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.
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Xiaojian Yang;
Chen Shao;
Ruoxiang Wang;
Chia-Yi Chu;
Peizhen Hu;
Viraj Master;
Adeboye O. Osunkoya;
Hyung L. Kim;
Haiyen E. Zhau;
Leland W. K. Chung
Purpose: We assessed the application of near infrared heptamethine carbocyanine dyes, including IR-783 and the synthetic analogue MHI-148, as optical imaging agents for the rapid detection of human kidney cancer. Materials and Methods: The uptake, retention and subcellular localization of these organic dyes were investiga ted in cultured kidney cancer cells. Tumor specificity of dye uptake and retention was evaluated by whole body imaging of mice bearing human kidney cancer xenografts or freshly harvested clinical kidney cancer specimens. In addition, dye accumulation at the tissue and cellular levels was confirmed by ex vivo studies with results confirmed by fluorescence imaging of frozen tissue sections. Peripheral blood spiked with kidney cancer cells was stained to simulate the detection of circulating tumor cells. Results: Preferential uptake and retention of carbocyanine near infrared dyes was observed in cultured human kidney cancer cells, human kidney cancer cell spiked whole blood, human kidney cancer xenografts and freshly harvested human kidney cancer tissues compared to normal kidney epithelial cells and normal host organs. Conclusions: We describe a new class of near infrared heptamethine carbocyanine dyes that show potential for detecting kidney cancer cells in circulating blood and kidney cancer cells in clinical specimens. Near infrared carbocyanine dyes can be further developed as dual modality agents for deep tissue imaging of localized and disseminated kidney cancer in patients.
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Christopher K. Hwang;
Thomas M. Aaberg, Jr.;
Patricia Chevez-Barrios;
Elizabeth Verner-Cole;
Dan S. Gombos;
Evelyn Paysse;
Murali Chintagumpala;
WeiQing Gao;
Hans Grossniklaus
Over the past decade, chemoreduction has become the treatment of choice for the initial management of retinoblastoma. Yet despite its established use, chemoreduction has not been able to treat diffuse infiltrating retinoblastoma, the least common subtype of retinoblastoma. Herein we report two cases in which residual intraretinal retinoblastoma exhibiting flat and diffuse infiltration was found on pathological exam after a chemoreduction failure. The chemoreduction reduced some of the tumor and unmasked the intraretinal component.
Background and Objectives Human neutrophil antibodies (HNA) have been associated with severe transfusion-related acute lung injury (TRALI). We identified HNA antibodies in a blood donor population and performed an observational lookback on patients who received products from these donors to determine whether TRALI was associated with these transfusions. Materials and Methods Human neutrophil antibodies were determined in 1171 blood donors (388 non-transfused males, 390 human leucocyte antigen (HLA) antibody-negative females and 393 HLA antibody-positive females) for IgG and IgM antibodies using a flow cytometric assay. Selected positive samples had a monoclonal antibody immobilization of granulocyte antigen (MAIGA) and neutrophil genotyping performed to confirm specificity. Lookback was performed on patients receiving blood from donors with positive samples by extracting recipient data from hospital medical records. An expert panel of three pulmonary critical care physicians reviewed the summarized data and assigned a diagnosis of TRALI, possible TRALI, cannot distinguish between TRALI and TACO, TACO and other. Results Eight donors had HNA antibodies of which five contributed to this lookback (3-HNA-specific antibodies, 2-HNA non-specific antibodies). Seventy-six blood products were transfused from these donors into individual patients. One patient developed TRALI that was associated with a donor with a non-specific HNA antibody as well as class-I and class-II HLA antibodies. Conclusion The incidence of TRALI in this lookback was low and combined with low frequency of HNA antibodies in the donor population suggests not screening donors for HNA antibodies at this time is acceptable.
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Erin M. Hill-Burns;
Navjot Singh;
Prabarna Ganguly;
Taye H. Hamza;
Jennifer Montimurro;
Denise M. Kay;
Dora Yearout;
Patricia Sheehan;
Devin Frodey;
Julie A. Mclear;
Mel B. Feany;
Steven D. Hanes;
William J. Wolfgang;
Cyrus P. Zabetian;
Stewart Factor;
Haydeh Payami
Prior studies have established an inverse association between cigarette smoking and the risk of developing Parkinson's disease (PD), and currently, the disease-modifying potential of the nicotine patch is being tested in clinical trials. To identify genes that interact with the effect of smoking/nicotine, we conducted genome-wide interaction studies in humans and in Drosophila. We identified SV2C, which encodes a synaptic-vesicle protein in PD-vulnerable substantia nigra (P=1 × 10 -7 for gene-smoking interaction on PD risk), and CG14691, which is predicted to encode a synaptic-vesicle protein in Drosophila (P=2 × 10 -11 for nicotine-paraquat interaction on gene expression). SV2C is biologically plausible because nicotine enhances the release of dopamine through synaptic vesicles, and PD is caused by the depletion of dopamine. Effect of smoking on PD varied by SV2C genotype from protective to neutral to harmful (P=5 × 10 -10). Taken together, cross-validating evidence from humans and Drosophila suggests SV2C is involved in PD pathogenesis and it might be a useful marker for pharmacogenomics studies involving nicotine.
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Gwendolyn Quinn;
Rebecca Pentz;
Teresita Muñoz-Antonia;
Theresa A. Boyle;
Matthew B. Schabath;
Christie Pratt;
Andrea Shaffer;
Luisa Duarte;
Meghan Bowman-Curci;
Scott Antonia;
Alberto Chiappori;
Benjamin Creelan;
Jhanelle E. Gray;
Charles Williams;
Eric Haura
Objective: The collection of posthumous tissue from advanced stage lung cancer patients is beneficial to medical science. Recruiting living patients to a Rapid Tissue Donation Program (RTD) poses several psychosocial challenges and little is known about perceptions of joining this type of program. This study qualitatively examined perceptions of advanced stage lung cancer patients (n = 14) participating in a lung cancer RTD program, their NoK (n = 11), and physicians (n = 6) at the Thoracic Oncology Clinic at H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida USA. Methods: Semi-structured interviews were conducted with participants and interview transcripts were analyzed using the constant comparison method. Results: Majority of patients joined to give back to research, discussed participation with family members, and desired for family to receive information about the use of the tissue after their death. All participating NoK were supportive of their family member's decision. Physicians described the program as running smoothly, but provided suggestions for process improvements. Conclusion: Participants joined with intention to give back to research community and families were supportive of loved one's participation in RTD. Physicians agreed with overall process. Practice implications: Key factors for a successful RTD program is tailoring to institutional and individual needs.