Time-Dependent Internalization of Polymer-Coated Silica Nanoparticles in Brain Endothelial Cells and Morphological and Functional Effects on the Blood-Brain Barrier

Nanoparticle (NP)-assisted procedures including laser tissue soldering (LTS) offer advantages compared to conventional microsuturing, especially in the brain. In this study, effects of polymer-coated silica NPs used in LTS were investigated in human brain endothelial cells (ECs) and blood-brain barrier models. In the co-culture setting with ECs and pericytes, only the cell type directly exposed to NPs displayed a time-dependent internalization. No transfer of NPs between the two cell types was observed. Cell viability was decreased relatively to NP exposure duration and concentration. Protein expression of the nuclear factor k-light-chain-enhancer of activated B cells and various endothelial adhesion molecules indicated no initiation of inflammation or activation of ECs after NP exposure. Differentiation of CD34+ ECs into brain-like ECs co-cultured with pericytes, blood-brain barrier (BBB) characteristics were obtained. The established endothelial layer reduced the passage of integrity tracer molecules. NP exposure did not result in alterations of junctional proteins, BBB formation or its integrity. In a 3-dimensional setup with an endothelial tube formation and tight junctions, barrier formation was not disrupted by the NPs and NPs do not seem to cross the blood-brain barrier. Our findings suggest that these polymer-coated silica NPs do not damage the BBB

Nestemäisten jätteiden vastaanotto ja käsittely Tarastenjärven jätekeskuksessa : Selvitys vaihtoehtoisista menetelmistä

Nestemäiset jätteet ovat erilaisia loka-autoilla kuljetettavia jätteitä, jotka koostuvat nesteestä ja kiintoaineksesta. Tällaisia jätteitä ovat esimerkiksi hiekan- ja rasvanerotuskaivoista imetyt nesteet sekä teollisuudessa syntyvät jätevesilietteet. Nestemäisiä jätteitä ei ole Valtioneuvoston päätöksestä enää vuoden 2002 jälkeen saanut sijoittaa sellaisenaan kaatopaikkapenkkaan, vaan ne on tullut esikäsitellä nesteen poistamiseksi. Tähän asti nestemäiset jätteet on käsitelty Tarastenjärven jätekeskuksessa painovoimaisesti erottelemalla. Menetelmä on ollut toimiva, mutta on päivityksen tarpeessa. Tämän opinnäytetyön tarkoituksena olikin tutkia erilaisia vaihtoehtoja nestemäisten jätteiden vastaanotolle sekä käsittelylle Tarastenjärven jätekeskuksessa, sekä arvioida eri vaihtoehtojen kustannuksia mikäli mahdollista. Myös tarvetta haitta-aineiden, kuten metallien ja öljyjen poistamiselle tutkittiin. Työn laatimisessa hyödynnettiin olemassa olevaa tieteellistä kirjallisuutta sekä asiantuntijahaastatteluja. Työn tilaajana toimi Pirkanmaan Jätehuolto Oy. Selvitystyön tuloksena kävi ilmi, että yleisimmät menetelmät nesteiden ja kiinteiden ainesten erottamiseen ovat painovoimainen erottelu ja suodatus. Painovoimaiset erottelijat ovat useimmiten kiinteäseinäisiä altaita, joissa kiintoaines laskeutuu painovoimaisesti altaan pohjalle ja pinnalle jäänyt kirkastunut neste ohjataan ylivuotona jatkokäsittelyyn. Kiintoaineksen ollessa kevyempää kuin vesi, se erottuu nesteen pinnalle. Suodattimien toiminta taas perustuu puoliläpäisevään suodatusmediaan, joka päästää nesteen läpi, mutta jättää kiintoaineksen suodattimeen. Suodattimet voivat olla esimerkiksi paineistettuja tai painovoimalla toimivia. Geotuubit ovat eräänlaisia paineistettuja säkkisuodattimia, joita on käytössä myös jätekeskuksissa. Useissa käsittelymenetelmissä käytetään apuna myös saostuskemikaaleja, jotka edistävät kiintoaineksen erottumista nesteestä. Haitta-aineiden osalta helpoin ratkaisu on poistaa ne kiintoaineksen mukana ja käsitellä ne pilaantuneiden maiden kanssa jätekeskuksessa. Mikäli haitta-aineet ovat nesteessä liukoisessa muodossa, voidaan apuna käyttää saostuskemikaaleja, kuten alumiini- tai rautasuoloja. Öljyjen erottamisessa olisi mahdollista hyödyntää öljynerotuskaivoa. Kirjallisuuden sekä haastattelujen perusteella päivitetty versio painovoimaisesta erottelujärjestelmästä sekä geotuubimenetelmä vaikuttavat parhailta käytettävissä olevilta ratkaisuilta. Suurin haaste jätekeskuksen nestemäisten jätteiden käsittelyssä on kuitenkin se, että nesteiden ominaisuudet eroavat kuormittain hyvin paljon toisistaan. Tähän voitaisiin vaikuttaa jätteiden tarkemmalla lajittelulla vastaanottovaiheessa. Käsittelymenetelmää valittaessa tulee kuitenkin pohtia järjestelmän kuluja sekä hyötyjä pitkällä aikavälillä niin, että päästään parhaaseen mahdolliseen puhdistuslopputulokseen.Liquid wastes are solid-liquid mixtures such as wastes from sand and grease separation wells and industrial wastewaters. Since 2002 liquid wastes have no longer been allowed to be placed at a waste disposal site according to the decree 1049/1999 of the Finnish Council of State. Therefore liquid wastes have to be dewatered before final disposal. At the moment liquid wastes are treated at the Tarastenjärvi waste management centre with gravitational separation but the system needs updating. The aim of this study was to provide Pirkanmaan Jätehuolto Oy with different methods of liquid waste handling as well as evaluate the costs of a new system if possible. Also the need for contaminant removal was taken into consideration. The study was carried out by literature reviews and interviewing professionals of the industry. The results showed that the most common methods of solid-liquid separation are gravitational separation and filtration. Gravitational separators are usually containers in which the solid matter is allowed to separate either by sedimentation or flotation. Filters have a semi-permeable filter media which traps the solid matter but allows the liquid to flow through. Geotubes are an example of filters that are currently being used in several waste management centres. Many separation methods also require the use of polymers or flocculants to operate effectively. When contaminants are in a settleable form they are rather easy to be removed and treated with the solid matter. If the contaminants are dissolved in the liquid it might be possible to make them settle by adding aluminium or iron sulphate. Also an oil separation well could be used to separate oils from the liquid phase. Based on the literature reviews and interviews an upgraded gravitational system or the geotube method seem to be the best options. However, more tests should be conducted to see if these methods would work in practice. The biggest challenge is that the incoming liquid waste loads at Tarastenjärvi are not homogenous which makes it difficult to choose a solution that works well for all of them. The situation could be improved with more precise classification of the waste loads and handling them separately according to their characteristics. The most important aspect is to find a long-term cost-effective solution that can provide sufficient handling of the liquid wastes

Viral population estimation using pyrosequencing

The diversity of virus populations within single infected hosts presents a major difficulty for the natural immune response as well as for vaccine design and antiviral drug therapy. Recently developed pyrophosphate based sequencing technologies (pyrosequencing) can be used for quantifying this diversity by ultra-deep sequencing of virus samples. We present computational methods for the analysis of such sequence data and apply these techniques to pyrosequencing data obtained from HIV populations within patients harboring drug resistant virus strains. Our main result is the estimation of the population structure of the sample from the pyrosequencing reads. This inference is based on a statistical approach to error correction, followed by a combinatorial algorithm for constructing a minimal set of haplotypes that explain the data. Using this set of explaining haplotypes, we apply a statistical model to infer the frequencies of the haplotypes in the population via an EM algorithm. We demonstrate that pyrosequencing reads allow for effective population reconstruction by extensive simulations and by comparison to 165 sequences obtained directly from clonal sequencing of four independent, diverse HIV populations. Thus, pyrosequencing can be used for cost-effective estimation of the structure of virus populations, promising new insights into viral evolutionary dynamics and disease control strategies.Comment: 23 pages, 13 figure

Having a pair: the key to immune evasion for the diploid pathogen Schistosoma japonicum

Schistosomes, unlike malaria parasites, are in their diploid stage when targeted by the human immune system. Diploids can be either homozygous or heterozygous. The difference has profound significance for developing immunity and yet has not previously been addressed. We examined the implications of zygosity on immunity to a diploid pathogen, Schistosoma japonicum and showed that the diploid state, and its associated heterozygous advantage, significantly affects the outcome of attack by the immune system and the accumulation of antigenic diversity in the parasite population. We demonstrate here that diploidy provides a novel means of immune evasion for diploid pathogens

Antibodies in HIV-1 Vaccine Development and Therapy

Despite 30 years of study, there is no HIV-1 vaccine and, until recently, there was little hope for a protective immunization. Renewed optimism in this area of research comes in part from the results of a recent vaccine trial and the use of single-cell antibody-cloning techniques that uncovered naturally arising, broad and potent HIV-1–neutralizing antibodies (bNAbs). These antibodies can protect against infection and suppress established HIV-1 infection in animal models. The finding that these antibodies develop in a fraction of infected individuals supports the idea that new approaches to vaccination might be developed by adapting the natural immune strategies or by structure-based immunogen design. Moreover, the success of passive immunotherapy in small-animal models suggests that bNAbs may become a valuable addition to the armamentarium of drugs that work against HIV-1

Protection of Macaques with Diverse MHC Genotypes against a Heterologous SIV by Vaccination with a Deglycosylated Live-Attenuated SIV

HIV vaccine development has been hampered by issues such as undefined correlates of protection and extensive diversity of HIV. We addressed these issues using a previously established SIV-macaque model in which SIV mutants with deletions of multiple gp120 N-glycans function as potent live attenuated vaccines to induce near-sterile immunity against the parental pathogenic SIVmac239. In this study, we investigated the protective efficacy of these mutants against a highly pathogenic heterologous SIVsmE543-3 delivered intravenously to rhesus macaques with diverse MHC genotypes. All 11 vaccinated macaques contained the acute-phase infection with blood viral loads below the level of detection between 4 and 10 weeks postchallenge (pc), following a transient but marginal peak of viral replication at 2 weeks in only half of the challenged animals. In the chronic phase, seven vaccinees contained viral replication for over 80 weeks pc, while four did not. Neutralizing antibodies against challenge virus were not detected. Although overall levels of SIV specific T cell responses did not correlate with containment of acute and chronic viral replication, a critical role of cellular responses in the containment of viral replication was suggested. Emergence of viruses with altered fitness due to recombination between the vaccine and challenge viruses and increased gp120 glycosylation was linked to the failure to control SIV. These results demonstrate the induction of effective protective immune responses in a significant number of animals against heterologous virus by infection with deglycosylated attenuated SIV mutants in macaques with highly diverse MHC background. These findings suggest that broad HIV cross clade protection is possible, even in hosts with diverse genetic backgrounds. In summary, results of this study indicate that deglycosylated live-attenuated vaccines may provide a platform for the elucidation of correlates of protection needed for a successful HIV vaccine against diverse isolates

Protection against Divergent Influenza H1N1 Virus by a Centralized Influenza Hemagglutinin

Influenza poses a persistent worldwide threat to the human population. As evidenced by the 2009 H1N1 pandemic, current vaccine technologies are unable to respond rapidly to this constantly diverging pathogen. We tested the utility of adenovirus (Ad) vaccines expressing centralized consensus influenza antigens. Ad vaccines were produced within 2 months and protected against influenza in mice within 3 days of vaccination. Ad vaccines were able to protect at doses as low as 107 virus particles/kg indicating that approximately 1,000 human doses could be rapidly generated from standard Ad preparations. To generate broadly cross-reactive immune responses, centralized consensus antigens were constructed against H1 influenza and against H1 through H5 influenza. Twenty full-length H1 HA sequences representing the main branches of the H1 HA phylogenetic tree were used to create a synthetic centralized gene, HA1-con. HA1-con minimizes the degree of sequence dissimilarity between the vaccine and existing circulating viruses. The centralized H1 gene, HA1-con, induced stronger immune responses and better protection against mismatched virus challenges as compared to two wildtype H1 genes. HA1-con protected against three genetically diverse lethal influenza challenges. When mice were challenged with 1934 influenza A/PR/8/34, HA1-con protected 100% of mice while vaccine generated from 2009 A/TX/05/09 only protected 40%. Vaccination with 1934 A/PR/8/34 and 2009 A/TX/05/09 protected 60% and 20% against 1947 influenza A/FM/1/47, respectively, whereas 80% of mice vaccinated with HA1-con were protected. Notably, 80% of mice challenged with 2009 swine flu isolate A/California/4/09 were protected by HA1-con vaccination. These data show that HA1-con in Ad has potential as a rapid and universal vaccine for H1N1 influenza viruses

Quantitative Deep Sequencing Reveals Dynamic HIV-1 Escape and Large Population Shifts during CCR5 Antagonist Therapy In Vivo

High-throughput sequencing platforms provide an approach for detecting rare HIV-1 variants and documenting more fully quasispecies diversity. We applied this technology to the V3 loop-coding region of env in samples collected from 4 chronically HIV-infected subjects in whom CCR5 antagonist (vicriviroc [VVC]) therapy failed. Between 25,000–140,000 amplified sequences were obtained per sample. Profound baseline V3 loop sequence heterogeneity existed; predicted CXCR4-using populations were identified in a largely CCR5-using population. The V3 loop forms associated with subsequent virologic failure, either through CXCR4 use or the emergence of high-level VVC resistance, were present as minor variants at 0.8–2.8% of baseline samples. Extreme, rapid shifts in population frequencies toward these forms occurred, and deep sequencing provided a detailed view of the rapid evolutionary impact of VVC selection. Greater V3 diversity was observed post-selection. This previously unreported degree of V3 loop sequence diversity has implications for viral pathogenesis, vaccine design, and the optimal use of HIV-1 CCR5 antagonists

Inferring viral quasispecies spectra from 454 pyrosequencing reads

<p>Abstract</p> <p>Background</p> <p>RNA viruses infecting a host usually exist as a set of closely related sequences, referred to as quasispecies. The genomic diversity of viral quasispecies is a subject of great interest, particularly for chronic infections, since it can lead to resistance to existing therapies. High-throughput sequencing is a promising approach to characterizing viral diversity, but unfortunately standard assembly software was originally designed for single genome assembly and cannot be used to simultaneously assemble and estimate the abundance of multiple closely related quasispecies sequences.</p> <p>Results</p> <p>In this paper, we introduce a new <b>Vi</b>ral <b>Sp</b>ectrum <b>A</b>ssembler (ViSpA) method for quasispecies spectrum reconstruction and compare it with the state-of-the-art ShoRAH tool on both simulated and real 454 pyrosequencing shotgun reads from HCV and HIV quasispecies. Experimental results show that ViSpA outperforms ShoRAH on simulated error-free reads, correctly assembling 10 out of 10 quasispecies and 29 sequences out of 40 quasispecies. While ShoRAH has a significant advantage over ViSpA on reads simulated with sequencing errors due to its advanced error correction algorithm, ViSpA is better at assembling the simulated reads after they have been corrected by ShoRAH. ViSpA also outperforms ShoRAH on real 454 reads. Indeed, 7 most frequent sequences reconstructed by ViSpA from a real HCV dataset are viable (do not contain internal stop codons), and the most frequent sequence was within 1% of the actual open reading frame obtained by cloning and Sanger sequencing. In contrast, only one of the sequences reconstructed by ShoRAH is viable. On a real HIV dataset, ShoRAH correctly inferred only 2 quasispecies sequences with at most 4 mismatches whereas ViSpA correctly reconstructed 5 quasispecies with at most 2 mismatches, and 2 out of 5 sequences were inferred without any mismatches. ViSpA source code is available at <url>http://alla.cs.gsu.edu/~software/VISPA/vispa.html</url>.</p> <p>Conclusions</p> <p>ViSpA enables accurate viral quasispecies spectrum reconstruction from 454 pyrosequencing reads. We are currently exploring extensions applicable to the analysis of high-throughput sequencing data from bacterial metagenomic samples and ecological samples of eukaryote populations.</p

A Novel Immunodominant CD8+ T Cell Response Restricted by a Common HLA-C Allele Targets a Conserved Region of Gag HIV-1 Clade CRF01_AE Infected Thais

Background: CD8+ T cell responses play an important role in the control of HIV-1. The extensive sequence diversity of HIV-1 represents a critical hurdle to developing an effective HIV-1 vaccine, and it is likely that regional-specific vaccine strains will be required to overcome the diversity of the different HIV-1 clades distributed world-wide. Unfortunately, little is known about the CD8+ T cell responses against CRF01_AE, which is responsible for the majority of infections in Southeast Asia. Methodology/Principal Findings: To identify dominant CD8+ T cell responses recognized in HIV-1 clade CRF01_AE infected subjects we drew upon data from an immunological screen of 100 HIV-1 clade CRF01_AE infected subjects using IFN-gamma ELISpot to characterize a novel immunodominant CD8+ T cell response in HIV-1 Gag restricted by HLA-Cw*0102 (p24, 277YSPVSILDI 285, YI9). Over 75% of Cw*0102+ve subjects targeted this epitope, representing the strongest response in more than a third of these individuals. This novel CD8 epitope was located in a highly conserved region of HIV-1 Gag known to contain immunodominant CD8 epitopes, which are restricted by HLA-B*57 and -B*27 in clade B infection. Nonetheless, viral escape in this epitope was frequently observed in Cw*0102+ve subjects, suggestive of strong selection pressure being exerted by this common CD8+ T cell response. Conclusions/Significance: As HLA-Cw*0102 is frequently expressed in the Thai population (allelic frequency of 16.8%), this immunodominant Cw*0102-restricted Gag epitope may represent an attractive candidate for vaccines specific to CRF01_AE and may help facilitate further studies of immunopathogenesis in this understudied HIV-1 clade. © 2011 Buranapraditkun et al