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Glioblastoma multiforme therapy and mechanisms of resistance

Wed, 02/11/2015 - 11:39am

Glioblastoma multiforme (GBM) is a grade IV brain tumor characterized by a heterogeneous population of cells that are highly infiltrative, angiogenic and resistant to chemotherapy. The current standard of care, comprised of surgical resection followed by radiation and the chemotherapeutic agent temozolomide, only provides patients with a 12-14 month survival period post-diagnosis. Long-term survival for GBM patients remains uncommon as cells with intrinsic or acquired resistance to treatment repopulate the tumor. In this review we will describe the mechanisms of resistance, and how they may be overcome to improve the survival of GBM patients by implementing novel chemotherapy drugs, new drug combinations and new approaches relating to DNA damage, angiogenesis and autophagy.

Evaluation of novel imidazotetrazine analogues designed to overcome temozolomide resistance and glioblastoma regrowth

Wed, 02/11/2015 - 11:39am

The cellular responses to two new temozolomide (TMZ) analogues, DP68 and DP86, acting against glioblastoma multiforme (GBM) cell lines and primary culture models are reported. Dose-response analysis of cultured GBM cells revealed that DP68 is more potent than DP86 and TMZ and that DP68 was effective even in cell lines resistant to TMZ. On the basis of a serial neurosphere assay, DP68 inhibits repopulation of these cultures at low concentrations. The efficacy of these compounds was independent of MGMT and MMR functions. DP68-induced interstrand DNA cross-links were demonstrated with H2O2-treated cells. Furthermore, DP68 induced a distinct cell-cycle arrest with accumulation of cells in S phase that is not observed for TMZ. Consistent with this biologic response, DP68 induces a strong DNA damage response, including phosphorylation of ATM, Chk1 and Chk2 kinases, KAP1, and histone variant H2AX. Suppression of FANCD2 expression or ATR expression/kinase activity enhanced antiglioblastoma effects of DP68. Initial pharmacokinetic analysis revealed rapid elimination of these drugs from serum. Collectively, these data demonstrate that DP68 is a novel and potent antiglioblastoma compound that circumvents TMZ resistance, likely as a result of its independence from MGMT and mismatch repair and its capacity to cross-link strands of DNA. Mol Cancer Ther; 14(1); 111-9. ©2014 AACR.

Evaluation of [In]-Labeled Zinc-Dipicolylamine Tracers for SPECT Imaging of Bacterial Infection

Tue, 02/10/2015 - 10:11pm

PURPOSE: This study prepared three structurally related zinc-dipicolylamine (ZnDPA) tracers with [111In] labels and conducted biodistribution and single-photon emission computed tomography/computed tomography (SPECT/CT) imaging studies of a mouse leg infection model.

PROCEDURES: Two monovalent tracers, ZnDPA-[111In]DTPA and ZnDPA-[111In]DOTA, each with a single zinc-dipicolylamine targeting unit, and a divalent tracer, Bis(ZnDPA)-[111In]DTPA, with two zinc-dipicolylamine units were prepared. Organ biodistribution and SPECT and CT imaging studies were performed on living mice with a leg infection created by injection of clinically relevant Gram positive Streptococcus pyogenes. Fluorescent and luminescent Eu3+-labeled versions of these tracers were also prepared and used to measure relative affinity for the exterior membrane surface of bacterial cells and mimics of healthy mammalian cells.

RESULTS: All three 111In-labeled radiotracers were prepared with a radiopurity of greater than 90%. The biodistribution studies showed that the two monovalent tracers were cleared from the body through the liver and kidney, with retained percentage injected dose for all organs of < 8% at 20h and infected leg target to non-target ratio (T/NT) ratio of greater than or equal to 3.0. Clearance of the divalent tracer from the bloodstream was slower and primarily through the liver, with a retained percentage injected dose for all organs greater than 37% at 20h and T/NT ratio rising to 6.2 after 20 h. The SPECT/CT imaging indicated the same large difference in tracer pharmacokinetics and higher accumulation of the divalent tracer at the site of infection.

CONCLUSIONS: All three [111In]-ZnDPA tracers selectively targeted the site of a clinically relevant mouse infection model that could not be discerned by visual external inspection of the living animal. The highest target selectivity, observed with a divalent tracer equipped with two zinc-dipicolylamine targeting units, compares quite favorably with the imaging selectivities previously reported for other nuclear tracers that target bacterial cell surfaces. The tracer pharmacokinetics depended heavily on tracer molecular structure suggesting that it may be possible to rapidly fine tune the structural properties for optimized in vivo imaging performance and clinical translation.

Her2/neu small interfering RNA delivered in culture by a streptavidin nanoparticle

Tue, 02/10/2015 - 10:11pm

A three-component nanoparticle consisting of biotinylated Trastuzumab antiHer2 antibody, tat transferring peptide and radiolabeled antisense oligomer, linked together through streptavidin, have shown promise in the delivery to Her2+ tumor in mice following intravenous administration and with evidence of radiotherapeutic efficacy. These results have encouraged us to consider the nanoparticle as a delivery vehicle for RNA interference therapy in which the radiolabeled antisense oligomer is replaced with an unlabeled siRNA duplex. The siRNA stability within the nanoparticle was first confirmed by incubation with RNase A. The interferon responses, that indicate off-target cytotoxicity, were evaluated by quantitative real-time RT-PCR in BT-474 (Her2+) human breast cancer cells by measuring the mRNA expression of 2', 5'-oligoadenylate synthetase (OAS1) and Stat-1, two key interferon-responsive genes. Thereafter the cytotoxicity induced by the siRNA nanoparticle was evaluated by a clonogenic survival assay in BT-474 cells while the Her2 expression of these target cells was evaluated for evidence of specific gene silencing. The siRNA within the three-component anti- Her2/neu siRNA nanoparticle was largely protected from RNase-dependent degradation and did not activate an interferon response. The nanoparticle effectively and significantly inhibited colony formation of the target cells and silenced the Her2 gene expression at 5 nM compared with the identical nanoparticle with a scrambled siRNA. Our delivery nanoparticle, with tumor targeting provided by the antibody and its accumulation without entrapment, possibly due to the transfecting peptide, delivered an siRNA duplex to the proper subcellular localization for specific and effective gene silencing in culture by what appears to be an siRNA mechanism.

Activin-like kinase 3 is important for kidney regeneration and reversal of fibrosis

Tue, 02/10/2015 - 10:11pm

Molecules associated with the transforming growth factor beta (TGF-beta) superfamily, such as bone morphogenic proteins (BMPs) and TGF-beta, are key regulators of inflammation, apoptosis and cellular transitions. Here we show that the BMP receptor activin-like kinase 3 (Alk3) is elevated early in diseased kidneys after injury. We also found that its deletion in the tubular epithelium leads to enhanced TGF-beta1-Smad family member 3 (Smad3) signaling, epithelial damage and fibrosis, suggesting a protective role for Alk3-mediated signaling in the kidney. A structure-function analysis of the BMP-Alk3-BMP receptor, type 2 (BMPR2) ligand-receptor complex, along with synthetic organic chemistry, led us to construct a library of small peptide agonists of BMP signaling that function through the Alk3 receptor. One such peptide agonist, THR-123, suppressed inflammation, apoptosis and the epithelial-to-mesenchymal transition program and reversed established fibrosis in five mouse models of acute and chronic renal injury. THR-123 acts specifically through Alk3 signaling, as mice with a targeted deletion for Alk3 in their tubular epithelium did not respond to therapy with THR-123. Combining THR-123 and the angiotensin-converting enzyme inhibitor captopril had an additive therapeutic benefit in controlling renal fibrosis. Our studies show that BMP signaling agonists constitute a new line of therapeutic agents with potential utility in the clinic to induce regeneration, repair and reverse established fibrosis.

Radiolabeled Zn-DPA as a potential infection imaging agent

Tue, 02/10/2015 - 10:11pm

INTRODUCTION: A zinc-dipicolylamine analog (Zn-DPA) conjugated with a fluorophore (PSVue(R)794) has been shown to image bacterial infections in mice. However, radiolabeled Zn-DPA has not previously been considered for nuclear imaging of infection.

METHODS: Both 111In-labeled DOTA-biotin and Zn-DPA-biotin were combined using streptavidin (SA) as a noncovalent linker. Mice injected intramuscularly with Streptococcus pyogenes (infection model) or with lipopolysaccharide (LPS) (inflammation model) were coinjected intravenously with 6 mug of DPA as PSVue794 and as 111In-DOTA-biotin/SA/biotin-Zn-DPA. Periodic fluorescent and SPECT (single photon emission computed tomography)/CT (computed tomography) images were acquired, and biodistributions were obtained at 22 h.

RESULTS: Histological examination confirmed the validity of both the infection and inflammation animal models. Both the whole-body optical and nuclear images showed obvious accumulations in the target thigh in both models at all time points. At 22 h, the average target thigh accumulation of 111In was 1.66%ID/g (S.D. 0.15) in the infection mice compared to 0.58%ID/g (S.D. 0.07) in the inflammation mice (P < .01), and the 111In target/normal thigh ratio was 2.8 fold higher in the infection animals compared to the inflammation animals.

CONCLUSIONS: These preliminary results show that Zn-DPA within streptavidin targets S. pyogenes-infected mice similarly to its free fluorescent analogue. The significantly higher accumulation in the live bacterial infection thigh compared to that of the LPS-induced inflammation thigh suggests that Zn-DPA may be a promising imaging agent to distinguish between bacterial infections and sterile inflammations.

Assessing antibody pharmacokinetics in mice with in vivo imaging

Tue, 02/10/2015 - 10:11pm

Recent advances in small-animal molecular imaging instrumentation combined with well characterized antibody-labeling chemistry have enabled detailed in vivo measurements of antibody distribution in mouse models. This article reviews the strengths and limitations of in vivo antibody imaging methods with a focus on positron emission tomography and single-photon emission computed tomography and a brief discussion of the role of optical imaging in this application. A description of the basic principles behind the imaging techniques is provided along with a discussion of radiolabeling methods relevant to antibodies. Practical considerations of study design and execution are presented through a discussion of sensitivity and resolution tradeoffs for these techniques as defined by modality, signaling probe (isotope or fluorophore) selection, labeling method, and radiation dosimetry. Images and analysis results from a case study are presented with a discussion of output data content and relevant informatics gained with this approach to studying antibody pharmacokinetics.

Comparison of 18F PET and 99mTc SPECT imaging in phantoms and in tumored mice

Tue, 02/10/2015 - 10:11pm

Our objective was to compare the performance of a micro-single photon emission computed tomography (micro-SPECT) with that of a micro-positron emission tomography (microPET) in a Her2+ tumored mice using an anti-Her2 nanoparticle radiolabeled with (99m)Tc and (18)F. Camera performance was first compared using phantoms; then a tumored mouse administered the (99m)Tc-nanoparticle was imaged on a Bioscan NanoSPECT/CT, while another tumored mouse received the identical nanoparticle, labeled now with (18)F, and was imaged on a Philips Mosaic HP PET camera. The nanoparticle was radiolabeled with (99m)Tc via MAG(3) chelation and with (18)F via SFB as an intermediate. Phantom imaging showed that the resolution of the SPECT camera was clearly superior, but even with 4 heads and multipinhole collimators, detection sensitivity was 15-fold lower. Radiolabeling of the nanoparticle by chelation with (99m)Tc was considerably easier and safer than manual covalent attachment of (18)F. Both cameras provided accurate quantitation of radioactivity over a broad range. In conclusion, when deciding between (99m)Tc vs (18)F, an advantage rests with the chelation of (99m)Tc over covalent attachment of (18)F, achieved manually or otherwise, but with these small animal cameras, this choice also results in trading lower sensitivity for higher resolution.

Auger-mediated cytotoxicity of cancer cells in culture by an 125I-antisense oligomer delivered as a three-component streptavidin nanoparticle

Tue, 02/10/2015 - 10:11pm

We reported recently that a three-component nanoparticle, consisting of a targeting antibody, a transfecting peptide and an 111In-antiRIalpha MORF antisense oligomer, provided Auger electron-mediated, antisense-mediated, cytotoxicity of cells in culture. We have now measured the cytotoxicity of the nanoparticle in culture with the 111In replaced by 125I, another attractive Auger electron emitter. The nanoparticle consisted of streptavidin linking the 125I labeled antiRIalpha mRNA antisense MORF oligomer, the tat transfecting peptide and the anti-Her2 Trastuzumab antibody. Cytotoxicity was evaluated by a clonogenic survival assay in BT-474 (Her2+) human breast cancer cells. In a dose escalation study, as measured by the surviving fraction, the cytotoxicity of tumor cells to the 125I-labeled antisense nanoparticle was significantly higher than that for the identical sense control. When compared with our previous study with 111In as label, a similar level of cytotoxicity was achieved but the observed minimal therapeutic dose for the 125I-labeled nanoparticle in BT-474 cells was lower than that for 111In-labeled nanoparticle in SK-BR-3 cells. Thus, a radiolabeled antisense MORF oligomer delivered into cells by a three-component nanoparticle is an effective vehicle for Auger radiotherapy when radiolabeled with 111In or 125I.

In vivo delivery of antisense MORF oligomer by MORF/carrier streptavidin nanoparticles

Tue, 02/10/2015 - 10:11pm

Tumor targeting by oligomers is largely limited by the pharmacokinetics and cell-membrane transport obstacles. In this article, we describe the use of a delivery nanoparticle, in which streptavidin served as a convenient bridge between a biotinylated oligomer and a biotinylated cell-membrane-penetrating peptide, to improve the delivery of an antisense phosphorodiamidate morpholino (MORF) oligomer in vivo. A biotinylated (99m)Tc-radiolabeled MORF oligomer with a base sequence antisense to the RIalpha mRNA and its sense control were incorporated separately into nanoparticles, along with biotinylated tat or polyarginine carrier. The streptavidin nanoparticles were administrated intravenously to both normal and nude mice bearing SUM149 breast tumor xenografts. The biodistributions showed much higher normal tissue levels for the radiolabeled MORFs, independent of antisense or sense or tat or polyarginine, when administered as the nanoparticles, compared to naked. A statistically significant higher accumulation of both antisense nanoparticles, compared to the respective sense control nanoparticles, was observed, along with much higher tumor accumulations, compared to historical naked controls. This study has provided evidence that the in vivo function of an antisense oligomer within the streptavidin nanoparticle is not impeded, and, as such, the MORF/streptavidin/carrier nanoparticles may be suitable for in vivo tumor delivery of antisense MORF and other oligomers.

Tumor delivery of antisense oligomer using trastuzumab within a streptavidin nanoparticle

Tue, 02/10/2015 - 10:11pm

PURPOSE: Trastuzumab (Herceptin) is often internalized following binding to Her2+ tumor cells. The objective of this study was to investigate whether trastuzumab can be used as a specific carrier to deliver antisense oligomers into Her2+ tumor cells both in vitro and in vivo.

METHODS: A biotinylated MORF oligomer antisense to RhoC mRNA and its biotinylated sense control were labeled with either lissamine for fluorescence detection or 99mTc for radioactivity detection and were linked to biotinylated trastuzumab via streptavidin. The nanoparticles were studied in SUM190 (RhoC+, Her2+) study and SUM149 (RhoC+, Her2-) control cells in culture and as xenografts in mice.

RESULTS: As evidence of unimpaired Her2+ binding of trastuzumab within the nanoparticle, accumulations were clearly higher in SUM190 compared to SUM149 cells and, by whole-body imaging, targeting of SUM190 tumor was similar to that expected for a radiolabeled trastuzumab. As evidence of internalization, fluorescence microscopy images of cells grown in culture and obtained from xenografts showed uniform cytoplasm distribution of the lissamine-MORF. An invasion assay showed decreased RhoC expression in SUM190 cells when incubated with the antisense MORF nanoparticles at only 100 nM.

CONCLUSION: Both in cell culture and in animals, the nanoparticle with trastuzumab as specific carrier greatly improved tumor delivery of the antisense oligomer against RhoC mRNA into tumor cells overexpressing Her2 and may be of general utility.

Auger radiation-induced, antisense-mediated cytotoxicity of tumor cells using a 3-component streptavidin-delivery nanoparticle with 111In

Tue, 02/10/2015 - 10:11pm

When antisense oligomers are intracellular, they migrate to and are retained in the nucleus of tumor cells and therefore may be used to carry Auger electron-emitting radionuclides such as (111)In for effective tumor radiotherapy.

METHODS: Our nanoparticle consists of streptavidin that links 3 biotinylated components: the antiHer2 antibody trastuzumab (to improve pharmacokinetics), the tat peptide (to improve cell membrane transport), and the (111)In-labeled antiRIalpha messenger RNA antisense morpholino (MORF) oligomer.

RESULTS: As evidence of unimpaired function, tumor cell and nuclear accumulations were orders of magnitude higher after incubation with (99m)Tc-MORF/tat/trastuzumab than after incubation with free (99m)Tc-MORF and significantly higher with the antisense than with the sense MORF. In mice, tumor and normal-tissue accumulations of the (99m)Tc-MORF/tat/trastuzumab nanoparticle were comparable to those of free (99m)Tc-trastuzumab, confirming the improved pharmacokinetics due to the trastuzumab component. Although kidneys, liver, and other normal tissues also accumulated the nanoparticle, immunohistochemical evaluation of tissue sections in mice receiving the Cy3-MORF/tat/trastuzumab nanoparticle showed evidence of nuclear accumulation only in tumor tissue. In a dose escalation study, as measured by the surviving fraction, the nanoparticle significantly increased the kill of SK-BR-3 breast cancer Her2+/RIalpha+ cells, compared with all controls.

CONCLUSION: Significant radiation-induced antisense-mediated cytotoxicity of tumor cells in vitro was achieved using an Auger electron-emitting antisense MORF oligomer administered as a member of a 3-component streptavidin-delivery nanoparticle.

Community health worker interventions for Latinos with type 2 diabetes: a systematic review of randomized controlled trials

Tue, 02/10/2015 - 5:28pm

This systematic review aimed to synthesize glucose (HbA1c) outcomes of community health worker (CHW)-delivered interventions for Latinos with type 2 diabetes that were tested in randomized controlled trials and to summarize characteristics of the targeted populations and interventions, including the background, training, and supervision of the CHWs. Searches of PubMed and Google Scholar databases and references from selected articles identified 12 studies that met the inclusion criteria. Of these, seven reported statistically significant improvements in HbA1c. Study participants were largely low-income, female, and Spanish-speaking and had uncontrolled diabetes. The CHWs led the interventions alone, in pairs, or as part of a team. Interventions varied considerably in session time, duration, and number. Most met standards for tailored, high-intensity interventions and half were theory-based. Overall, methodological quality was good but there were inconsistencies in the reporting of key information. Future research should report in greater detail CHW background, training, and supervision; examine factors associated with intervention effectiveness; and provide data on cost and cost-effectiveness.

Medical school curriculum characteristics associated with intentions and frequency of tobacco dependence treatment among 3rd year U.S. medical students

Tue, 02/10/2015 - 4:30pm

OBJECTIVE: Physicians play a critical role in addressing tobacco dependence, yet report limited training. Tobacco dependence treatment curricula for medical students could improve performance in this area. This study identified student and medical school tobacco treatment curricula characteristics associated with intentions and use of the 5As for tobacco treatment among 3rd year U.S. medical students.

METHODS: Third year medical students (N = 1065, 49.3% male) from 10 U.S. medical schools completed a survey in 2009-2010 assessing student characteristics, including demographics, tobacco treatment knowledge, and self-efficacy. Tobacco curricula characteristics assessed included amount and type of classroom instruction, frequency of tobacco treatment observation, instruction, and perception of preceptors as role models.

RESULTS: Greater tobacco treatment knowledge, self-efficacy, and curriculum-specific variables were associated with 5A intentions, while younger age, tobacco treatment self-efficacy, intentions, and each curriculum-specific variable were associated with greater 5A behaviors. When controlling for important student variables, greater frequency of receiving 5A instruction (OR = 1.07; 95%CI 1.01-1.12) and perception of preceptors as excellent role models in tobacco treatment (OR = 1.35; 95%CI 1.04-1.75) were significant curriculum predictors of 5A intentions. Greater 5A instruction (B = .06 (.03); p< .05) and observation of tobacco treatment (B = .35 (.02); p< .001) were significant curriculum predictors of greater 5A behaviors.

CONCLUSIONS: Greater exposure to tobacco treatment teaching during medical school is associated with both greater intentions to use and practice tobacco 5As. Clerkship preceptors, or those physicians who provide training to medical students, may be particularly influential when they personally model and instruct students in tobacco dependence treatment.

Evaluating and selecting mobile health apps: strategies for healthcare providers and healthcare organizations

Tue, 02/10/2015 - 4:30pm

Mobile applications (apps) to improve health are proliferating, but before healthcare providers or organizations can recommend an app to the patients they serve, they need to be confident the app will be user-friendly and helpful for the target disease or behavior. This paper summarizes seven strategies for evaluating and selecting health-related apps: (1) Review the scientific literature, (2) Search app clearinghouse websites, (3) Search app stores, (4) Review app descriptions, user ratings, and reviews, (5) Conduct a social media query within professional and, if available, patient networks, (6) Pilot the apps, and (7) Elicit feedback from patients. The paper concludes with an illustrative case example. Because of the enormous range of quality among apps, strategies for evaluating them will be necessary for adoption to occur in a way that aligns with core values in healthcare, such as the Hippocratic principles of nonmaleficence and beneficence.

Tobacco Use, Quitting Behavior, and Health Characteristics Among Current Electronic Cigarette Users in a National Tri-Ethnic Adult Stable Smoker Sample

Tue, 02/10/2015 - 4:30pm

INTRODUCTION: The present study characterizes the tobacco use, quitting behaviors, and health characteristics of cigarette smokers who did not change their smoking pattern over the past 6 months and have used electronic cigarettes (ECs) in the past 30 days. This is an important subpopulation to characterize if EC dual use with cigarettes continues to grow.

METHODS: Participants (N = 2,376) from a research survey panel completed an online cross-sectional survey between June and August 2012. Sampling was stratified to recruit equal numbers of cigarette smoking participants by race/ethnicity (Black, Hispanic, and Caucasian) and smoking frequency (nondaily and daily). All displayed a stable rate of smoking for the past 6 months and were not currently in treatment. Bivariate and multivariate analyses were used to examine correlates of current EC use (any use within the past 30 days).

RESULTS: Current EC use was reported by 9.2% (n = 219) of the total sample. Of current EC users, 44% reported having used ECs as a quit method. Bivariate and multivariate analyses showed that current EC use was significantly associated with greater nicotine dependence, concurrent poly-tobacco use, more past-year quit attempts, past use of multiple cessation methods, and more depressive symptoms. No demographic variables were significantly associated with current EC use.

CONCLUSIONS: This study suggests that stable smokers who currently use ECs possess characteristics that are associated with difficulty in achieving smoking cessation. These characteristics should be considered when examining the effectiveness of ECs on cessation and in designing future cessation trials using ECs. Society for Research on Nicotine and Tobacco. All rights reserved.

Real-Time Mobile Detection of Drug Use with Wearable Biosensors: A Pilot Study

Tue, 02/10/2015 - 4:30pm

While reliable detection of illicit drug use is paramount to the field of addiction, current methods involving self-report and urine drug screens have substantial limitations that hinder their utility. Wearable biosensors may fill a void by providing valuable objective data regarding the timing and contexts of drug use. This is a preliminary observational study of four emergency department patients receiving parenteral opioids and one individual using cocaine in a natural environment. A portable biosensor was placed on the inner wrist of each subject, to continuously measure electrodermal activity (EDA), skin temperature, and acceleration. Data were continuously recorded for at least 5 min prior to drug administration, during administration, and for at least 30 min afterward. Overall trends in biophysiometric parameters were assessed. Injection of opioids and cocaine use were associated with rises in EDA. Cocaine injection was also associated with a decrease in skin temperature. Opioid tolerance appeared to be associated with a blunted physiologic response as measured by the biosensor. Laterality may be an important factor, as magnitude of response varied between dominant and nondominant wrists in a single patient with bilateral wrist measurements. Changes in EDA and skin temperature are temporally associated with intravenous administration of opioids and cocaine; the intensity of response, however, may vary depending on history and extent of prior use.

A research agenda for gender and substance use disorders in the emergency department

Tue, 02/10/2015 - 4:30pm

For many years, gender differences have been recognized as important factors in the etiology, pathophysiology, comorbidities, and treatment needs and outcomes associated with the use of alcohol, drugs, and tobacco. However, little is known about how these gender-specific differences affect ED utilization; responses to ED-based interventions; needs for substance use treatment and barriers to accessing care among patients in the ED; or outcomes after an alcohol-, drug-, or tobacco-related visit. As part of the 2014 Academic Emergency Medicine consensus conference on "Gender-Specific Research in Emergency Care: Investigate, Understand and Translate How Gender Affects Patient Outcomes," a breakout group convened to generate a research agenda on priority questions related to substance use disorders.

Profiles of acute cytokine and antibody responses in patients infected with avian influenza A H7N9

Mon, 02/09/2015 - 10:00pm

The influenza A H7N9 virus outbreak in Eastern China in the spring of 2013 represented a novel, emerging avian influenza transmission to humans. While clinical and microbiological features of H7N9 infection have been reported in the literature, the current study investigated acute cytokine and antibody responses in acute H7N9 infection. Between March 27, 2013 and April 23, 2013, six patients with confirmed H7N9 influenza infection were admitted to Drum Tower Hospital, Nanjing, China. Acute phase serum cytokine profiles were determined using a high-throughput multiplex assay. Daily H7 hemagglutinin (HA)-specific IgG, IgM, and IgA responses were monitored by ELISA. Neutralizing antibodies specific for H7N9 viruses were determined against a pseudotyped virus expressing the novel H7 subtype HA antigen. Five cytokines (IL-6, IP-10, IL-10, IFNgamma, and TNFalpha) were significantly elevated in H7N9-infected patients when compared to healthy volunteers. Serum H7 HA-specific IgG, as well as IgM and IgA responses, were detected within 8 days of disease onset and increased in a similar pattern during acute infection. Neutralizing antibodies developed shortly after the appearance of binding antibody responses and showed similar kinetics as a fraction of the total H7 HA-specific IgG responses. H7N9 infection resulted in hallmark serum cytokine increases, which correlated with fever and disease persistence. The novel finding of simultaneous development of IgG, IgM, and IgA responses in acute H7N9 infection points to the potential for live influenza viruses to elicit fast and potent protective antibodies to limit the infection.

Evaluation of the contribution of multiple DAMPs and DAMP receptors in cell death-induced sterile inflammatory responses

Mon, 02/09/2015 - 10:00pm

When cells die by necrosis in vivo they stimulate an inflammatory response. It is thought that this response is triggered when the injured cells expose proinflammatory molecules, collectively referred to as damage associated molecular patterns (DAMPs), which are recognized by cells or soluble molecules of the innate or adaptive immune system. Several putative DAMPs and/or their receptors have been identified, but whether and how much they participate in responses in vivo is incompletely understood, and they have not previously been compared side-by-side in the same models. This study focuses on evaluating the contribution of multiple mechanisms that have been proposed to or potentially could participate in cell death-induced inflammation: The third component of complement (C3), ATP (and its receptor P2X7), antibodies, the C-type lectin receptor Mincle (Clec4e), and protease-activated receptor 2 (PAR2). We investigate the role of these factors in cell death-induced inflammation to dead cells in the peritoneum and acetaminophen-induced liver damage. We find that mice deficient in antibody, C3 or PAR2 have impaired inflammatory responses to dying cells. In contrast there was no reduction in inflammation to cell death in the peritoneum or liver of mice that genetically lack Mincle, the P2X7 receptor or that were treated with apyrase to deplete ATP. These results indicate that antibody, complement and PAR2 contribute to cell death-induced inflammation but that Mincle and ATP- P2X7 receptor are not required for this response in at least 2 different in vivo models.