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El objetivo del presente artículo es, en un primer momento analizar las condiciones de violencia que se presentan al interior y periferia de las instituciones escolares, para después, evidenciar la necesidad de diseñar una propuesta de atención a reportes de violencia escolar en la entidad mexiquense, desde la cual se canalicen acciones por medio de diferentes instancias gubernamentales. La investigación documental es de corte cualitativo, comprende el periodo de 2016 al 2020, se emplearon técnicas de recolección de datos que permitieron registrar la realidad en torno a las diferentes líneas de atención a reportes de violencia en el Estado de México, mismas que se describen en matrices de datos. Como resultados preliminares, se presenta el proyecto de Centro de Atención para la Convivencia Escolar Mexiquense (CACEM).

Cryptosporidium spp. are apicomplexan protozoa associated with chronic diarrhea in AIDS and other immunocompromised patients, and one of the commonest causes of childhood diarrhea and malnutrition, particularly in low-income settings. In Colombia, there are few molecular epidemiological studies on Cryptosporidium spp.; thereby, the transmission dynamics of this parasite in the country is poorly known. This study evaluated the diversity of Cryptosporidium at species, subtype family, and subtype level in children attending various day-care centers in Medellin, Colombia. Two hundred and ninety stool samples from children

Physiological blood-tissue barriers play a critical role in separating the circulation from immune-privileged sites and denying access to blood-borne viruses. The mechanism of virus restriction by these barriers is poorly understood. We utilize induced pluripotent stem cell (iPSC)-derived human brain microvascular endothelial cells (iBMECs) to study virus-blood-brain barrier (BBB) interactions. These iPSC-derived cells faithfully recapitulate a striking difference in in vivo neuroinvasion by two alphavirus isolates and are selectively permissive to neurotropic flaviviruses. A model of cocultured iBMECs and astrocytes exhibits high transendothelial electrical resistance and blocks non-neurotropic flaviviruses from getting across the barrier. We find that iBMECs constitutively express an interferon-induced gene, IFITM1, which preferentially restricts the replication of non-neurotropic flaviviruses. Barrier cells from blood-testis and blood-retinal barriers also constitutively express IFITMs that contribute to the viral resistance. Our application of a renewable human iPSC-based model for studying virus-BBB interactions reveals that intrinsic immunity at the barriers contributes to virus exclusion.

TOPIC: Chest Infections TYPE: Medical Student/Resident Case Reports INTRODUCTION: Coronavirus Disease 2019 (COVID-19) has affected over 140 million with over 3 million deaths worldwide. As the virus continues, we face a new challenge;to discern between post COVID syndromes or COVID reinfection. We present a case with this medical dilemma. CASE PRESENTATION: 64 y/o F with Diabetes, Hypertension & history of asymptomatic COVID infection eight weeks prior to presentation with follow-up negative testing, presented with fever & SOB. She initially developed progressive dyspnea on exertion for which bronchodilators were initiated as an outpatient. She then presented to our hospital with fevers & progressive SOB. Her initial workup revealed normal blood work & inflammatory markers, however her COVID RNA test was positive with seronegative COVID antibodies. Imaging revealed increased bilateral ground-glass opacities & inter-septal thickening when compared to imaging one month prior. Her positive COVID tests were attributed to viral shedding & COVID therapy was not initiated. On hospital day 6 she began to decompensate requiring rapid escalation of oxygen supplementation to high flow nasal cannula. At this time high dose steroids were initiated as post COVID syndrome was thought to be the disease pathology. On hospital day 12 retesting revealed seroconversion of COVID antibodies. Her hospital course was complicated by severe hypoxia requiring prolonged steroid taper & oxygen supplementation. She was ultimately discharged on home oxygen with outpatient management. DISCUSSION: Complications of COVID-19 disease include reinfection & post-COVID syndrome. Reinfection was considered in this patient due to repeated negative tests & an asymptomatic period. This remains a rare phenomenon. There are only 5 reported known cases thus far. It was ruled out according to WHO guidelines, since there were 3 months after infection & despite neutralizing antibodies. We believe she developed severe ARDS due to COVID pneumonitis with possible progression to ILD. Typical disease course is still unclear. There is increasing evidence of fibrotic-like changes in the lung in high rate of patients for up to 6 months. Reversibility of these findings is unknown. Post-mortem evaluation of lung tissue reveals diffuse alveolar damage from chronic inflammation. Obesity, ARDS, prolonged hospitalization & NIMV are reported risk factors. CONCLUSIONS: This case shows the importance of considering COVID reinfection in patients with atypical presentation. It is imperative that we differentiate between viral shedding & reinfection in patients with post-acute COVID symptoms. Both presentations may be similar, therefore it is pivotal that we continue to investigate these two-potentially fatal COVID related processes. REFERENCE #1: Pan American Health Organization / World Health Organization. Interim guidelines for detecting cases of reinfection by SARS-CoV-2. 29 October 2020, Washington, D.C.: PAHO/WHO;2020 REFERENCE #2: Qian Li, Xiao-Shuang Zheng, Xu-Rui Shen, Hao-Rui Si, Xi Wang, Qi Wang, Bei Li, Wei Zhang, Yan Zhu, Ren-Di Jiang, Kai Zhao, Hui Wang, Zheng-Li Shi, HuiLan Zhang, Rong-Hui Du & Peng Zhou (2020): Prolonged shedding of severe acute respiratory syndrome coronavirus 2 in patients with COVID-19, Emerging Microbes & Infections, DOI: 10.1080/22221751.2020.1852058 REFERENCE #3: Han X, Fan Y, Alwalid O, et al. Six-month Follow-up Chest CT Findings after Severe COVID-19 Pneumonia. Radiology. 2021;299(1):E177-E186. doi:10.1148/radiol.2021203153 DISCLOSURES: No relevant relationships by Angelica Medina-Pena, source=Web Response No relevant relationships by Anna-Belle Robertson, source=Web Response No relevant relationships by Noura Semreen, source=Web Response no disclosure on file for David Shiu;No relevant relationships by Ro-Kaye Simmonds, source=Web Re ponse No relevant relationships by Jodi-Ann Smith, source=Web Response

Glycated hemoglobin (HbA1c) is an invaluable tool in diabetes mellitus (DM) management. Conventionally obtained via venous blood sampling, point-of-care (POCT) capillary HbA1c measurement offers an opportunity for immediate treatment modification, reduced cost & increased patient satisfaction. While previous studies using the POCT HbA1c test A1cNow+ have shown accuracy within a 0.5% range from the gold standard venous HbA1c, we noted discrepancy in our community health clinic & sought to evaluate the accuracy of POCT HbA1c levels compared to venous HbA1c levels to guide our clinical decision-making. In this 2-part study, we compared POCT HbA1c levels measured via a single use A1CNow+ HbA1c monitoring device & venous HbA1c samples measured by a standardized lab. Part1: after retrospective chart review, we identified 262 patients with prediabetes, Type1 or Type2 DM based on ADA guidelines who attended our clinic from January 2019-June 2019 & received POCT HbA1c with A1cNow+ testing during their visit. Of those cases, 47 patients also had a venous HbA1c at a standardized laboratory within 1 month of having their POCT HbA1c performed in our clinic. Part2: We noted variability in the temperature storage of A1CNow+ test strips. Storage was standardized to room temperature as per device instructions in June 2019. We subsequently reviewed charts from June 2019-December 2019 & identified 118 patients who had both POCT HbA1c & venous HbA1c measurement within a 1 month period. Patients was categorized into subgroups per ACP guidelines for DM control: prediabetic (HbA1c 5.7–6.4%), controlled DM (HbA1c 6.5 to 8.0%) & uncontrolled DM (HbA1c >8.0%). The average difference between POCT & venous HbA1c tests was calculated & analyzed for statistical significance using paired t test analysis. Part1: For patients in prediabetic, controlled & uncontrolled DM subgroups, the mean difference between A1cNow+ & standardized venous HbA1c testing was 0.68% (p= 0.004), 1.15% (p= POCT HbA1c provides a unique opportunity to immediately address glycemic control. Its advantages are especially apparent in a patient population with limited resources & poor follow up, as in our clinic. Although standardizing test storage improved overall concordance between A1cNow+ HbA1c testing & venous HbA1c, there was still a statistically significant larger mean difference in uncontrolled DM patients. In prediabetic & controlled DM patients, however, POCT HbA1c was accurate within previously published reports of a 0.5% range when compared to venous HbA1c. An algorithm has since been developed to guide our clinical decision making with these findings.

Introduction: Hypogammaglobulinemia is a known potential adverse event in patients with CLL receiving anti-CD20 monoclonal antibody (mAb) therapy. However, real-world immunoglobulin G (IgG) testing rates have not been extensively studied. We used a real-world database to investigate IgG testing rates in patients with CLL before and after anti-CD20 exposure. We further examined factors that increased the likelihood of new onset hypogammaglobulinemia following anti-CD20 therapy. Methods: The Flatiron Health EHR-derived de-identified database was used to select patients with abstraction-confirmed CLL diagnosed before 6/1/2020 with: (1) Exposure to an anti-CD20 mAb, (2) At least one IgG lab within 180 days prior to first anti-CD20 line start, and (3) No evidence of hypogammaglobulinemia (IgG < 7g/L) within 6 months prior to first anti-CD20 line start. A "hypogammaglobulinemia detection window" was defined for each patient as the time between first anti-CD20 administration through to 365 days thereafter. Patients were grouped into 3 categories: (A) "Unknown status": those with no IgG testing during the window, (B) "Confirmed hypogam": those with hypogammaglobulinemia detected on any test during the window, and (C) "No detected hypogam": those tested for IgG during the window with 0 tests meeting the hypogammaglobulinemia threshold. Demographic and clinical baseline characteristics were compared across the groups. A logistic regression was performed to compare factors associated with higher likelihood of detecting hypogammaglobulinemia among patients with an IgG test in the detection window. Factors included were: patient age at start of detection window, exposure to chemotherapy before detection window (Y/N), concurrent chemotherapy to anti-CD20 during the detection window (Y/N), and whether anti-CD20 was given in the first line (1L) setting (Y/N). We also performed a separate scenario analysis with outcome of severe hypogammaglobulinemia (IgG < 5 g/L, compared to < 7 g/L in primary analysis). Results: We found 5,838 anti-CD20 mAb-exposed patients with CLL, of which 1,927 (33%) had at least 1 IgG lab within 180 days of anti-CD20 start. Of those, 922 (48%) had hypogammaglobulinemia within 6 months prior to starting anti-CD20 therapy, leaving 1,005 patients in the cohort. 526 patients (52%) fell into the 'unknown status' category. 182 (18%) patients fell into the 'confirmed hypogam' category, and 297 (30%) fell into the 'no hypogam detected' category. Patients across these 3 categories had similar baseline characteristics (Table 1). Among the patients with an IgG test in the detection window (N=479), patients with 'confirmed hypogam' had more IgG tests during the window than the patients with 'no detected hypogam' (mean 3.9 vs. 2.8 tests, p Results of the logistic regression for IgG < 7 g/L did not yield any factors significantly associated with higher likelihood of hypogammaglobulinemia (Table 2). However, the regression at IgG < 5 g/L (incl. changing cohort inclusion criteria 3, N=703) found that concurrent exposure to chemotherapy was associated with higher likelihood of severe documented hypogammaglobulinemia (odds ratio 1.89; 95% CI 1.23-2.96; p = 0.004). Conclusions: In this real-world cohort of patients with CLL with at least one IgG test within 6 months prior to anti-CD20 mAb initiation, hypogammaglobulinemia was common. A majority of the cohort was not tested for IgG post anti-CD20 mAb initiation, which likely reflects clinical practice of infrequently obtaining IgG levels in patients with normal pre-treatment IgG levels. As clinically expected, patients with hypogammaglobulinemia were tested for IgG more frequently than those who were not. This hypothesis-generating study suggests that administering chemotherapy concurrently to anti-CD20 mAbs increases the likelihood of documented severe hypogammaglobulinemia (IgG < 5g/L) in patients with CLL. Further research should confirm this. Our analyses did not account for patients who may have had transient hypogammaglobulinemia during anti-CD20 treatment, and did not stratify by intravenous immunoglobulin (IVIG) usage in this population. Further research should examine the effect of anti-CD20-associated hypogammaglobulinemia on outcomes, and determine if IgG surveillance should be standardized as part of clinical guidelines. Disclosures Hooley: Flatiron Health Inc: Current Employment, Research Funding; Roche Group: Current equity holder in publicly-traded company. Maignan:Flatiron Health Inc: Current Employment, Research Funding; Roche Group: Current equity holder in publicly-traded company. Jacob:Flatiron Health Inc: Current Employment, Research Funding; Roche Group: Current equity holder in publicly-traded company. Medina:Flatiron Health Inc: Current Employment, Research Funding; Roche Group: Current equity holder in publicly-traded company. Benderoff:Roche Group: Current equity holder in publicly-traded company; Flatiron Health Inc: Current Employment, Research Funding. Chen:Flatiron Health Inc: Current Employment, Research Funding; Roche Group: Current equity holder in publicly-traded company. Huntington:AbbVie: Consultancy; Astrazeneca: Honoraria; Bayer: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; DTRM: Research Funding; Genentech: Consultancy; Novartis: Consultancy; Pharmacyclics: Honoraria; TG Therapeutics: Research Funding.

Companies need to become more environmentally conscious in their actions and business-based operations. In developed countries, there is an increased pressure towards this direction enforced by legal requirements. Moreover, a notion of sustainability is more and more perceived as a business advantage and as part of a successful company’s business models [1, 2]. Companies have various options on how to innovate their products, operations and business models to be more sustainable by e.g. addressing growing income inequality [3] and minimizing their own impact on the deterioration of our natural livelihood [4]. To identify suitable options companies often collaborate with higher education institutions to develop innovative ideas. And within this realm, lies option for collaboration in the form of hackathons and e.g. code camps. These events are great sources of innovation and these special cases they also did support collaboration driven sustainability innovation seeding too. This work is based on case studies and experience-based evidence on how companies were successful in generating ideas for their operations and new potential options for new implementation directions to support higher levels of circularity in their supply chains. Additionally, companies have been able to improve their services to support their customers’ actions to minimize energy consumption and improve operations to positively affect CO₂ emissions thus reducing their carbon footprint. The original version of this article supplied to AIP Publishing contained an error in the author’s name. The name originally appeared as, Maria Angelica and Medina Angarita, but the correct name is Maria Angelica Medina Angarita. An updated version of this article, with the author’s name corrected, was published on 27 July 2020.

Rare diseases defined by genetic mutations are classic targets for gene therapy. More recently, researchers expanded the use of gene therapy in non-clinical studies to infectious diseases through the delivery of vectorized antibodies to well-defined antigens. Here, we further extend the utility of gene therapy beyond the "accepted" indications to include organophosphate poisoning. There are no approved preventives for the multi-organ damage resulting from acute or chronic exposure to organophosphates. We show that a single intramuscular injection of adeno-associated virus vector produces peak expression (~0.5 mg/ml) of active human butyrylcholinesterase (hBChE) in mice serum within 3-4 weeks post-treatment. This expression is sustained for up to 140 days post-injection with no silencing. Sustained expression of hBChE provided dose-dependent protection against VX in male and female mice despite detectable antibodies to hBChE in some mice, thereby demonstrating that expression of hBChE in vivo in mouse muscle is an effective prophylactic against organophosphate poisoning.

Zika virus (ZIKV) infection attenuates the growth of human neural progenitor cells (hNPCs). As these hNPCs generate the cortical neurons during early brain development, the ZIKV-mediated growth retardation potentially contributes to the neurodevelopmental defects of the congenital Zika syndrome. Here, we investigate the mechanism by which ZIKV manipulates the cell cycle in hNPCs and the functional consequence of cell cycle perturbation on the replication of ZIKV and related flaviviruses. We demonstrate that ZIKV, but not dengue virus (DENV), induces DNA double-strand breaks (DSBs), triggering the DNA damage response through the ATM/Chk2 signaling pathway while suppressing the ATR/Chk1 signaling pathway. Furthermore, ZIKV infection impedes the progression of cells through S phase, thereby preventing the completion of host DNA replication. Recapitulation of the S-phase arrest state with inhibitors led to an increase in ZIKV replication, but not of West Nile virus or DENV. Our data identify ZIKV’s ability to induce DSBs and suppress host DNA replication, which results in a cellular environment favorable for its replication. IMPORTANCE Clinically, Zika virus (ZIKV) infection can lead to developmental defects in the cortex of the fetal brain. How ZIKV triggers this event in developing neural cells is not well understood at a molecular level and likely requires many contributing factors. ZIKV efficiently infects human neural progenitor cells (hNPCs) and leads to growth arrest of these cells, which are critical for brain development. Here, we demonstrate that infection with ZIKV, but not dengue virus, disrupts the cell cycle of hNPCs by halting DNA replication during S phase and inducing DNA damage. We further show that ZIKV infection activates the ATM/Chk2 checkpoint but prevents the activation of another checkpoint, the ATR/Chk1 pathway. These results unravel an intriguing mechanism by which an RNA virus interrupts host DNA replication. Finally, by mimicking virus-induced S-phase arrest, we show that ZIKV manipulates the cell cycle to benefit viral replication.

Zika virus (ZIKV) infection represents a significant threat to the global health system, and the search for efficient antivirals to ZIKV remains necessary and urgent. In this study, we extended the exploration of our previously discovered scaffold of 1H-pyrrolo[1,2-c]imidazol-1-one and revealed that two trans isomers of compounds 2 and 7 and one mixture with major trans isomer of compound 3 as novel tetrahydroquinoline-fused imidazolone derivatives are active against ZIKV infection but they are not virucidal. Western Blot and ELISA analyses of ZIKV NS5 and NS1 further demonstrate that compounds of (±)-2, (±)-3 and (±)-7 act as effective agents against ZIKV infection. We show that the N10′s basicity is not the basic requirement for these compounds’ antiviral activity in the current work. Importantly, tuning of some pharmacophores including substituents at arene can generate promising candidates for anti-ZIKV agents.

The seroprevalence of neutralizing antibodies (NAbs) to adeno-associated viral (AAV) vector capsids may preclude a percentage of the population from receiving gene therapy, particularly following systemic vector administration. We hypothesized that the use of intramuscular (IM) administration of AAV vectors might circumvent this issue. IM injections were used to administer AAV8 vectors expressing either secreted or non-secreted transgenes into mice and the influence of NAbs supplied by pre-administration of pooled human IgG on transgene expression was evaluated. We then studied the impact of naturally occurring pre-existing AAV8 NAbs on expression of a secreted transgene following IM vector delivery in rhesus macaques. Finally, we evaluated the ability to readminister AAV vectors via IM injections in rhesus macaques. In mice, the presence of AAV8 NAbs had no effect on gene expression in the injected skeletal muscle. However, liver transgene expression following hepatic distribution of the vector was ablated. In rhesus macaques, naturally occurring pre-existing AAV8 NAb titers of ⩽1:160 had no effect on expression levels of a secreted transgene after IM delivery of the vector. Additionally, readministration of AAV vectors was possible by IM injection into the previously injected muscle groups, with no effect on transgene expression by the original vector. Therefore, the presence of pre-existing NAbs in the human population should not preclude subjects from receiving gene therapy by IM administration of the vector so long as sufficient levels of secreted transgene expression can be produced without the involvement of liver.

Parvovirus B19 is the causative agent of fifth disease in children, aplastic crisis in those with blood dyscrasias, and hydrops fetalis. Previous parvovirus B19 virus-like-particle (VLP) vaccine candidates were produced by co-infection of insect cells with two baculoviruses, one expressing wild-type VP1 and the other expressing VP2. In humans, the VLPs were immunogenic but reactogenic. We have developed new VLP-based parvovirus B19 vaccine candidates, produced by co-expressing VP2 and either wild-type VP1 or phospholipase-negative VP1 in a regulated ratio from a single plasmid in Saccharomyces cerevisiae. These VLPs are expressed efficiently, are very homogeneous, and can be highly purified. Although VP2 alone can form VLPs, in mouse immunizations, VP1 and the adjuvant MF59 are required to elicit a neutralizing response. Wild-type VLPs and those with phospholipase-negative VP1 are equivalently potent. The purity, homogeneity, yeast origin, and lack of phospholipase activity of these VLPs address potential causes of previously observed reactogenicity.

Broad, multispecific CD4 + and CD8 + T-cell responses to the hepatitis C virus (HCV), as well as virus-cross-neutralizing antibodies, are associated with recovery from acute infection and may also be associated in chronic HCV patients with a favorable response to antiviral treatment. In order to recapitulate all of these responses in an ideal vaccine regimen, we have explored the use of recombinant HCV polypeptides combined with various Th1-type adjuvants and replication-defective alphaviral particles encoding HCV proteins in various prime/boost modalities in BALB/c mice. Defective chimeric alphaviral particles derived from the Sindbis and Venezuelan equine encephalitis viruses encoding either the HCV envelope glycoprotein gpE1/gpE2 heterodimer (E1E2) or nonstructural proteins 3, 4, and 5 (NS345) elicited strong CD8 + T-cell responses but low CD4 + T helper responses to these HCV gene products. In contrast, recombinant E1E2 glycoproteins adjuvanted with MF59 containing a CpG oligonucleotide elicited strong CD4 + T helper responses but no CD8 + T-cell responses. A recombinant NS345 polyprotein also stimulated strong CD4 + T helper responses but no CD8 + T-cell responses when adjuvanted with Iscomatrix containing CpG. Optimal elicitation of broad CD4 + and CD8 + T-cell responses to E1E2 and NS345 was obtained by first priming with Th1-adjuvanted proteins and then boosting with chimeric, defective alphaviruses expressing these HCV genes. In addition, this prime/boost regimen resulted in the induction of anti-E1E2 antibodies capable of cross-neutralizing heterologous HCV isolates in vitro. This vaccine formulation and regimen may therefore be optimal in humans for protection against this highly heterogeneous global pathogen.

On our initial discovery that prion protein (PrP)-derived peptides were capable of capturing the pathogenic prion protein (PrP Sc ), we have been interested in how these peptides interact with PrP Sc . After screening peptides from the entire human PrP sequence, we found two peptides (PrP 19–30 and PrP 100–111 ) capable of binding full-length PrP Sc in plasma, a medium containing a complex mixture of other proteins including a vast excess of the normal prion protein (PrP C ). The limit of detection for captured PrP Sc was calculated to be 8 amol from a ≈10 5 -fold dilution of 10% (wt/vol) human variant Creutzfeldt–Jakob disease brain homogenate, with >3,800-fold binding specificity to PrP Sc over PrP C . Through extensive analyses, we show that positively charged amino acids play an important, but not exclusive, role in the interaction between the peptides and PrP Sc . Neither hydrophobic nor polar interactions appear to correlate with binding activity. The peptide–PrP Sc interaction was not sequence-specific, but amino acid composition affected binding. Binding occurs through a conformational domain that is only present in PrP Sc , is species-independent, and is not affected by proteinase K digestion. These and other findings suggest a mechanism by which cationic domains of PrP C may play a role in the recruitment of PrP C to PrP Sc .

Although approximately 3 % of the world's population is infected withHepatitis C virus(HCV), there is no prophylactic vaccine available. This study reports the design, cloning and purification of a single polyprotein comprising the HCV core protein and non-structural proteins NS3, NS4a, NS4b, NS5a and NS5b. The immunogenicity of this polyprotein, which was formulated in alum, oil-in-water emulsion MF59 or poly(dl-lactide co-glycolide) in the presence or absence of CpG adjuvant, was then determined in a murine model for induction of B- and T-cell responses. The addition of adjuvants or a delivery system to the HCV polyprotein enhanced serum antibody and T-cell proliferative responses, as well as IFN-γresponses, by CD4+T cells. The antibody responses were mainly against the NS3 and NS5 components of the polyprotein and relatively poor responses were elicited against NS4 and the core components. IFN-γresponses, however, were induced against all of the individual components of the polyprotein. These data suggest that the HCV polyprotein delivered with adjuvants induces broad B- and T-cell responses and could be a vaccine candidate against HCV.

Epithelial ovarian cancer is the most frequent cause of gynecologic malignancy-related mortality in women. To identify genes up-regulated in ovarian cancer, PCR-select cDNA subtraction was done and Drosophila Eyes Absent Homologue 2 (EYA2) was isolated as a promising candidate. The transcriptional coactivator eya controls essential cellular functions during organogenesis of Drosophila. EYA2 mRNA was found to be up-regulated in ovarian cancer by real-time reverse transcription–PCR, whereas its protein product was detected in 93.6% of ovarian cancer specimens by immunohistochemistry (n = 140). EYA2 was amplified in 14.8% of ovarian carcinomas, as detected by array-based comparative genomic hybridization (n = 88). Most importantly, EYA2 overexpression was significantly associated with short overall survival in advanced ovarian cancer (n = 99, P = 0.0361). EYA2 was found to function as transcriptional activator in ovarian cancer cells by Gal4 assay and to promote tumor growth in vivo in xenograft models. Therefore, this study suggests an important role of EYA2 in ovarian cancer and its potential application as a therapeutic target.

Recent clinical studies suggest that hepatitis B virus (HBV) load and genotype may be independent predictors of responses to antiviral therapies. However, it is difficult for clinicians to accurately determine viral loads in patient samples because results—both the values and the units of measure—can vary greatly among different tests. Accordingly, the World Health Organization (WHO) has produced the first international standard for HBV DNA for nucleic acid amplification technology (NAT) assays. In the present study, we describe the performance of the target-capture PCR HBV DNA quantitative assay for the quantitation of HBV DNA in clinical samples and reference panels. The range of quantitation was between 50 and 10 10 IU/ml. The sensitivity and accuracy of the target-capture PCR assay were demonstrated by using the HBV panel from Quality Control for Medical Diagnostics (QCMD) and the WHO HBV DNA standard. The target-capture PCR assay quantitated the six genotype A members of the QCMD panel and dilutions of the WHO HBV DNA standard within an accuracy of 74 to 142%. Compared to current serological methods, the assay offers window period reductions of 19 days prior to HBV surface antigen and 26 days prior to HBV e antigen detection. The target-capture PCR assay was also compared with four commercially available NAT assays, and the various units of measure were standardized with respect to the international units of the WHO HBV DNA standard. The target-capture PCR assay is a sensitive, accurate, high-throughput, rapid, and reproducible assay for the determination of HBV loads.

Naturally occurring hepatitis C virus (HCV) infection has long been thought to induce a weak immunity which is insufficient to protect an individual from subsequent infections and has cast doubt on the ability to develop effective vaccines. A series of intrahepatic genetic inoculations (IHGI) with type 1a HCV RNA were performed in a chimpanzee to determine whether a form of genetic immunization might stimulate protective immunity. We demonstrate that the chimpanzee not only developed protective immunity to the homologous type 1a RNA after rechallenge by IHGI but was also protected from chronic HCV infection after sequential rechallenge with 100 50% chimpanzee infectious doses of a heterologous type 1a (H77) and 1b (HC-J4) whole-virus inoculum. These results offer encouragement to pursue the development of HCV vaccines.

Current commercial hepatitis B virus (HBV) anti-HBe immunoassays are designed so that anti-HBe is detectable only in the absence of excess HBeAg. Recently, with the use of direct anti-HBe assays, anti-HBe was detected in individuals who had been seropositive for several years for HBeAg [Maruyama et al. (1993) J. Clin. Invest. 91:2586-2595]. Although anti-HBe seroconversion does not necessarily indicate subsequent HBeAg clearance, the ability to detect earlier anti-HBe seroconversion could have clinical significance for monitoring patients undergoing HBV immunotherapy (e.g., alpha interferon therapy). Because the HBeAg and the HBcAg share 149 amino acids, an anti-HBe assay must distinguish anti-HBe from anti-HBc antibodies. Although the HBV HBeAg and HBcAg display distinct immunogenic determinants, much remains unknown regarding the complete epitope spectrum specific to each antigen. The goal of this study was 3-fold. The first objective was to identify HBeAg specific linear epitopes. The second objective was to design an anti-HBe immunoassay capable of detecting anti-HBe specific antibody in the presence of excess HBeAg. The third objective was to characterize early anti-HBe seroconversion antibodies. The major linear epitope residing in the HBeAg amino acid sequence was mapped and 2 novel minor epitopes (delta, gamma) which appear to be HBeAg specific have been identified. An anti-HBe immunoassay capable of detecting anti-HBe specific antibody in the presence of excess HBeAg was designed. Finally, it was found that early anti-HBe seroconversion antibodies appear to be conformational, whereas later seroconversion, more typically associated with the clearance of HBeAg, is characterized by the presence of antibodies to the linear HBeAg epitopes.

The trkB family of transmembrane proteins serves as receptors for BDNF and NT-4/5. The family is composed of a tyrosine kinase-containing isoform as well as several alternatively spliced “truncated receptors” with identical extracellular ligand-binding domains but very small intracellular domains. The two best-characterized truncated trkB receptors, designated as trkB.T1 and trkB.T2, contain intracellular domains of only 23 and 21 amino acids, respectively. Although it is known that the tyrosine kinase isoform (trkB.FL) is capable of initiating BDNF and NT-4/5-induced signal transduction, the functional role or roles of the truncated receptors remain enigmatic. At the same time, the potential importance of the truncated receptors in the development, maintenance, and regeneration of the nervous system has been highlighted by recent developmental and injury paradigm investigations. Here we have used trkB cDNA transfected cell lines to demonstrate that both trkB.T1 and trkB.T2 are capable of mediating BDNF-induced signal transduction. More specifically, BDNF activation of either trkB.T1 or trkB.T2 increases the rate of acidic metabolite release from the cell, a common physiological consequence of many signaling pathways. Further, these trkB.T1- and trkB.T2-mediated changes occur with kinetics distinct from changes mediated by trkB.FL, suggesting the participation of at least some unique rate-limiting component or components. Mutational analysis demonstrates that the isoform-specific sequences within the intracellular domains of each receptor are essential for signaling capability. Finally, inhibitor studies suggest that kinases are likely to be involved in the trkB.T1 and trkB.T2 signaling pathways.

Age-related macular degeneration, otherwise known as AMD, is a progressive, irreversible eye condition in which the retina is permanently damaged by either atrophy of its pigment epithelial layer (dry AMD) or abnormal blood vessel growth in the retina's membrane (wet AMD). Wet AMD, the more advanced, severe version of the disease, is induced by overexposure to vascular endothelial growth factor (VEGF) in the eye. This VEGF-induced blood vessel growth leads to rupture and leaking of the retina's membrane, endangering the integrity of the macula, which is vital for acute central vision. Once the macula is scarred and central vision is lost, it cannot be recovered. Current treatments for wet AMD include several drugs (i.e. lucentis) that inhibit VEGF, yielding a reduction in retinal thickness and moderate increases in visual acuity. While these treatments succeed in prohibiting disease progression, they require frequent intravitreal injections. In the future, the goal is to prevent disease progression while avoiding repeated injections with the use of therapeutic gene transfer.To develop this new generation of AMD therapeutic, we sought to create an efficacious AAV vector expressing anti-VEGF antibody via systematic optimization of all vector elements. Six promoters, 12 coding sequence variants, an F2A linker, and 3 internal ribosome entry sites (IRES) were evaluated for tissue-specific expression in mouse eye. Mean expression from the 2 best promoters, UbC and CB7, was 400 and 450 ng/mg of eye lysate, respectively, compared with the CMV promoter at 10 ng/mg. In the context of a CMV promoter, mean expression for best-performing IRES (EMCV) was 2 ng/mg of tissue lysate, which was 5-fold lower than that from the construct containing an F2A linker (10 ng/mg). The best-expressing coding sequence variants, named AMD11 and AMD42, yielded 12 and 13 ng/mg of tissue lysate, respectively.In order to identify a clinical candidate vector, the best-performing elements were combined to generate 4 additional vectors, which were evaluated in both eyes of 8 rhesus macaques for each vector. Following subretinal injection with 1×1011 GC/eye, anterior chamber fluid was sampled on days 15 and 29. Among the 16 injected eyes, average expression of AAV-UbC-AMD11 was 473.6 ng/ml (ranging from 123.5 to 933 ng/ml) of anterior chamber fluid on day 15 after vector administration and 546.2 ng/ml (262 to 1263 ng/ml) on day 29. Average expression of AAV-UbC-AMD42 was 360.6 ng/ml (144.6 to 646.8 ng/ml) and 536.8 ng/ml (238.3 to 920.7 ng/ml) on days 15 and 29, respectively. For AAV-CB7-AMD11, average expression on days 15 and 29 was 972.4 ng/ml (495.9 to 1684.7 ng/ml) and 1100.3 ng/ml (422.3 to 2187.8 ng/ml). The final vector, AAV-CB7-AMD42, expressed at 1922.2 ng/ml (707.9 to 2892.7 ng/ml) on day 15 and 1396 ng/ml (431.2 to 2346.4 ng/ml) on day 29. After final selection, the clinical candidate vector will be evaluated in formal nonclinical studies prior to a clinical trial. Selection criteria will include expression levels and vector yields.

To transition the AAV-mediated passive immunization strategy for protection against influenza A and B from mice to humans, an efficient vector delivery strategy is needed. Here, we demonstrate the ability of AAV9 expressing anti-influenza A and B antibodies (FI6 and CR8033, respectively) coupled with an intranasal mucosal atomization device (IMAD) to effectively protect mice against challenge with pandemic and seasonal influenza. First, we determined that an equimolar combination of AAV9.FI6 and AAV9.CR8033 vectors delivered intranasally at a dose of 5×1010 GC/KG per mouse effectively protected mice against lethal intranasal challenge with influenza A (PR8) or influenza B (B/Lee/40), respectively. In an effort to progress AAV9 into the clinic, we next determined vector compatibility with the IMAD for loss in volume, vector titer and vector potency. Groups of mice were intranasally administered a mixture of AAV9.FI6 and AAV9.CR8033 as well as a mixture of AAV9.FI6 and AAV9.CR8033 that was processed through the IMAD. Seven days later, the mice were challenged with 5LD50 of PR8 or B/Lee/40. No difference in effectiveness of protection against influenza A or B was observed when the vector mixture was processed through the IMAD. We next translated this approach to the nose of macaques, a model more closely related to the human nose that is the target tissue. In this model, we assessed the kinetics of the onset of transgene expression and stability of expression when vector was delivered via the IMAD compared with traditional, direct liquid delivery. At the conclusion of the study, vector biodistribution was also examined. Overall, no significant differences in the kinetics and profile of gene expression or vector biodistribution were observed between the two delivery methods. In conclusion, we demonstrate the safety and effectiveness of AAV-mediated prophylaxis against influenza A and B when vectors are delivered via the IMAD, an easy-to-use device that localizes transduction to the site of influenza infection, the nasal mucosa.

Human butyrylcholinesterase (hBuChE) is a widely-distributed enzyme that may play a role in nerve conduction, can hydrolyze various toxic esters (e.g., cocaine), and acts as a potent scavenger of organophosphates (OP) including pesticides and chemical warfare agents. Major limitations prohibiting the development of purified hBuChE as a mainstream biologic drug include: limited availability of human plasma, limited scalability of manufacturing, high cost of purified human-derived product, and the need for intravenous administration. In addition, the preventive uses of hBuChE are limited by its relatively short half-life. In an attempt to address some of these limitations, several methods for the production of recombinant hBuChE in vitro (cell lines) and in vivo (transgenic animals, plants) were developed; however, the resulting recombinant proteins presented with unfavorable pharmacokinetic profiles making them unsuitable for preventive uses. Alternatively, adenovirus-mediated delivery of hBuChE is very effective, but is not a candidate for development due to the inherent immunogenicity of the vector. Expression of hBuChE peaks at around 4 days post IV administration of the adenoviral vector, and the peak is followed by a rapid decline, with minimal expression being detectable 10 days post-administration.AAV vectors present a radically different, non-immunogenic hBuChE delivery platform, which is not reliant on protein purification and has the flexibility to deliver the product into both liver and muscle. AAV technology engineers the recipient's own cells to produce sustainable pharmacologic amounts of hBuChE on demand and/or replenish the exhausted scavenger via a single dose of hBuChE-expressing vector under the control of a constitutive promoter. Through the systematic optimization of AAV-hBuChE vectors, we achieved not only rapid onset of hBuChE expression in mice, but also sustainable, long-term expression for at least 2 months with no decline. AAV-hBuChE expressed at 100 units/ml of serum at 24 hours post vector administration into mouse muscle, with steady-state expression levels of up to 30,000 units/ml of serum at 1 month. The optimization parameters included selection of the protein isoform, tetramerization partner, coding sequence, as well as promoters and other regulatory elements. Overall, approximately 40 vectors were evaluated in comparative studies. Based on previous findings, the levels of hBuChE expressed by our platform are predicted to be protective in OP challenges, which we plan to perform in future studies. In the future, the use of our platform can be expanded to include populations that are occupationally exposed to pesticides. This approach may also be applicable for the delivery of other protective human enzymes, such as the use of rhodanese to protect against cyanides or glutathione against 2-chloro-acetophenone.

The complete nucleotide sequence of an isolate of simian adenovirus 7 (SAdV-7) was determined. The genome organization of this isolate was found to be similar to that of other primate adenoviruses with two principal notable points: severe truncation of the E1A and E1B 19K proteins and an E3 region encoding only the 12.5K homologue. The viral gene products of SAdV-7 are most closely related to simian adenovirus 1 (SAdV-1), and like SAdV-1, are related to the human adenovirus species HAdV-F, such as the enteric adenoviruses HAdV-40 and HAdV-41 and the recently defined HAdV-G (HAdV-52). Two kinds of gene transfer vectors were made: a replication-competent SAdV-7-based vector with no genomic deletion, and a standard replication-incompetent vector deleted for E1. Importantly, the E1-deleted vector could be propagated to high titre by trans-complementation in human HEK 293 cells.

Objective Persistent adenoviral shedding in stools is known to occur past convalescence following acute adenoviral infections. We wished to establish the frequency with which adenoviruses may colonize the gut in normal human subjects. Methods The presence of adenoviral DNA in intestinal specimens obtained at surgery or autopsy was tested using a nested PCR method. The amplified adenoviral DNA sequences were compared to each other and to known adenoviral species. Lamina propria lymphocytes (LPLs) were isolated from the specimens and the adenoviral copy numbers in the CD4+ and CD8+ fractions were determined by quantitative PCR. Adenoviral gene expression was tested by amplification of adenoviral mRNA. Results Intestinal tissue from 21 of 58 donors and LPLs from 21 of 24 donors were positive for the presence of adenoviral DNA. The majority of the sequences could be assigned to adenoviral species E, although species B and C sequences were also common. Multiple sequences were often present in the same sample. Forty-one non-identical sequences were identified from 39 different tissue donors. Quantitative PCR for adenoviral DNA in CD4+ and CD8+ fractions of LPLs showed adenoviral DNA to be present in both cell types and ranged from a few hundred to several million copies per million cells on average. Active adenoviral gene expression as evidenced by the presence of adenoviral messenger RNA in intestinal lymphocytes was demonstrated in 9 of the 11 donors tested. Conclusion Adenoviral DNA is highly prevalent in lymphocytes from the gastro-intestinal tract indicating that adenoviruses may be part of the normal gut flora.

Background We recently reported the isolation and sequencing of 30 novel adenoviruses from chimpanzees, bonobos and gorillas. These adenoviruses are promising candidates for the purpose of expanding the repertoire of adenoviral serotypes that can be used to create vectors for circumventing pre-existing neutralizing antibodies in human populations. We thus aimed to create vectors from 20 of the newly isolated adenoviruses. Methods Plasmid molecular clones were created that harbored the complete E1-deleted genomes from 20 of the newly isolated ape adenoviruses belonging to species B, C and E. The plasmids were transfected into human embryonic kidney (HEK) 293 cells to rescue vectors. We also tested normal human sera to determine the extent of pre-existing cross-neutralizing anti-adenovirus neutralizing antibodies. Results Twelve vectors could be rescued and expanded following transfection into HEK 293 cells with yields (from fifty 150-mm culture dishes) that ranged from 3 × 1011 to 7 × 1013 viral particles. Sera from 50 normal human donors were tested for the presence of neutralizing activity against 21 of the newly isolated ape adenoviruses. Cross-neutralizing activity was generally low, although outliers with high neutralizing activity were frequently detected. Species B ape adenoviruses generally showed the least cross-neutralization with antibodies present in the human sera that were tested. Conclusions E1-deleted adenovirus vectors can be created from a wide variety of ape adenoviruses that can be rescued and propagated in HEK 293 cells. The prevalence of pre-existing antibodies that can neutralize these adenoviruses in human populations is low. Copyright © 2010 John Wiley & Sons, Ltd.

A majority of malignant melanomas harbor an oncogenic mutation in either BRAF or NRAS .I fBRAF and NRAS transform melanoma cells by a similar mechanism, then additional genetic aberrations would be similar (or random). Alternatively, distinct mutation-associated changes would suggest the existence of unique cooperating requirements for each mutation group. We first analyzed a panel of 52 melanoma cell lines (n ¼ 35, 11, 6 for BRAF*, NRAS*, and BRAF/NRAS wt/wt , respectively) by array-based comparative genomic hybridization for unique alterations that associate with each mutation subgroup. Subsequently, those DNA copy number changes that correlated with a mutation subgroup were used to predict the mutation status of an independent panel of 43 tumors (n ¼ 17, 13, 13 for BRAF*, NRAS*, and BRAF/NRAS wt/wt , respectively). BRAF mutant tumors were classified with a high rate of success (74.4%, P ¼ 0.002), whereas NRAS mutants were not significantly distinguished from wild types (26/43, P ¼ 0.12). Copy number gains of 7q32.1-36.3, 5p15.31, 8q21.11, and 8q24.11 were most strongly associated with BRAF* tumors and cell lines, as were losses of 11q24.2-24.3. BRAF* melanomas appear to be associated with a specific profile of DNA copy number aberrations that is distinct from those found in NRAS* and BRAF/NRAS wt/wt tumors. These findings suggest that although both BRAF and NRAS appear to function along the same signal transduction pathway, each may have different requirements for cooperating oncogenic events. The genetic loci that make up this profile may harbor therapeutic targets specific for tumors with BRAF mutations. V

Broad, multispecific CD4(+) and CD8(+) T-cell responses to the hepatitis C virus (HCV), as well as virus-cross-neutralizing antibodies, are associated with recovery from acute infection and may also be associated in chronic HCV patients with a favorable response to antiviral treatment. In order to recapitulate all of these responses in an ideal vaccine regimen, we have explored the use of recombinant HCV polypeptides combined with various Th1-type adjuvants and replication-defective alphaviral particles encoding HCV proteins in various prime/boost modalities in BALB/c mice. Defective chimeric alphaviral particles derived from the Sindbis and Venezuelan equine encephalitis viruses encoding either the HCV envelope glycoprotein gpE1/gpE2 heterodimer (E1E2) or nonstructural proteins 3, 4, and 5 (NS345) elicited strong CD8(+) T-cell responses but low CD4(+) T helper responses to these HCV gene products. In contrast, recombinant E1E2 glycoproteins adjuvanted with MF59 containing a CpG oligonucleotide elicited strong CD4(+) T helper responses but no CD8(+) T-cell responses. A recombinant NS345 polyprotein also stimulated strong CD4(+) T helper responses but no CD8(+) T-cell responses when adjuvanted with Iscomatrix containing CpG. Optimal elicitation of broad CD4(+) and CD8(+) T-cell responses to E1E2 and NS345 was obtained by first priming with Th1-adjuvanted proteins and then boosting with chimeric, defective alphaviruses expressing these HCV genes. In addition, this prime/boost regimen resulted in the induction of anti-E1E2 antibodies capable of cross-neutralizing heterologous HCV isolates in vitro. This vaccine formulation and regimen may therefore be optimal in humans for protection against this highly heterogeneous global pathogen.

Mutations in the BRAF gene are found in the majority of cutaneous malignant melanomas and subsets of other tumors. These mutations lead to constitutive activation of BRAF with increased downstream ERK (extracellular signal-regulated kinase) signaling; therefore, the development of RAF kinase inhibitors for targeted therapy is being actively pursued. A methodology that allows sensitive, cost-effective, high-throughput analysis of BRAF mutations will be needed to triage patients for specific molecular-based therapies. Pyrosequencing is a high-throughput, sequencing-by-synthesis method that is particularly useful for analysis of single nucleotide polymorphisms or hotspot mutations. Mutational analysis of BRAF is highly amenable to pyrosequencing because the majority of mutations in this gene localize to codons 600 and 601 and consist of single or dinucleotide substitutions. In this study, DNAs from a panel of melanocyte cell lines, melanoma cell lines, and melanoma tumors were used to validate a pyrosequencing assay to detect BRAF mutations. The assay demonstrates high accuracy and precision for detecting common and variant exon 15 BRAF mutations. Further, comparison of pyrosequencing data with 100K single nucleotide polymorphism microarray data allows characterization of BRAF amplification events that may accompany BRAF mutation. Pyro-sequencing serves as an excellent platform for BRAF genotyping of tumors from patients entering clinical trial.

MicroRNAs (miRNAs) are endogenous noncoding RNAs, which negatively regulate gene expression. To determine genomewide miRNA DNA copy number abnormalities in cancer, 283 known human miRNA genes were analyzed by high-resolution array-based comparative genomic hybridization in 227 human ovarian cancer, breast cancer, and melanoma specimens. A high proportion of genomic loci containing miRNA genes exhibited DNA copy number alterations in ovarian cancer (37.1%), breast cancer (72.8%), and melanoma (85.9%), where copy number alterations observed in >15% tumors were considered significant for each miRNA gene. We identified 41 miRNA genes with gene copy number changes that were shared among the three cancer types (26 with gains and 15 with losses) as well as miRNA genes with copy number changes that were unique to each tumor type. Importantly, we show that miRNA copy changes correlate with miRNA expression. Finally, we identified high frequency copy number abnormalities of Dicer1 , Argonaute2 , and other miRNA-associated genes in breast and ovarian cancer as well as melanoma. These findings support the notion that copy number alterations of miRNAs and their regulatory genes are highly prevalent in cancer and may account partly for the frequent miRNA gene deregulation reported in several tumor types.

The current commercially licensed enzyme-linked immunosorbent assays (ELISAs) for hepatitis C virus (HCV) mainly use recombinant proteins containing linear epitopes. There is evidence, however, that conformational epitopes of HCV are more immunoreactive. Thus, we have designed an HCV antibody assay that employs a conformational protein, NS3NS4a PI (with functional protease and helicase activities), and a linear fusion protein, multiple-epitope fusion antigen 7.1 (MEFA 7.1) or MEFA 7.2. We have shown that NS3NS4a PI detects early-seroconversion conformation-sensitive antibodies better than c33c antigen. The correct conformation of NS3NS4a PI also cross-reacts with different genotype samples better than the c33c antigen. MEFA 7.1 and MEFA 7.2 incorporate all the major immunodominant and genotype-specific epitopes of HCV core, E1, E2 hypervariable region 1 (HVR1), E2 HVR1-plus-HVR2 consensus, NS3, NS4, and NS5. Since MEFA 7.1 is degraded by the active NS3NS4a PI protease, we designed a second MEFA 7.2 construct in which the six protease cleavage sites found in MEFA 7.1 were eliminated by amino acid mutation. We demonstrate here that MEFA 7.2 remains intact in the presence of NS3NS4a PI and preserves the epitopes present in MEFA 7.1. Compared to currently licensed assays, an ELISA incorporating a combination of the two antigens NS3NS4a PI and MEFA 7.1 or 7.2 demonstrates better serotype sensitivity and detects seroconversion earlier in many commercially available panels. We believe that an assay using NS3NS4a PI and MEFA 7.1 or 7.2 may have the potential to replace current HCV immunoassays for better sensitivity.

Several vaccine technologies were evaluated for their abilities to induce anti-human immunodeficiency virus Gag immune responses in rhesus macaques. While no vaccine alone was able to induce broad and strong immune responses, these were achieved by priming with Gag DNA and boosting with Gag protein adsorbed to polylactide coglycolide microparticles. This regimen elicited strong antibodies, helper T cells, and cytotoxic T lymphocytes and thus holds promise as an effective vaccination scheme.