An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behaviour.

Serpins are important regulators of proteolytic pathways with an anti-protease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an inter-molecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of a number of pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39, and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A - on the opposite face of the molecule - more liable to adopt an 'open' state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of pre-formed serpin-enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the 'open' state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for rational design of ligands that is able to dynamically influence α1-AT polymerisation

Birtamimab plus standard of care in light-chain amyloidosis: the phase 3 randomized placebo-controlled VITAL trial

Amyloid light-chain (AL) amyloidosis is a rare, typically fatal disease characterized by the accumulation of misfolded immunoglobulin light chains (LCs). Birtamimab is an investigational humanized monoclonal antibody designed to neutralize toxic LC aggregates and deplete insoluble organ-deposited amyloid via macrophage-induced phagocytosis. VITAL was a phase 3 randomized, double-blind, placebo-controlled clinical trial assessing the efficacy and safety of birtamimab + standard of care (SOC) in 260 newly diagnosed, treatment-naive patients with AL amyloidosis. Patients received 24 mg/kg IV birtamimab + SOC or placebo + SOC every 28 days. The primary composite end point was the time to all-cause mortality (ACM) or centrally adjudicated cardiac hospitalization ≥91 days after the first study drug infusion. The trial was terminated early after an interim futility analysis; there was no significant difference in the primary composite end point (hazard ratio [HR], 0.826; 95% confidence interval [CI], 0.574-1.189; log-rank P = .303). A post hoc analysis of patients with Mayo stage IV AL amyloidosis, those at the highest risk of early mortality, showed significant improvement in the time to ACM with birtamimab at month 9 (HR, 0.413; 95% CI, 0.191-0.895; log-rank P = .021). At month 9, 74% of patients with Mayo stage IV AL amyloidosis treated with birtamimab and 49% of those given placebo survived. Overall, the rates of treatment-emergent adverse events (TEAEs) and serious TEAEs were generally similar between treatment arms. A confirmatory phase 3 randomized, double-blind, placebo-controlled clinical trial of birtamimab in patients with Mayo stage IV AL amyloidosis (AFFIRM-AL; NCT04973137) is currently enrolling. The VITAL trial was registered at www.clinicaltrials.gov as #NCT02312206

Generation of lung epithelial-like tissue from human embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>Human embryonic stem cells (hESC) have the capacity to differentiate <it>in vivo </it>and <it>in vitro </it>into cells from all three germ lineages. The aim of the present study was to investigate the effect of specific culture conditions on the differentiation of hESC into lung epithelial cells.</p> <p>Methods</p> <p>Undifferentiated hESC, grown on a porous membrane in hESC medium for four days, were switched to a differentiation medium for four days; this was followed by culture in air-liquid interface conditions during another 20 days. Expression of several lung markers was measured by immunohistochemistry and by quantitative real-time RT-PCR at four different time points throughout the differentiation and compared to appropriate controls.</p> <p>Results</p> <p>Expression of <it>CC16 </it>and <it>NKX2.1 </it>showed a 1,000- and 10,000- fold increase at day 10 of differentiation. Other lung markers such as <it>SP-C </it>and <it>Aquaporin 5 </it>had the highest expression after twenty days of culture, as well as two markers for ciliated cells, <it>FOXJ1 </it>and <it>β-tubulin IV</it>. The results from qRT-PCR were confirmed by immunohistochemistry on paraffin-embedded samples. Antibodies against CC16, SP-A and SP-C were chosen as specific markers for Clara Cells and alveolar type II cells. The functionality was tested by measuring the secretion of CC16 in the medium using an enzyme immunoassay.</p> <p>Conclusion</p> <p>These results suggest that by using our novel culture protocol hESC can be differentiated into the major cell types of lung epithelial tissue.</p

Development of a Multi-Step Leukemogenesis Model of MLL-Rearranged Leukemia Using Humanized Mice

Mixed-lineage-leukemia (MLL) fusion oncogenes are intimately involved in acute leukemia and secondary therapy-related acute leukemia. To understand MLL-rearranged leukemia, several murine models for this disease have been established. However, the mouse leukemia derived from mouse hematopoietic stem cells (HSCs) may not be fully comparable with human leukemia. Here we developed a humanized mouse model for human leukemia by transplanting human cord blood-derived HSCs transduced with an MLL-AF10 oncogene into a supra-immunodeficient mouse strain, NOD/Shi-scid, IL-2Rγ−/− (NOG) mice. Injection of the MLL-AF10-transduced HSCs into the liver of NOG mice enhanced multilineage hematopoiesis, but did not induce leukemia. Because active mutations in ras genes are often found in MLL-related leukemia, we next transduced the gene for a constitutively active form of K-ras along with the MLL-AF10 oncogene. Eight weeks after transplantation, all the recipient mice had developed acute monoblastic leukemia (the M5 phenotype in French-American-British classification). We thus successfully established a human MLL-rearranged leukemia that was derived in vivo from human HSCs. In addition, since the enforced expression of the mutant K-ras alone was insufficient to induce leukemia, the present model may also be a useful experimental platform for the multi-step leukemogenesis model of human leukemia

Caveolin-1 expression in benign and malignant lesions of the breast

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Alarm Pheromones and Chemical Communication in Nymphs of the Tropical Bed Bug Cimex hemipterus (Hemiptera: Cimicidae)

The recent resurge of bed bug infestations (Cimex spp.; Cimicidae) and their resistance to commonly used pesticides calls for alternative methods of control. Pheromones play an important role in environmentally sustainable methods for the management of many pest insects and may therefore be applicable for the control of bed bugs. The tropical bed bug, Cimex hemipterus, is a temporary ectoparasite on humans and causes severe discomfort. Compared to the common bed bug, Cimex lectularius, little is known about the chemical signalling and pheromone-based behaviour of the tropical species. Here, we show that the antennal morphology and volatile emission of C. hemipterus closely resembles those of C. lectularius and we test their behavioural responses to conspecific odour emissions. Two major volatiles are emitted by male, female and nymph C. hemipterus under stress, (E)-2-hexenal and (E)-2-octenal. Notably, nymph emissions show contrasting ratios of these compounds to adults and are further characterized by the addition of 4-oxo-(E)-2-hexenal and 4-oxo-(E)-2-octenal. The discovery of this nymph pheromone in C. hemipterus is potentially the cause of a repellent effect observed in the bio-tests, where nymph odours induce a significantly stronger repellent reaction in conspecifics than adult odours. Our results suggest that pheromone-based pest control methods developed for C. lectularius could be applicable to C. hemipterus, with the unique nymph blend showing promising practical properties

Parallel Evolution under Chemotherapy Pressure in 29 Breast Cancer Cell Lines Results in Dissimilar Mechanisms of Resistance

Background: Developing chemotherapy resistant cell lines can help to identify markers of resistance. Instead of using a panel of highly heterogeneous cell lines, we assumed that truly robust and convergent pattern of resistance can be identified in multiple parallel engineered derivatives of only a few parental cell lines. Methods: Parallel cell populations were initiated for two breast cancer cell lines (MDA-MB-231 and MCF-7) and these were treated independently for 18 months with doxorubicin or paclitaxel. IC50 values against 4 chemotherapy agents were determined to measure cross-resistance. Chromosomal instability and karyotypic changes were determined by cytogenetics. TaqMan RT-PCR measurements were performed for resistance-candidate genes. Pgp activity was measured by FACS. Results: All together 16 doxorubicin- and 13 paclitaxel-treated cell lines were developed showing 2-46 fold and 3-28 fold increase in resistance, respectively. The RT-PCR and FACS analyses confirmed changes in tubulin isofom composition, TOP2A and MVP expression and activity of transport pumps (ABCB1, ABCG2). Cytogenetics showed less chromosomes but more structural aberrations in the resistant cells. Conclusion: We surpassed previous studies by parallel developing a massive number of cell lines to investigate chemoresistance. While the heterogeneity caused evolution of multiple resistant clones with different resistance characteristics, the activation of only a few mechanisms were sufficient in one cell line to achieve resistance. © 2012 Tegze et al

Importance of pre-analytical steps for transcriptome and RT-qPCR analyses in the context of the phase II randomised multicentre trial REMAGUS02 of neoadjuvant chemotherapy in breast cancer patients

<p>Abstract</p> <p>Background</p> <p>Identification of predictive markers of response to treatment is a major objective in breast cancer. A major problem in clinical sampling is the variability of RNA templates, requiring accurate management of tumour material and subsequent analyses for future translation in clinical practice. Our aim was to establish the feasibility and reliability of high throughput RNA analysis in a prospective trial.</p> <p>Methods</p> <p>This study was conducted on RNA from initial biopsies, in a prospective trial of neoadjuvant chemotherapy in 327 patients with inoperable breast cancer. Four independent centres included patients and samples. Human U133 GeneChips plus 2.0 arrays for transcriptome analysis and quantitative RT-qPCR of 45 target genes and 6 reference genes were analysed on total RNA.</p> <p>Results</p> <p>Thirty seven samples were excluded because <it>i) </it>they contained less than 30% malignant cells, or <it>ii) </it>they provided RNA Integrity Number (RIN) of poor quality. Among the 290 remaining cases, taking into account strict quality control criteria initially defined to ensure good quality of sampling, 78% and 82% samples were eligible for transcriptome and RT-qPCR analyses, respectively. For RT-qPCR, efficiency was corrected by using standard curves for each gene and each plate. It was greater than 90% for all genes. Clustering analysis highlighted relevant breast cancer phenotypes for both techniques (ER+, PR+, HER2+, triple negative). Interestingly, clustering on trancriptome data also demonstrated a "centre effect", probably due to the sampling or extraction methods used in on of the centres. Conversely, the calibration of RT-qPCR analysis led to the centre effect withdrawing, allowing multicentre analysis of gene transcripts with high accuracy.</p> <p>Conclusions</p> <p>Our data showed that strict quality criteria for RNA integrity assessment and well calibrated and standardized RT-qPCR allows multicentre analysis of genes transcripts with high accuracy in the clinical context. More stringent criteria are needed for transcriptome analysis for clinical applications.</p

High ALDH Activity Identifies Chemotherapy-Resistant Ewing's Sarcoma Stem Cells That Retain Sensitivity to EWS-FLI1 Inhibition

Cancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor, thereby causing relapse and patient death. Ewing's sarcoma, the second most common form of bone tumor in adolescents and young adults, follows a clinical pattern consistent with the Cancer Stem Cell model - remission is easily achieved, even for patients with metastatic disease, but relapse remains frequent and is usually fatal.We have isolated a subpopulation of Ewing's sarcoma cells, from both human cell lines and human xenografts grown in immune deficient mice, which express high aldehyde dehydrogenase (ALDH(high)) activity and are enriched for clonogenicity, sphere-formation, and tumor initiation. The ALDH(high) cells are resistant to chemotherapy in vitro, but this can be overcome by the ATP binding cassette transport protein inhibitor, verapamil. Importantly, these cells are not resistant to YK-4-279, a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewing's sarcoma cells both in vitro and in vivo.Ewing's sarcoma contains an ALDH(high) stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewing's sarcoma stem cells that survive standard chemotherapy