Protein aggregation (literature review) : Soluble expression of human Ciliary neurotrophic factor in Escherichia coli (experimental work)

Proteins are endogenous molecules that carry out most biological functions in vivo. They are called as the biological workhorses. Proteins are made up of polypeptide chains that usually fold in the three dimensional space to adopt a native stable conformation. Stability of proteins is dependent on the interplay of environmental factors (pH, temperature, ionic strength). For most proteins, the biological function closely relates to the structural attributes of the protein. Misfolding or unfolding of proteins often result in aggregation. Protein aggregation in vivo is known to cause debilitating and fatal diseases such as Alzheimer's, Huntington's, Parkinson's and age related macular degeneration (AMD). Instability (physical and chemical) of proteins in vitro is believed to result in aggregation. This is a huge concern for the biopharmaceutical industry as it not only limits the effectiveness of the manufacturing process but also poses a great risk of fatality in vivo due to the immunogenic nature of the aggregates. Mechanisms of protein aggregation are complex and not well understood. Regulatory requirements for patient safety in biopharmaceutical products require characterization and analysis of aggregates in protein drug formulations. This review provides an overview of protein aggregation in general and highlights the different analytical methods used to characterize protein aggregates in biopharmaceuticals. Neurotrophic factors influence survival, differentiation, proliferation and death of neuronal cells within the central nervous system. Human ciliary neurotrophic factor (hCNTF) has neuroprotective properties and is also known to influence energy balance. Consequently, hCNTF has potential therapeutic applications in neurodegenerative, obesity and diabetes related disorders. Clinical and biological applications of CNTF necessitate a recombinant expression system to produce large amounts of functional protein. Previous studies have reported that recombinant expression of CNTF in Escherichia coli (E. coli) was limited by low yields and the need to refold the protein from inclusion bodies. In this report, we describe a strategy to effectively screen fusion constructs and expression conditions for soluble hCNTF production in E. coli. Most conditions tested with the codon optimized hCNTF sequence in fusion with soluble tags resulted in soluble expression of the protein. The construct 6-His-CNTF showed soluble expression in all the conditions tested. Our results suggest that codon optimization of the hCNTF sequence is sufficient for soluble expression in E. coli. The recombinant hCNTF was found to bind to CNTFRα with an EC50 = 36 nM.Proteiinit ovat endogeenisiä molekyylejä, jotka suorittavat lähes kaikki solun toiminnot ja ovat solun normaalille toiminnalle välttämättömiä. Proteiinit muodostuvat polypeptidiketjuista, jotka laskostuessaan normaalisti muodostavat stabiilin ja toimintakykyisen kolmiulotteisen rakenteen. Proteiinien stabiilius on riippuvainen ympäristön tekijöistä (pH, lämpötila, ionivahvuus, jne.) ja niiden keskinäisistä vuorovaikutuksista. Useimmilla proteiineilla niiden biologinen toiminta on läheisesti kytköksissä niiden rakenteeseen. Proteiinien vääränlainen laskostuminen tai laskoksen aukeaminen johtaa usein proteiinien ryvästymiseen eli aggregaatioon. In vivo proteiinien aggregaation tiedetään olevan osallisena aiheuttajana vaikeissa ja tappavissa sairauksissa, kuten Alzheimerin ja Parkinsonin taudissa, sekä verkkokalvon ikärappeumassa. In vitro, proteiinien fysikaalisen ja kemiallisen epästabiiliuden uskotaan johtavan aggregaatioon. Tämä on suuri huolenaihe varsinkin biofarmaseuttiselle teollisuudelle, sillä se rajoittaa proteiinilääkkeiden valmistuprosessin tehokkuutta. Tärkeämpänä syynä on kuitenkin proteiiniaggregaattien vahva immunogeeninen luonne, joka voi aiheuttaa vakavia seurauksia potilaissa. Proteiinien aggregaation mekanismit ovat monimutkaisia ja huonosti ymmärrettyjä. Biologisten lääkkeiden potilasturvallisuutta koskevat säännökset vaativat proteiinilääkeformulaatioilta proteiiniaggregaattien analyysiä ja karakterisointia. Tämä katsaus antaa yleiskuvan proteiiniaggregaatiosta ja keskittyy biologisten lääkkeiden proteaggregaattien analyysissa ja karakterisoinnissa käytettyihin metelmiin. Neurotrofiset tekijät vaikuttavat keskushermoston neuronien eloonjäämiseen, erilaistumiseen, leviämiseen ja säädeltyyn kuolemaan. Ihmisen siliaarisella hermokasvutekijällä (human Ciliary neurotrophic factor, hCNTF) on neuroneja suojaava ominaisuus ja sen tunnetaan vaikuttavan myös energiatasapainoon. Tästä syystä CNTF:llä onkin potentiaalista terapeuttista käyttöä neurodegeneratiivisten tautien ja aineenvaihduntatautien hoidossa. Tälläisia tauteja ovat esimerkiksi liikalihavuus ja diabetes. CNTF:n kliininen ja biologinen käyttö vaatii rekombinanttien ilmennysmenetelmien käyttöä, jotta voidaan tuottaa suuria määriä toiminnallista proteiinia. Aikaisemmissa tutkimuksissa on raportoitu rekombinantin CNTF:n Eschrecchia coli (E. coli) -soluissa tapahtuva ilmentäminen on rajoittunut alhaisen saannon vuoksi ja vaatii usein proteiinin uudelleenlaskostusta inkluusiojyväsistä. Tässä raportissa kuvaamme menetelmän, jolla voi seuloa tehokkaasti eri konstrukteja ja ilmentämisolosuhteita liukoisen hCNTF:n tuottamiseen E. coli-soluissa. Liukoisen proteiinien ilmentymistä havaittiin useimmilla liukoisen fuusioproteiinin ja kodonioptimoidulla hCNTF-sekvenssin yhdistelmillä ja tutkituissa ilmentämisolosuhteissa. Fuusiokonstruktin 6-His-CNTF havaittiin ilmentyvän liukoisena kaikissa tutkituissa olosuhteissa. Havaintomme viittaavat hCNTF:n sekvenssin kodonioptimoinnin olevan riittävä aikaansaamaan hCNTF:n ilmentyminen liukoisena E. coli-soluissa. Rekombinantin hCNTF:n havaittiin sitoutuvan CNTFRα:aan EC50-arvolla 36 nM

Therapeutic proteins and their use in posterior eye segment diseases

Since their introduction in the late 20th century, therapeutic proteins have become an irreplaceable class of pharmaceuticals and are today used to treat a wide variety of diseases ranging from arthritis, diabetes, and various cancers to more recently, for example, asthma and migraine. In ophthalmology, the treatment of certain neurodegenerative diseases of the retina, such as age-related macular degeneration and diabetic retinopathy, has been revolutionized by therapeutic proteins that combat the pathological growth of abnormal blood vessels in the retina. As retinal diseases are some of the leading causes of vision loss and blindness globally, and expected to grow in prevalence with aging populations, the importance and need for such ophthalmologic therapeutic proteins is expected to increase. During the early development of therapeutic protein candidates, the production of functional protein in adequate amounts can often be a significant roadblock. In this thesis, the expression of soluble recombinant human ciliary neurotrophic factor (rhCNTF) – a neuroprotective protein with therapeutic potential against retinal neurodegeneration – in Escherichia coli was enhanced. Codon optimization of the hCNTF gene was combined with screening of different culture media, culture conditions, and fusion partners to pinpoint ideal expression conditions for rhCNTF. Following expression in the determined optimal conditions, the protein was purified with immobilized metal-ion affinity chromatography and gel filtration, and the in vitro activity of purified rhCNTF was demonstrated in a binding assay with its cognate receptor, CNTFRα. Overall, an 8–9 fold increase in soluble rhCNTF fraction and a 10–20 fold increase in yield was achieved, whereas earlier efforts to produce CNTF have commonly required purification from insoluble inclusion bodies and/or yielded low protein amounts. Furthermore, such a combinatorial approach is successful as a screening strategy for soluble expression and could be applied to other proteins of pharmaceutical interest. When taken out of their natural biological milieus, most proteins are only marginally stable and susceptible to environmental perturbations. As such, the formulation of a therapeutic protein aims to protect the protein and retain its stability and biological activity, and ultimately to guarantee the therapeutic efficacy and safety throughout the lifetime of the pharmaceutical. Here, further characterization, formulation, and stability studies were carried out with purified rhCNTF. The proper folding of purified rhCNTF was observed with circular dichroism spectroscopy and the biological activity of the protein was verified in a cell proliferation study with a CNTFα expressing cell-line. After screening, two buffers were chosen as storage buffers for rhCNTF. Whereas minute changes in rhCNTF’s oligomeric status were observed in only of these buffers, no changes in rhCNTF’s thermal stability were observed in either buffer during the study period. As such, these results provide a basis for further formulation development for rhCNTF. Although intravitreally injected therapeutic proteins have become the cornerstone in the management of retinal neovascularization, how and to what extent these and other proteins penetrate into the retina remains poorly understood. Here, permeation into the neural retina was observed with fluorescently labeled rhCNTF in ex vivo retinal explant models. Our results indicate that permeation to the CNTF-responsive target cells in the retina is not a limitation to exogenous CNTF’s direct neuroprotective actions. Moreover, our results provide further impetus to utilize ex vivo methods to systematically elucidate the retinal permeation and ocular pharmacokinetics of therapeutic proteins by and large. Therapeutic proteins have not only held the helm of best-selling pharmaceuticals for some time now, but currently also represent more than 40% of new pharmaceuticals in the development pipeline. Regardless, protein drug development has been plagued with ever increasing development costs yet with fewer new drugs entering the market, and there is an urgent call to disrupt this unsustainable cycle. While reasons for failure are diverse, for therapeutic proteins the most reported are poor therapeutic efficacy and immune responses, issues which are often encountered relatively late during the drug development workflow. Therefore, it would be of utmost utility to develop methods for detecting such susceptibility before significant effort and funds are spent in the development of less than ideal candidates. In this thesis, by integrating cell-free protein synthesis in small volumes together with split-intein mediated capture and light-triggered release, a streamlined platform for rapid protein production and screening was developed. Our results provide a proof-of-principle, with successful capture and release of protein of interest, as well as protein bioconjugation achieved using hCNTF as a model protein. The developed platform can be used for the rapid screening of therapeutic protein candidate producibility, and acts as a first module to be coupled to in-line assays for monitoring e.g. the candidate’s immunogenic potential, enabling such issues to be addressed/resolved already during early development stages.  Ensimmäisten proteiinilääkkeiden tultua markkinoille 1900-luvun loppupuolella ovat ne sittemmin ottaneet paikkansa keskeisenä lääkeaineluokkana ja niitä on kehitetty laajalti lääketieteen eri osa-alueille esim. niveltulehduksen, diabeteksen ja useiden eri syöpien sekä nyttemmin myös jopa astman ja migreenin lääkehoitoon. Tiettyjen verkkokalvon hermorappeumasairauksien kuten silmänpohjan ikärappeuman ja diabeettisen retinopatian hoito on viimeisen 15 vuoden aikana mullistunut verkkokalvon patologista uudelleensuonittumista estävien proteiinilääkkeiden tultua silmälääkäreiden käyttöön. Verkkokalvon sairaudet ovat maailmanlaajuisesti yksi merkittävimmistä näkövammaisuuteen ja sokeutumiseen johtavista syistä, ja yleistyessään väestön ikääntymisen myötä niiden hoitoon käytettävien proteiinilääkkeiden merkityksen ja tarpeen voidaan olettaa ainoastaan lisääntyvän. Varsinkin kehitystyön varhaisemmissa vaiheissa proteiinilääkkeiden tuottaminen riittävissä määrin voi olla merkittävä, ellei jopa suurin rajoite. Tässä väitöstutkimuksessa tehostimme hermosolujen toimintaa suojaavan ja siten verkkokalvon hermorappeumien hoidossa terapeuttisesti lupaavan proteiinin – ihmisen sädekehän hermokasvutekijän (human ciliary neurotrophic factor, hCNTF) – tuotantoa liukoisena rekombinanttiproteiinina Escherichia coli-bakteerisoluissa. rhCNTF:n geenin kodonioptimointia yhdistettynä mm. elatusliuosten, elatusolosuhteiden ja geneettisten fuusiopartnerien seulontaan käytettiin proteiinin ideaalisten tuotanto-olosuhteiden havaitsemiseksi. Määritetyissä optimiolosuhteissa tapahtuneen tuotannon jälkeen liukoisa rhCNTF puhdistettiin immobilisoitua metalli-ioni affiniteettikromatografiaa ja kokoerottelukromatografiaa käyttäen, ja puhdistetun proteiinin in vitro-aktiivisuus osoitettiin sitoutumiskokeessa CNTF:lle spesifin reseptorialayksikön, CNTFRα:n kanssa. Kaiken kaikkiaan, rhCNTF:n liukoisaa osuutta saatiin lisättyä 8–9 kertaisesti ja tuotetun proteiini määrää 10–20 kertaisesti verrattuna aiempiin julkaisuihin, joissa CNTF:n tuotettu määrä on ollut alhainen ja/tai on useasti vaatinut sen puhdistamista ei-liukoisista inkluusiojyväsistä. Lisäksi osoitimme, että käyttämämme tuotanto-olosuhteiden seulonta on strategiana rekombinanttiproteiinien liukoisan osuuden tuotannon lisäämiseksi käyttökelpoinen ja sovellettavissa myös muihin lääkinnällisesti potentiaalisiin proteiineihin. Valtaosa proteiineista on epästabiileja ja herkkiä ympäristönsä muutoksille, erityisesti itselleen ominaisten, biologisten ympäristöjen ulkopuolella. Proteiinilääkkeiden formuloinnilla pyritään suojaamaan proteiinien stabiiliutta ja biologista aktiivisuutta ennen kaikkea terapeuttisen tehon ja käytön turvallisuuden takaamiseksi koko lääkevalmisteen elinkaaren ajan. Tässä väitöstutkimuksessa jatkoimme puhdistetun rhCNTF:n kehittämistä karakterisoimalla ja formuloimalla proteiinia sekä tekemällä stabiiliuskokeita. Puhdistetun rhCNTF:n oikeanlainen laskostuminen osoitettiin sirkulaarista dikroismia hyödyntäen ja proteiinin biologinen aktiivisuus varmistettiin CNTFRα-reseptoria ilmentävän solulinjan proliferaation kautta. Seulonnan tuloksena valikoimme kaksi eri puskuria rhCNTF:n pitkäaikaista säilytystä varten. Vaikka havaitsimme vähäisiä muutoksia rhCNTF:n oligomeerisessa tilassa toisessa näistä puskureista, emme toisaalta havainneet muutoksia proteiinin lämpöstabiiliudessa kummassakaan puskurissa stabiiliuskokeiden aikana. Tulokset tarjoavat lähtökohdan rhCNTF:n formulaation jatkokehitykselle. Vaikka patologisen uudelleensuonittumista estävät proteiinilääkkeet ovat nousseet keskiöön tiettyjen verkkokalvon sairauksien hoidossa, millä tavoin, kuinka syvälle ja miten paljon nämä ja muut lasiaiseen annostellut proteiinilääkkeet permeoituvat verkkokalvoon tunnetaan edelleen huonosti. Tässä väitöstutkimuksessa selvitimme myös leima-aineella merkityn rhCNTF;n permeoitumista hermosolukerroksiin ex vivo verkkokalvoeksplanteissa. Tulosten perusteella ulkoperäisen, lasiaiseen annostellun CNTF:n permeoituminen verkkokalvoon ja sen CNTF-vasteisille soluille ei tulisi olla esteenä proteiinin hermosoluja suojaaville vaikutuksille silmässä. Lisäksi, toivomme näiden tulosten toimivan yleisemminkin sysäyksenä ex vivo-menetelmien käyttöönotossa proteiinilääkkeiden verkkokalvoon tunkeutumisen sekä niiden silmäfarmakokinetiikan selvittämisessä. Proteiinilääkkeet eivät ole pelkästään pysytelleet kärkisijoilla viimeaikaisissa tilastoissa parhaiten myyvistä lääkkeistä, vaan ne myös edustavat nykyään yli 40% osuutta kehitettävistä lääkkeistä. Tästä huolimatta myös proteiinilääkkeiden kehityksessä on ollut havaittavissa jatkuva kehityskulujen kasvu ja toisaalta yhä harvempien valmisteiden tulo markkinoille, mikä on kehityssuuntana kestämätön ja jonka katkaisemiseksi tarvitaan kipeästi ratkaisuja. Syyt kliinisten kokeiden epäonnistumiseen vaihtelevat; proteiinilääkkeiden kohdalla yleisimmin raportoituja epäonnistumisia ovat huono terapeuttinen teho ja lääkkeen aiheuttamat ja/tai niihin kohdistuvat immuunivasteet, jotka molemmat tulevat usein esiin vasta isommilla potilasaineistoilla ja suhteellisen myöhäisessä vaiheessa kliinisiä kokeita ja lääkekehitystä. Näistä syistä olisi ensiarvoisen hyödyllistä kehittää menetelmiä ja keinoja, joilla alttius tällaisiin epäonnistumisiin voitaisiin havaita jo aiemmissa vaiheissa ennen kuin aikaa ja resursseja on menetetty vähemmän ideaalisten proteiinilääkekandidaattien kehitykseen. Tässä väitöstutkimuksessa kehitimme alustan proteiinien nopeutettua tuotantoa ja seulontaa varten yhdistämällä 1) proteiinien pienessä tilavuudessa tapahtuvan soluvapaan tuotannon puhdistukseen, joka hyödyntää 2) inteiini-välitteistä kiinniottoa ja 3) valo-aktivoitavaa vapauttamista. Proteiinin puhdistus ja tuotetun proteiinin biokonjugoiminen onnistuivat hCNTF:ää malliproteiinina käyttäen, osoittaen konseptin toimivan. Kehittämäämme menetelmää voidaan hyödyntää mm. proteiinilääkekandidaattien tuotettavuuden nopeassa seulonnassa. Sellaisenaan kehittämämme menetelmä muodostaa ensimmäisen osan suunnitellusta alustasta, jonka avulla esim. proteiinilääkekandidaatin immunogeenisen potentiaalin tuotannonaikaisella tarkkailulla em. ongelmia voidaan huomioida/ratkaista jo tuotannon alkuvaiheissa

Assessment of recombinant protein production in E. coli with Time-Gated Surface Enhanced Raman Spectroscopy (TG-SERS)

Time-Gated Surface-Enhanced Raman spectroscopy (TG-SERS) was utilized to assess recombinant protein production in Escherichia coli. TG-SERS suppressed the fluorescence signal from the biomolecules in the bacteria and the culture media. Characteristic protein signatures at different time points of the cell cultivation were observed and compared to conventional continuous wave (CW)-Raman with SERS. TG-SERS can distinguish discrete features of proteins such as the secondary structures and is therefore indicative of folding or unfolding of the protein. A novel method utilizing nanofibrillar cellulose as a stabilizing agent for nanoparticles and bacterial cells was used for the first time in order to boost the Raman signal, while simultaneously suppressing background signals. We evaluated the expression of hCNTF, hHspA1, and hHsp27 in complex media using the batch fermentation mode. HCNTF was also cultivated using EnBase in a fed-batch like mode. HspA1 expressed poorly due to aggregation problems within the cell, while hCNTF expressed in batch mode was correctly folded and protein instabilities were identified in the EnBase cultivation. Time-gated Raman spectroscopy showed to be a powerful tool to evaluate protein production and correct folding within living E. coli cells during the cultivation.Peer reviewe

Accelerated pharmaceutical protein development with integrated cell free expression, purification, and bioconjugation

The use of living cells for the synthesis of pharmaceutical proteins, though state-of-the-art, is hindered by its lengthy process comprising of many steps that may affect the protein’s stability and activity. We aimed to integrate protein expression, purification, and bioconjugation in small volumes coupled with cell free protein synthesis for the target protein, ciliary neurotrophic factor. Split-intein mediated capture by use of capture peptides onto a solid surface was efficient at 89–93%. Proof-of-principle of light triggered release was compared to affinity chromatography (His6 fusion tag coupled with Ni-NTA). The latter was more efficient, but more time consuming. Light triggered release was clearly demonstrated. Moreover, we transferred biotin from the capture peptide to the target protein without further purification steps. Finally, the target protein was released in a buffer-volume and composition of our choice, omitting the need for protein concentration or changing the buffer. Split-intein mediated capture, protein trans splicing followed by light triggered release, and bioconjugation for proteins synthesized in cell free systems might be performed in an integrated workflow resulting in the fast production of the target protein.Peer reviewe

Analysis of Biologics Molecular Descriptors towards Predictive Modelling for Protein Drug Development Using Time-Gated Raman Spectroscopy

Pharmaceutical proteins, compared to small molecular weight drugs, are relatively fragile molecules, thus necessitating monitoring protein unfolding and aggregation during production and post-marketing. Currently, many analytical techniques take offline measurements, which cannot directly assess protein folding during production and unfolding during processing and storage. In addition, several orthogonal techniques are needed during production and market surveillance. In this study, we introduce the use of time-gated Raman spectroscopy to identify molecular descriptors of protein unfolding. Raman spectroscopy can measure the unfolding of proteins in-line and in real-time without labels. Using K-means clustering and PCA analysis, we could correlate local unfolding events with traditional analytical methods. This is the first step toward predictive modeling of unfolding events of proteins during production and storage

Codon optimization and factorial screening for enhanced soluble expression of human ciliary neurotrophic factor in Escherichia coli

Peer reviewe

Electron dispersive X-ray spectroscopy and degradation properties of hyaluronic acid decorated microparticles

The purpose of this study was to produce poly(DL-lactic-co-glycolic acid) (PLGA) – based microparticles (MPs), externally decorated with hyaluronic acid (HA). The MPs are intended for intravitreal injections in the treatment of posterior eye segment and have been designed to prolong the release of growth factors into the vitreous body, therefore aiming to increase the time interval between two consecutive injections. The MPs, prepared by a modified double emulsion-solvent evaporation technique and loaded with bovine serum albumins (BSA) and ciliary neurotrophic factor (CNTF), were spherical, with a diameter around 70 μm and a > 90% encapsulation efficiency. Energy Dispersive Spectroscopy (EDS) outcomes indicated that HA presence in the external aqueous phase of the emulsion did affect the surface properties of MPs. Moreover, poloxamers drastically slowed down MP degradation properties which are, in turn, closely related to their ability to prolong drug release. This is promising for the envisaged application of the produced MPs. Further work will be devoted to optimizing MP formulation with respect to the envisaged intravitreal route of administration.The purpose of this study was to produce poly(DL-lactic-co-glycolic acid) (PLGA) - based microparticles (MPs), externally decorated with hyaluronic acid (HA). The MPs are intended for intravitreal injections in the treatment of posterior eye segment and have been designed to prolong the release of growth factors into the vitreous body, therefore aiming to increase the time interval between two consecutive injections. The MPs, prepared by a modified double emulsion-solvent evaporation technique and loaded with bovine serum albumins (BSA) and ciliary neurotrophic factor (CNTF), were spherical, with a diameter around 70 mu m and a > 90% encapsulation efficiency. Energy Dispersive Spectroscopy (EDS) outcomes indicated that HA presence in the external aqueous phase of the emulsion did affect the surface properties of MPs. Moreover, poloxamers drastically slowed down MP degradation properties which are, in turn, closely related to their ability to prolong drug release. This is promising for the envisaged application of the produced MPs. Further work will be devoted to optimizing MP formulation with respect to the envisaged intravitreal route of administration.Peer reviewe

Characterization, Stability, and In Vivo Efficacy Studies of Recombinant Human CNTF and Its Permeation into the Neural Retina in Ex Vivo Organotypic Retinal Explant Culture Models

Ciliary neurotrophic factor (CNTF) is one of the most studied neuroprotective agents with acknowledged potential in treating diseases of the posterior eye segment. Although its efficacy and mechanisms of action in the retina have been studied extensively, it is still not comprehensively understood which retinal cells mediate the therapeutic effects of CNTF. As with therapeutic proteins in general, it is poorly elucidated whether exogenous CNTF administered into the vitreous can enter and distribute into the retina and hence reach potentially responsive target cells. Here, we have characterized our purified recombinant human CNTF (rhCNTF), studied the protein’s in vitro bioactivity in a cell-based assay, and evaluated the thermodynamic and oligomeric status of the protein during storage. Biological activity of rhCNTF was further evaluated in vivo in an animal model of retinal degeneration. The retinal penetration and distribution of rhCNTF after 24 h was studied utilizing two ex vivo retina models. Based on our characterization findings, our rhCNTF is correctly folded and biologically active. Moreover, based on initial screening and subsequent follow-up, we identified two buffers in which rhCNTF retains its stability during storage. Whereas rhCNTF did not show photoreceptor preservative effect or improve the function of photoreceptors in vivo, this could possibly be due to the used disease model or the short duration of action with a single intravitreal injection of rhCNTF. On the other hand, the lack of in vivo efficacy was shown to not be due to distribution limitations; permeation into the retina was observed in both retinal explant models as in 24 h rhCNTF penetrated the inner limiting membrane, and being mostly observed in the ganglion cell layer, distributed to different layers of the neural retina. As rhCNTF can reach deeper retinal layers, in general, having direct effects on resident CNTF-responsive target cells is plausible

Do we eat what we buy? : Relative validity of grocery purchase data as an indicator of food consumption in the LoCard study

The validity of grocery purchase data as an indicator of food consumption is uncertain. This paper investigated 1) the associations between food consumption and grocery purchases using automatically accumulated purchase data, and 2) whether the strength of the associations differed in certain sub-populations. The participants filled in a food frequency questionnaire (FFQ), and a major Finnish retailer issued us with their loyalty-card holders grocery purchase data covering the 1- and 12-month periods preceding the FFQ. We used gamma statistics to study the association between thirds/quarters of FFQ and grocery purchase data (frequency/amount) separately for 18 food groups among the 11,983 participants. Stratified analyses were conducted for subgroups based on gender, family structure, educational level, household income and self-estimated share of purchases from the retailer. We also examined the proportion of participants classified into the same, adjacent, subsequent and opposite categories using the FFQ and purchase data. The gammas ranged from 0.12 (cooked vegetables) to 0.75 (margarines). Single households had stronger gammas than two-adult families, and participants with >60% of purchases from the retailer had stronger gammas. For most food groups, the proportion of participants classified into the same or adjacent category was >70%. Most discrepancies were observed for fresh/cooked vegetables, berries, and vegetable oils. Even though the two methods did not categorize all food groups similarly, we conclude that grocery purchase data are able to describe food consumption in an adult population, and future studies should consider purchase data as a resource-saving and moderately valid measure in large samples.acceptedVersionPeer reviewe