Design and Expression of a Dimeric Form of the Human Immunodeficiency Virus Type 1 Antibody 2G12 with Increased Neutralization Potency

The antigen-binding fragment of the broadly neutralizing Human Immunodeficiency Virus Type 1 (HIV-1) antibody 2G12 has an unusual 3D domain-swapped structure with two aligned combining sites that facilitates recognition of its carbohydrate epitope on gp120. When expressed as an intact IgG, 2G12 formed typical IgG monomers containing two combining sites and a small fraction of a higher molecular weight species, which showed a significant increase in neutralization potency (50- to 80-fold compared to 2G12 monomer) across a range of clade A and B strains of HIV-1. Here we show that the higher molecular weight species corresponds to a 2G12 dimer containing four combining sites, and present a model for how intermolecular 3D domain swapping could create a 2G12 dimer. Based on the structural model for a 3D domain-swapped 2G12 dimer, we designed and tested a series of 2G12 mutants predicted to increase the ratio of 2G12 dimer to monomer. We report a mutation that effectively increases the 2G12 dimer/monomer ratio without decreasing the expression yield. Increasing the proportion of 2G12 dimer compared with monomer could lead to a more potent reagent for gene therapy or passive immunization

The effect of acute maximal exercise on the regional distribution of ventilation using ventilation MRI in CF

Background The importance of exercise in the management of people with CF is well recognised, yet the effect of exercise on lung function is not well understood. FEV1 is insensitive to the detection of small changes in lung function. Ventilation MRI and LCI are both more sensitive to mild lung disease than FEV1 and may be better suited to assess the effects of exercise. Here we assessed the short-term effects of maximal exercise on the distribution of ventilation using ventilation MRI and LCI. Methods Patients with CF and a range of lung disease were assessed. Baseline LCI and ventilation MRI was followed by a maximal cardio-pulmonary exercise test (CPET). Repeated ventilation MRI was performed within 30 minutes of exercise termination, followed by LCI and finally by FEV1. Results 13 patients were recruited and completed all assessments. Mean (SD) age was 25 (10) years and mean (SD) FEV1 z-score was -1.8 (1.7). Mean LCI at baseline was 8.2, mean ventilation defect percentage on MRI (VDP) was 7.3%. All patients performed maximal CPET. Post-exercise, there was a visible change in lung ventilation in 85% of patients, including two patients with increased ventilation heterogeneity post-CPET who had normal FEV1. VDP and LCI were significantly reduced post-exercise (p < 0.05) and 45% of patients had a significant change in VDP. Conclusions Acute maximal exercise directly affects the distribution of ventilation on ventilation MRI in patients with CF. This suggests that exercise is beneficial in CF and that ventilation MRI is suitable to assess airway clearance efficacy

Data exploration in phylogenetic inference: scientific, heuristic, or neither

The methods of data exploration have become the centerpiece of phylogenetic inference, but without the scientific importance of those methods having been identified. We examine in some detail the procedures and justifications of Wheeler's sensitivity analysis and relative rate comparison (saturation analysis). In addition, we review methods designed to explore evidential decisiveness, clade stability, transformation series additivity, methodological concordance, sensitivity to prior probabilities (Bayesian analysis), skewness, computer‐intensive tests, long‐branch attraction, model assumptions (likelihood ratio test), sensitivity to amount of data, polymorphism, clade concordance index, character compatibility, partitioned analysis, spectral analysis, relative apparent synapomorphy analysis, and congruence with a “known” phylogeny. In our review, we consider a method to be scientific if it performs empirical tests, i.e., if it applies empirical data that could potentially refute the hypothesis of interest. Methods that do not perform tests, and therefore are not scientific, may nonetheless be heuristic in the scientific enterprise if they point to more weakly or ambiguously corroborated hypotheses, such propositions being more easily refuted than those that have been more severely tested and are more strongly corroborated. Based on common usage, data exploration in phylogenetics is accomplished by any method that performs sensitivity or quality analysis. Sensitivity analysis evaluates the responsiveness of results to variation or errors in parameter values and assumptions. Sensitivity analysis is generally interpreted as providing a measure of support, where conclusions that are insensitive (robust, stable) to perturbations are judged to be accurate, probable, or reliable. As an alternative to that verificationist concept, we define support objectively as the degree to which critical evidence refutes competing hypotheses. As such, degree of support is secondary to the scientific optimality criterion of maximizing explanatory power. Quality analyses purport to distinguish good, reliable, accurate data from bad, misleading, erroneous data, thereby assessing the ability of data to indicate the true phylogeny. Only the quality analysis of character compatibility can be judged scientific—and a weak test at that compared to character congruence. Methods judged to be heuristic include Bremer support, long‐branch extraction, and safe taxonomic reduction, and we underscore the great heuristic potential of a posteriori analysis of patterns of transformations on the total‐evidence cladogram. However, of the more than 20 kinds of data exploration methods evaluated, the vast majority is neither scientific nor heuristic. Given so little demonstrated cognitive worth, we conclude that undue emphasis has been placed on data exploration in phylogenetic inference, and we urge phylogeneticists to consider more carefully the relevance of the methods that they employ. [T]he cult of impressive technicalities or the cult of precision may get the better of us, and interfere with our search for clarity, simplicity, and truth [Popper, 1983, p. 60. Empirical papers chosen for publication are judged to be of interest to a broad systematics audience because they represent exemplary case studies involving some important contemporary issue or issues. These may be unusually thorough explorations of data , applications of new methodology, illustrations of fundamental principles, and/or investigations of interesting evolutionary questions. [Systematic Biology: Instructions for authors, 2002; italics added]Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/93511/1/j.1096-0031.2003.tb00311.x.pd

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Tension pneumatocoele in a child with an empyema

A previously well 13-month-old child presented to our institution with a pneumonia which developed into a tension pneumatocoele. Management was conservative and the patient fully recovered. Streptococcus pneumoniae type 7F was isolated and is not covered by the current heptavalent vaccine