or suppressed under optimal cutting conditions. In the present study, compression and thickness were measured for different cutting speeds and knife angles. It was found that compression decreased with feed and that crevasses appeared only above a certain thickness. The optimal feed for vitreous sections was between 50 and 80 nm. The thickness, calculated by two independent methods, was quantitatively related to feed and compression.

The lipophilic hapten parthenolide induces interferon-gamma and interleukin-13 production by peripheral blood-derived CD8+ T cells from contact allergic subjects in vitro.

BACKGROUND: Peripheral blood mononuclear cells (PBMC) from persons with contact allergy to nickel react in vitro predominantly with nickel-induced CD4+ T cell-mediated production of both T-cell type 1 and 2 cytokines. OBJECTIVES: The aim of the present study was to investigate if the contact allergen parthenolide, a sesquiterpene lactone of lipophilic character, elicits an immune response which differs from that induced by water-soluble nickel ions. PATIENTS AND METHODS: Ten allergic subjects with strong (n = 6), moderate (n = 2), or weak (n = 2) patch-test reactivity to parthenolide and five patch test-negative control subjects participated in the study. PBMC from the subjects were analysed for in vitro reactivity with parthenolide by an enzyme-linked immunospot (ELISpot) assay, measuring cytokine production at the single-cell level. RESULTS: The allergic group, but not the control group, responded to parthenolide with increased numbers of cells producing interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-5 (P < 0.05 for all) and IL-13 (P < 0.01). The responses manifested by T-cell type 1 (IFN-gamma and IL-2) and type 2 (IL-4, IL-5 and IL-13) cytokines were positively correlated between cytokines. Subjects with a strong or moderate, but not weak or negative, patch-test reaction displayed detectable in vitro responses. In contrast to the CD4+ T cell-mediated peripheral reactivity induced by nickel, cell depletion experiments identified the parthenolide-reactive IFN-gamma- and IL-13-producing cells as CD8+ T cells. CONCLUSIONS: The finding that the PBMC reactivity to parthenolide in humans involves a CD8+ T cell-mediated type 1 and 2 cytokine response warrants further studies on the relationship between the chemical nature of a hapten and the resulting immune response.

Nickel-induced IL-10 down-regulates Th1- but not Th2-type cytokine responses to the contact allergen nickel.

Whereas the involvement of Th1- and Th2-type cytokines in contact allergy to nickel (Ni) is well documented, the role of the regulatory cytokine IL-10 is less clear. We therefore investigated the impact of IL-10 on Ni-induced Th1- (IFN-gamma) and Th2-type (IL-4 and IL-13) cytokine responses in human peripheral blood mononuclear cells (PBMC). PBMC from 15 blood donors with reactivity to Ni (Ni-PBMC) and 8 control donors devoid of reactivity (control PBMC) were stimulated with Ni and the frequency of cytokine-producing cells and the levels of secreted cytokines were analysed by ELISpot (IL-4, IL-13 and IFN-gamma) and ELISA (IL-10, IL-13 and IFN-gamma), respectively. The Ni-induced response was further assessed in the presence of recombinant IL-10 (rIL-10) or neutralizing antibody to IL-10 and the phenotype of the Ni-specific cytokine-producing cells regulated by IL-10 was determined by cell depletion experiments. Ni induced IL-10 production in Ni-PBMC (mean, (range); 33.1 pg/ml (0-93.4 pg/ml)) but not control PBMC (2.2 pg/ml (0-14.9 pg/ml)) (P = 0.002). Ni also induced significant production of IL-4, IL-13 and IFN-gamma that correlated with the IL-10 response. Addition of rIL-10 down-regulated the Ni-induced production of all cytokines but with a more pronounced effect on IFN-gamma. However, neutralization of Ni-induced IL-10 enhanced the levels of IFN-gamma induced by Ni (P = 0.004) but did not affect the number of IFN-gamma-producing cells or the production of other cytokines. Cell depletion experiments suggested that the Ni-specific IFN-gamma (and Th2-type cytokine) producing cells were CD4(+) T cells. The impact of IL-10 on Ni-induced IFN-gamma responses by CD4(+) T cells suggests that an important role of IL-10 in vivo is to counteract the allergic reactions mediated by Th1-type cytokines.

Multi-subtype gp160 DNA immunization induces broadly neutralizing anti-HIV antibodies.

A highly desirable feature for an human immunodeficiency virus type 1 (HIV-1) vaccine is the ability to induce broadly reactive anti-envelope antibodies that can neutralize primary HIV-1 isolates. Two immunizations with an HIV-1 envelope-encoding plasmid together with recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) resulted in high antibody titers in mice. The antibody induction was further enhanced after immunization with genes encoding HIV-1 envelopes originating from subtypes A, B and C. The sera from these animals were able to neutralize A, B and C viral isolates, whereas the sera from animals immunized solely with subtype B DNA neutralized only subtype B virus. The combined DNA vaccine gave serum antibodies with broad recognition of HIV-1 envelope epitopes as determined by peptide mapping. Cell-mediated immunity was not compromised by the increased humoral immunity. This demonstrates the ability of multiple envelope genes to induce the desired antibody response against several subtypes. Moreover, it documents the ability of rGM-CSF to enhance the potency of such a vaccine when given simultaneously. The strategy may be useful for making an HIV vaccine more potent and broadly effective against strains of different clades.

Genetic immunization is augmented by murine polyomavirus VP1 pseudocapsids.

To improve immune responses induced by DNA immunization, murine polyomavirus major capsid protein (VP1) pseudocapsids were complexed with a DNA plasmid encoding the p37 (p24 and p17) nucleocapsid proteins of the human immunodeficiency virus type 1 (HIV-1). A 10-fold increase in antibody titer was noted in mice given DNA plasmid together with VP1 pseudocapsids in comparison to animals that received DNA plasmid alone. Cell mediated responses to HIV-1 p24 occurred, but were not significantly augmented by delivering the DNA as a VP1 complex. We have consequently for the first time shown a carrier/adjuvant effect of polyomavirus pseudocapsids that strongly increased the humoral immune response in DNA immunization.

HIV subtypes and recombination strains--strategies for induction of immune responses in man.

Clinical and experimental studies of HIV-1 subcomponents were made in order to increase their immunogenicity. HIV subtype envelopes A, B and C have been compared and a detailed analysis made by peptides of the coreceptor-ligand interactions. We identified a direct interaction between HIV-1 envelope and a cellular receptor at the amino acid level. Both the viral subtype and its tropism appeared to influence inhibition of infection. Genetic immunization induced new cytotoxic responses while proteins appeared to efficiently boost previous responses. One HIV-1 subtype B antigen was strongly immunogenic in a human immunotherapeutic trial and permitted better survival at 2 years of the study in patients with poor prognosis.

An in vivo model for HIV resistance development.

Treatment of human immunodeficiency virus (HIV-1) with drugs targeted to the reverse transcriptase (RT) rapidly selects for drug-resistant virus. It is essential to develop a suitable animal model that allows the study of the emergence and reversal of drug resistance. A monkey model was previously developed on the basis of a hybrid virus (RT-SHIV) of simian immunodeficiency virus (SIV) with its RT exchanged for HIV-1 RT. In the present study cynomolgus monkeys infected with RT-SHIV were treated with varying doses of the non-nucleoside RT inhibitor nevirapine. The drug was administered for 2-3 weeks, in agreement with clinical experience of resistance development during nevirapine monotherapy. This resulted in the selection of mutants with Y181C and K103N changes in RT, which correspond to the HIV-1 mutations in nevirapine-resistant HIV-1 patients. The mutants coexisted at varying levels with wild-type virus and fluctuations in the proportion of mutants could be closely monitored. Low-dose treatment was not more efficient in induction of mutations than a virus-inhibiting dose. Structured therapy interruptions could be performed. The monkey RT-SHIV infection offers an in vivo model to determine effects of therapies on resistance development.

Induction of immune responses and break of tolerance by DNA against the HIV-1 coreceptor CCR5 but no protection from SIVsm challenge.

An inactivating mutation in the human CCR5 gene reduces the risk of HIV-1 infection in individuals with homozygous alleles. We explored whether genetic immunization would induce an immune response directed to CCR5 structures and if immunological tolerance toward endogenous CCR5 could be broken. We also studied whether this immunization approach could protect cynomolgus monkeys from an infection, with SIVsm, which primarily uses CCR5 as a coreceptor. Epidermal but not intramuscular delivery of the CCR5 gene to mice elicited strong IgG antibody binding responses to CCR5. Intramucosal immunization of cynomolgus macaques with CCR5 DNA followed by boosts with CCR5 peptides induced prominent IgG and IgA antibody responses in serum and vaginal washings. The CCR5-specific antibodies neutralized the infectivity of primary human R5 HIV-1 strains, and the macaque SIVsm but not that of a tissue culture-adapted X4 HIV-1 strain. The consecutive CCR5 gene and CCR5 peptide immunizations induced B- and T-cell responses to peptides representing both human and macaque amino acid sequences of the respective CCR5 proteins. This indicates that tolerance was broken against endogenous macaque CCR5, which has a 98% homology to the human CCR5 gene. After the final boost, the vaccinated monkeys together with two control monkeys were challenged with SIVsm. Neither protection against nor enhancement of SIVsm infection was achieved.

DNA-encoding enzymatically active HIV-1 reverse transcriptase, but not the inactive mutant, confers resistance to experimental HIV-1 challenge.

The present study was undertaken to examine the immunogenicity of a single plasmid DNA representing the reverse transcriptase (RT) of HIV-1. Plasmids containing the enzymatically active RT as well as a mutated nonenzymatically active RT with nucleotide (nt)-binding motifs of YMDD and YMLL, respectively, were used to immunize mice. Both constructs induced similar good antibody and T cell responses, with a tendency towards antibody directed to peptides representing the active and mutated sites. Immunized mice were challenged with a murine pseudotype HIV-1/MuLV infected spleen cells. Seven out of 10 mice immunized with RT had no recoverable HIV-1, while 10 individuals immunized with the RT mutant and all the 18 controls had high levels of recoverable HIV-1. This indicates that mutation of RT reduces the desired immunogenicity.