Here, we report improved solubility and enhanced colonic delivery of reduced bromonoscapine (Red-Br-Nos), a cyclic ether brominated analogue of noscapine, upon encapsulation of its cyclodextrin (CD) complexes in bioresponsive guar gum microspheres (GGM). Phase-solubility analysis suggested that Red-Br-Nos complexed with β-CD and methyl-β-CD in a 1:1 stoichiometry, with a stability constant (Kc) of 2.29 × 103 M-1 and 4.27 × 103 M-1. Fourier transforms infrared spectroscopy indicated entrance of an O-CH2 or OCH3-C6H4-OCH3 moiety of Red-Br-Nos in the β-CD or methyl-β-CD cavity. Furthermore, the cage complex of Red-Br-Nos with β-CD and methyl-β-CD was validated by several spectral techniques. Rotating frame Overhauser enhancement spectroscopy revealed that the Ha proton of the OCH3-C6H4-OCH3 moiety was closer to the H5 proton of β-CD and the H3 proton of the methyl-β-CD cavity. The solubility of Red-Br-Nos in phosphate buffer saline (PBS, pH ∼ 7.4) was improved by ∼10.7-fold and ∼21.2-fold when mixed with β-CD and methyl-β-CD, respectively. This increase in solubility led to a favorable decline in the IC50 by ∼2-fold and ∼3-fold for Red-Br-Nos-β-CD-GGM and Red-Br-Nos-methyl-β-CD-GGM formulations respectively, compared to free Red-Br-Nos-β-CD and Red-Br-Nos-methyl-β-CD in human colon HT-29 cells. GGM-bearing drug complex formulations were found to be highly cytotoxic to the HT-29 cell line and further effective with simultaneous continuous release of Red-Br-Nos from microspheres. This is the first study to showing the preparation of drug-complex loaded GGMS for colon delivery of Red-Br-Nos that warrants preclinical assessment for the effective management of colon cancer.
Objective-The platelet storage lesion accelerates platelet clearance after transfusion, but the underlying molecular mechanism remains elusive. Although inhibiting sheddase activity hampers clearance of platelets with storage lesion, the target platelet protein responsible for ectodomain shedding-induced clearance is not definitively identified. Monocl onal antibody 5G6 was developed recently to bind specifically human platelet receptor glycoprotein (GP)Iba and inhibit its shedding but not shedding of other receptors. Here, the role of GPIbα shedding in platelet clearance after transfusion was addressed.
Approach and Results-Both human leukoreduced apheresis-derived platelets and transgenic mouse platelets expressing human GPIbα were stored at room temperature in the presence and absence of 5G6 Fab fragment. At various time points, aliquots of stored platelets were analyzed and compared. 5G6 Fab inhibited GPIbα shedding in both platelets during storage and preserved higher level of GPIbα on the platelet surface. Compared with age-matched control platelets, 5G6 Fab-stored platelets exhibited similar levels of platelet activation, degranulation, and agonist-induced aggregation. 5G6 Fab-stored human GPIbα platelets exhibited significantly higher post-Transfusion recovery and in vivo hemostatic function in recipient mice than control platelets. Consistently, 5G6 Fab-stored, 8-day-old human platelets produced similar improvement in post-Transfusion recovery in immunodeficient mice and in ex vivo thrombus formation over collagen under shear flow.
Conclusions-Specific inhibition of GPIbα shedding in the stored platelets improves post-Transfusion platelet recovery and hemostatic function, providing clear evidence for GPIbα shedding as a cause of platelet clearance. These results suggest that specific inhibition of GPIbα shedding may be used to optimize platelet storage conditions.
Purpose: Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) carries a distinct clinical behaviour. c-Met oncogene is an important driver for tumour progression and its relationship with HPV in OPSCC was explored in the present study.
Experimental design: Knockdown of HPV oncogene E6 or p53 alone and in combination was performed to examine their effects on c-Met expression by Western blot and quantitative real-time polymerase chain reaction. The effects of c-Met inhibition on cell proliferation, migration, and colony formation were examined in HPV-positive head and neck squamous cell carcinoma (HNSCC) cells. Retrospectively collected OPSCC patient specimens (N = 78) were stained for c-Met by immunohistochemistry and the staining levels were correlated with HPV status and patient outcomes.
Results: E6 knockdown decreased c-Met protein and mRNA expression in HPV-positive HNSCC cells, which was partially abolished by the elimination of p53. Reducing c-Met decreased cell proliferation, migration, and colony formation in HPV-positive HNSCC cells. In OPSCC patient samples, high c-Met expression was associated with HPV-positive status (OR = 4.11, 95%CI: 1.16–14.55, P = 0.028) and tumour stage (OR = 0.27, 95%CI: 0.08–0.93, P = 0.039) by multivariable analysis. In T3/T4 stage patients, high c-Met expression was associated with HPV positivity and low p53 levels, supporting an axis of E6-p53-c-Met regulation. Furthermore, high c-Met expression was marginally associated with poor disease-free survival in HPV-positive patients.
Conclusions: Our results suggest that c-Met may serve as a novel target for treating HPV-associated OPSCC. The data also demonstrate that HPV E6 upregulates c-Met expression partially through p53 downregulation.
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Dmitri Kazmin;
Helder Nakaya;
Eva K. Lee;
Matthew J. Johnson;
Robbert van der Most;
Robert A. van den Berg;
W. Ripley Ballou;
Erik Jongert;
Ulrike Wille-Reece;
Christian Ockenhouse;
Alan Aderem;
Daniel E. Zak;
Jerald Sadoff;
Jenny Hendriks;
Jens Wrammert;
Rafi Ahmed;
Bali Pulendran
RTS,S is an advanced malaria vaccine candidate and confers significant protection against Plasmodium falciparum infection in humans. Little is known about the molecular mechanisms driving vaccine immunity. Here, we applied a systems biology approach to study immune responses in subjects receiving three consecutive immunizations with RTS,S (RRR), or in those receiving two immunizations of RTS,S/AS01 following a primary immunization with adenovirus 35 (Ad35) (ARR) vector expressing circumsporozoite protein. Subsequent controlled human malaria challenge (CHMI) of the vaccinees with Plasmodium-infected mosquitoes, 3 wk after the final immunization, resulted in ∼50% protection in both groups of vaccinees. Circumsporozoite protein (CSP)-specific antibody titers, prechallenge, were associated with protection in the RRR group. In contrast, ARR-induced lower antibody responses, and protection was associated with polyfunctional CD4 + T-cell responses 2 wk after priming with Ad35. Molecular signatures of B and plasma cells detected in PBMCs were highly correlated with antibody titers prechallenge and protection in the RRR cohort. In contrast, early signatures of innate immunity and dendritic cell activation were highly associated with protection in the ARR cohort. For both vaccine regimens, natural killer (NK) cell signatures negatively correlated with and predicted protection. These results suggest that protective immunity against P. falciparum can be achieved via multiple mechanisms and highlight the utility of systems approaches in defining molecular correlates of protection to vaccination.
Recent advances in microscopy imaging and genomics have created an explosion of patient data in the pathology domain. Whole-slide images (WSIs) of tissues can now capture disease processes as they unfold in high resolution, recording the visual cues that have been the basis of pathologic diagnosis for over a century. Each WSI contains billions of pixels and up to a million or more microanatomic objects whose appearances hold important prognostic information. Computational image analysis enables the mining of massive WSI datasets to extract quantitative morphologic features describing the visual qualities of patient tissues. When combined with genomic and clinical variables, this quantitative information provides scientists and clinicians with insights into disease biology and patient outcomes. To facilitate interaction with this rich resource, we have developed a web-based machine-learning framework that enables users to rapidly build classifiers using an intuitive active learning process that minimizes data labeling effort. In this paper we describe the architecture and design of this system, and demonstrate its effectiveness through quantification of glioma brain tumors.
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Samantha Reiss;
Amy E. Baxter;
Kimberly M. Cirelli;
Jennifer M. Dan;
Antigoni Morou;
Audrey Daigneault;
Nathalie Brassard;
Guido Silvestri;
Jean-Pierre Routy;
Colin Havenar-Daughton;
Shane Crotty;
Daniel E. Kaufmann
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells.
The anomalous folding and polymerization of the β-amyloid (Aβ) peptide is thought to initiate the neurodegenerative cascade in Alzheimer's disease pathogenesis1. Aβ is predominantly a 40- or 42-amino acid peptide that is prone to self-aggregation into β-sheet-rich amyloid fibrils that are found in the cores of cerebral senile plaques in Alzheimer's disease. Increasing evidence suggests that low molecular weight, soluble Aβ multimers are more toxic than fibrillar Aβ amyloid2. The identification and quantification of low- and high-molecular weight multimeric Aβ species in brain tissue is an essential objective in Alzheimer's disease research, and the methods employed also can be applied to the identification and characterization of toxic multimers in other proteopathies3. Naturally occurring Aβ multimers can be detected by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with Aβ-specific antibodies. However, the separation and detection of multimeric Aβ requires the use of highly concentrated cortical homogenates and antigen retrieval in small pore-size nitrocellulose membranes. Here we describe a technique for the preparation of clarified human cortical homogenates, separation of proteins by SDS-PAGE, and antigen-epitope retrieval/Western blotting with antibody 6E10 to the N-terminal region of the Aβ peptide. Using this protocol, we consistently detect Aβ monomers, dimers, trimers, tetramers, and higher molecular weight multimers in cortical tissue from humans with Alzheimer's pathology.
Purpose. Meningioangiomatosis (MA) is a rare, benign lesion that commonly mimics other intracranial malformations in clinical presentation and appearance on imaging. The case presented and the literature review performed highlight the importance of combining MRI and CT results to better characterize intracranial lesions and including MA on the list of differential diagnoses of patients presenting with seizures. Methods. The case described is of a 19-year-old male with a 10-year history of worsening seizures refractory to multiple drug regimens. MRI revealed an atypical vascular malformation. The patient underwent surgical resection of the epileptogenic cortex. Results. Although the radiologic impression of the lesion was a vascular malformation, pathological examination revealed MA. A literature search performed highlights the variability of the appearance of MA on CT and MRI and suggests the utility of the T2 GRE sequence in illustrating the presence of calcification and, in a lesion with other characteristic features, the diagnosis of MA. Conclusion. MA can be a difficult diagnosis to make based on imaging findings alone. However, in a patient with a characteristic history and presentation, the presence of a calcified mass on CT and MRI brain susceptibility artifact on a T2 GRE sequence may suggest MA.
Administration of the bacterial protein flagellin to mice activates innate immune signaling that protects against an array of challenges, including ionizing radiation. Herein, we define the underlying mechanism for this protection. We report that flagellin treatment induces proliferation and mobilization of bone marrow cells that aid survival following irradiation. Specifically, treatment of mice or bone marrow cells ex vivo with flagellin induced Toll-like receptor 5 (TLR5)-dependent and NOD-like receptor C4-independent proliferation of Lin-Sca-1+Kit+ (LSK) cells, which includes both hematopoietic stem cells that provide long-term repopulation (LTR) and multipotent progenitor cells (MPPs) that transiently proliferate and differentiate into a range of blood cell types. TLR5 expression on bone marrow cells was necessary and sufficient for flagellin-induced LSK proliferation. Flagellin treatment stimulated LSK proliferation by inducing a 10-fold increase in type 3 MPP (MPP3) without a concomitant increase in LTR cells. Cotransfer of 5 × 103 fluorescence-activated cell sorted flagellin-induced MPP3 cells along with 1 × 105 whole bone marrow cells to lethally irradiated mice revealed that such cells predominantly repopulated the neutrophil compartment for up to 4 week, and dramatically increased the survival rate of the bone marrow transplantation procedure. Hence, we propose the administration of MPP3 cells, elicited by flagellin, as a novel approach to prevent life-threatening neutropenia that can accompany bone marrow transplant and other myeloablative therapeutic procedures.