51 research outputs found
Network Topologies and Dynamics Leading to Endotoxin Tolerance and Priming in Innate Immune Cells
The innate immune system, acting as the first line of host defense, senses
and adapts to foreign challenges through complex intracellular and
intercellular signaling networks. Endotoxin tolerance and priming elicited by
macrophages are classic examples of the complex adaptation of innate immune
cells. Upon repetitive exposures to different doses of bacterial endotoxin
(lipopolysaccharide) or other stimulants, macrophages show either suppressed or
augmented inflammatory responses compared to a single exposure to the
stimulant. Endotoxin tolerance and priming are critically involved in both
immune homeostasis and the pathogenesis of diverse inflammatory diseases.
However, the underlying molecular mechanisms are not well understood. By means
of a computational search through the parameter space of a coarse-grained
three-node network with a two-stage Metropolis sampling approach, we enumerated
all the network topologies that can generate priming or tolerance. We
discovered three major mechanisms for priming (pathway synergy, suppressor
deactivation, activator induction) and one for tolerance (inhibitor
persistence). These results not only explain existing experimental
observations, but also reveal intriguing test scenarios for future experimental
studies to clarify mechanisms of endotoxin priming and tolerance.Comment: 15 pages, 8 figures, submitte
Examining the Acute and Chronic Effects of Sepsis on the Circadian Clock in the Mouse
Circadian rhythms are recurring patterns (~24hrs) in behaviour and physiology that are driven primarily by an endogenous biological timekeeping system, with the master pacemaker located in the suprachiasmatic nucleus. Studies have indicated bidirectional relationships between the circadian and the immune systems, however while there is much evidence regarding the regulation of immune function by the circadian system, information regarding the impact of immune processes on the timekeeping system is more limited, including that regarding the long-term modulation of the circadian system following immune challenge. The current set of studies address this gap in the literature by examining the long-term impact of sepsis, a substantial immune challenge, on circadian timekeeping processes, following sepsis induction by peripheral treatment with lipopolysaccharide (5mg/kg). Following recovery, post-septic circadian behaviour, SCN molecular oscillations and SCN responsiveness were assessed. SCN neurochemistry was also assessed both in the acute phase and in the long-term post LPS treatment.
LPS induced sepsis did not affect core circadian locomotor rhythmicity parameters, but did result in long-term attenuations in post-septic resetting in response to phase advancing photic stimulation, and alterations in re-entrainment to advances of the photoperiod. Perturbations were observed in SCN neurochemistry in the acute phase following septic LPS treatment, and chronic attenuations were also found in post-septic SCN clock gene protein product expression. LPS induced sepsis caused attenuations in SCN functional activation in response to both photic and immune stimulation, as well as alterations in circadian resetting in response to phase resetting immune stimuli.
Overall, these data provide further insight into immune circadian communication, and the long-term impact of immune challenge on timekeeping processes, and describe a previously unknown impact of the chronic effects of experimental sepsis on the circadian timekeeping system
Effects of chronic LPS stimulation on the response of macrophages to subsequent stimuli
Wound healing constitutes a brief inflammatory phase, followed by proliferative phase and ends with a longer period of tissue remodeling. Post tissue injury, macrophages initiate an inflammatory cascade to propagate wound healing. Macrophages initially adapt a pro-‐ inflammatory "M1" activation state, followed an anti-‐inflammatory "M2" macrophages that are associated with the resolution of initial inflammation. The M1-‐to-‐M2 transition has been shown to be crucial to facilitate healing. Impairment of this phenotypic switch is associated with chronic inflammation. Chronic inflammation is characterized by a sustained M1. Noteworthy, patients suffering from chronic inflammatory conditions have a systemically higher that normal LPS levels, which is thought to trigger chronic and systemic pro-‐inflammatory activation of macrophages. Surprisingly, while normal wounds posses an initial and robust inflammatory reaction, recent studies have highlighted that chronic wounds suffer lower than normal initial inflammatory state in response to otherwise potent pro-‐inflammatory stimulation. The goal of this work is to study the impact of chronic LPS stimulation on macrophages pro-‐inflammatory reaction to a fresh LPS treatment and its subsequent capacity to respond to IL4/IL13, "M2" promoting cytokines. This work highlighted that chronic LPS stimulation rendered macrophages hypo-‐responsive to fresh LPS stimulation. However, it did not impact their "M2" switching capacity. IFNg, a pro-‐ inflammatory cytokine, treatment of chronic "M1" macrophages did not improve their responsiveness to LPS.M.S., Biomedical Engineering -- Drexel University, 201
Novel therapeutic targets in uveitis
Autoimmune posterior uveitis is a potentially blinding ocular disorder characterized by inflammation of the choroid and retina. Etiology is unknown and is assumed to be autoimmune and proposed to involve activation of autoreactive retinal-peptide specific T-cells, blood-retinal barrier breakdown, further leukocyte recruitment and ensuing ocular inflammation. The P2X7 receptor is a transmembrane purinergic receptor activated by high concentrations of ATP. Expression is ubiquitous with highest expression in immune cells. Stimulation results in the production and release of pro-inflammatory cytokines and potentiates leukocyte recruitment. Differences in mechanisms of P2X7 regulation in macrophages, dendritic cells and T cells were explored in vitro. Dendritic cells and macrophages have been proposed to release IL-1β through the NLRP3 inflammasome, requiring TLR4 stimulation followed by P2X7 stimulation. Dendritic cells were found to release IL-1β with TLR4 stimulation only, unlike macrophages which required additional P2X7 stimulus. Potential mechanisms of P2X7 regulation were explored, and it was found that T cells and not macrophages or dendritic cells exhibited significantly potentiated P2X7 mediated dye uptake upon lipid raft disruption. These results suggested a role for lipid rafts in P2X7 regulation in T cells. In vivo experiments utilized animal models of anterior, posterior and pan-uveitis. Mice deficient in P2X7 were protected against developing severe posterior uveitis, and treatment of mice with established EAU with P2X7 specific antagonist A438079 prevented the development of severe panuveitis. Finally, the role of the spleen tyrosine kinase (SYK) was investigated in murine posterior uveitis. Recent research implicates potential crossover of the P2X7 and SYK signalling pathways. SYK inhibition with the specific inhibitor fostamatinib did not prevent the development of uveitis in mice.Open Acces
P2X7 Activation of Non-Primed Myeloid Cells Promotes the Shedding of Stimulatory Materials Within Microvesicles
There is an increasing need to understand how inflammation is initiated by endogenous factors in the absence of infection. Various diseases such as atherosclerosis and arthritis are shaped by endogenous mediators. In situations such as transplant or trauma where there is extensive amount of tissue damage, endogenous factors can be released to influence inflammation. The modes of activation in which immune cells liberate endogenous factors for incurring immune responses remain elusive.Adenosine triphosphate (ATP) activation of the puringeric receptor P2X7 has been implicated in several immune responses. P2X7 promotes the shedding of microvesicles (MV) and the secretion of inflammatory mediators. I hypothesized that P2X7-induced MV containing some of these inflammatory mediators would promote the activation of innate immune cells such as macrophages.Using murine bone marrow derived macrophages as a model for macrophage function, I describe that harvested P2X7-induced MV from myeloid cells promote macrophage activation including pro-inflammatory cytokine secretion and co-stimulatory ligand upregulation. Phospholipids from P2X7-induced MV are partially responsible for the observed macrophage activation. Isolated phospholipids from P2X7-induced MV activate TLR4. Secondly, I describe mature cathepsin D release into P2X7-induced MV from myeloid cells.P2X7-induced MV from myeloid cells contain both intermediate and mature forms of cathepsin D. Furthermore, P2X7 stimulation of myeloid cells promotes the peripheral displacement of cathepsin D and dynamin. Dynasore, a selective and potent dynamin inhibitor, significantly reduced the secretion of mature but not intermediate cathepsin D.Lastly, I describe a novel morphological alteration following P2X7 activation of myeloid cells. ATP stimulates de novo filopodia production. These filopodia are the result of actin polymerization, Rho kinases, and phospholipases. Furthermore, P2X7 promotes the re-localization of lipids and actin-based machinery to the periphery of ATP treated cells.Collectively, these results demonstrate that P2X7-induced MV possess stimulatory cargo including phospholipids that can activate macrophages and cathepsins that are potentially capable of degrading extracellular matrix components. This data would suggest a provocative role for P2X7-induced MV and actin-based processes in promoting sterile disease
A role for a leaky gut and the intestinal microbiota in the pathophysiology of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)
The field of ME/CFS research is challenged by many often confusing and conflicting reports
of immune, neuroendocrine, autonomic, neurological dysfunction. During the prodromal
phase of this condition patients often report flu-like symptoms, persistent chronic fatigue
and gastro-intestinal symptoms including abdominal pain and discomfort.
Its study is complicated by the lack of specific biomarkers and criteria to accurately define
the illness, relying on the exclusion of other fatiguing illnesses. Recent publications suggest
an altered intestinal microbiota and increased intestinal permeability are associated with
ME/CFS. Further evidence is accumulating for dysfunctional energy, lipid and amino acid
metabolism that may indicate oxidative stress and/or immune-mediated damage to
mitochondria, disrupting the efficiency of aerobic respiration, explaining the effect of postexternal
malaise (PEM), a unique characteristic for the diagnosis of ME.
In this study, Next Generation Sequencing (NGS) and Nuclear Magnetic Resonance (NMR)
spectroscopy probed the composition of the intestinal microbiota and faecal and serum
microbiomes in 17 severe, house-bound patients and house-hold healthy controls (HHC).
Severe, house-bound patients account for 0.5% of all ME/CFS research, yet it is estimated
they represent 25% of the patient population. We found Faecalibacterium prausnitzii was
significantly reduced in severe patients (p = 0.018) but did not replicate individual
differences in faecal and serum metabolites that others have previously reported. We
further enhanced a flow cytometry technique for detecting IgA coated bacteria in faecal
suspensions and analysed the proportional differences between patients and HHCs. This
demonstrated a trend for increased IgA-coated bacteria in most patients; however, this
trend was reversed when repeated with a second sample produced a year on.
Since the initial concept for this study, several advances have been made in sequencing
methods and quality control standards for metagenomic and metabolomic studies. Based
on these, we conclude further investigations are warranted using whole genome
sequencing and targeted metabolomics to address the emerging hypotheses in ME/CFS
research, with an emphasis on the study of severe, house-bound ME patients
TMEM203 is a putative co-receptor of innate immune adaptor protein STING
Acute inflammation is the innate immune defence against environmental disturbances. Macrophages are one of the central immune cells that react to infections and maintain tissue homeostasis, and they exhibit their functions via numerous inflammatory signalling regulators. In addition to previously identified immune mediators, novel proteins involved in inflammation continue to emerge.
A previous cDNA functional screening in murine macrophages has identified a novel protein named transmembrane protein 203 (Tmem203) displaying pro-inflammatory characteristics. Tmem203-promoted inflammatory activities were found to be TLR independent but dependent on STING, a cytosolic innate immune adaptor for DNA detection. STING responds to upstream DNA sensors and microbial cyclic dinucleotides, and instigates type I interferon response via TBK1-IRF3 axis.
The work in this thesis investigated the function of TMEM203 in STING-dependent type I interferon responses. TMEM203 has been found to colocalise, interact and migrate with STING. Further studies revealed a critical role for TMEM203 in STING-dependent type I interferon response in both human and mouse primary macrophages. We showed that TMEM203-STING association was highly dependent on STING’s N-terminal transmembrane domains. Finally, TMEM203 showed a distinct regulation of STING-interferon signalling between stimulation by natural and synthetic STING ligands, and this difference was also reflected in TMEM203-STING interaction. Thus, this novel mechanism of TMEM203-dependent STING regulation has brought new insights to better understand critical regulators of pathogen infections and interferon-associated autoimmune diseases.
Additionally, a brief research was conducted to explore STING regulation in flavivirus infected primary macrophages. Flaviviruses Dengue virus and Zika virus infect humans to cause global pandemics. Dengue virus is known to specifically and potently interrupt STING-interferon pathway. The emerging flavivirus Zika virus is genetically-closely related to Dengue virus and thus it has been hypothesised to adopt similar strategies in STING antagonism. We have investigated Dengue and Zika virus-induced type I interferon stimulated ISG response in the M-CSF differentiated primary macrophage model, and tested the role of STING in such conditions. Contradictory to previous report, our experiments showed a potent and persistent ISG induction in virus-infected macrophages. Prior virus infections were unable to intercept ISG induction cause by STING ligands, whereas the downregulation of STING dampens virus-induced ISG response. Therefore, this primary macrophage model highlights alternative regulatory mechanisms via STING in response to Dengue and Zika virus
Monoliths for purification of a DNA vaccine against Influenza
O vírus influenza é responsável por uma infeção muito contagiosa que afeta o sistema
respiratório, convencionalmente designada por Gripe. Este vírus é constituído por uma cadeia
simples de RNA “negative sense” pertencente à família Orthomyxoviridae. O vírus influenza
apresenta vários antigénios de superfície como a hemaglutinina (HA) e a neuraminidase (NA).
A HA é o glicopéptido responsável pela entrada do vírus nas células do hospedeiro e tem
correlação direta com a proteção contra o vírus, uma vez que induz a produção de anticorpos
neutralizantes. Vários estudos têm demonstrado que o insert HA contido nas vacinas de DNA
usadas esperimentalmente permitem conferir protecção cruzada contra várias estirpes do vírus,
o que justifica a sua escolha para este projecto. Por outro lado, a NA é responsável pela saída
do vírus da célula infetada permitimdo a sua propagação. É de referir ainda a proteína M2, um
canal proteico transmembranar responsável pela replicação do vírus.
Segundo dados da organização mundial de saúde em dezembro de 2015, as infeções
respiratórias são mais frequentemente causadas por influenza do tipo A e B, sendo que a
elevada propagação do vírus pelos vários continentes contribui para a ocorrência de mutações
e aparecimento de novas estirpes. Assim, todos os anos vários milhares de pessoas são
hospitalizadas devido a complicações da gripe, havendo uma taxa de mortalidade considerável,
em especial em indivíduos com idade superior a 65 anos e com patologias preexistentes. Deste
modo, a prevenção passa pelo reforço anual do sistema imunitário o que é conseguido de forma
mais rápida e efetiva mediante a aplicação de vacinas. O objetivo da vacinação é controlar ou
diminuir os sinais de infeção e é especialmente importante para indivíduos de elevado risco.
As vacinas convencionais contra influenza são preparadas sazonalmente e são específicas para
uma determinada estirpe, ou seja, a imunidade gerada não é particularmente ampla e a sua
eficácia depende do grau de correspondência entre a vacina e a estirpe circulante. Como
alternativa, a utilização do DNA plasmídico (pDNA) como uma vacina não viral tem-se tornado
numa potencial estratégia terapêutica para prevenir ou tratar determinadas doenças de forma
menos invasiva e segura comparando com os vetores virais.
A vacinação de DNA consiste na injecção de DNA bacteriano geneticamente modificado com o
gene de interesse que expressa proteínas antigénicas responsáveis por desencadear uma
resposta imunológica, evitando a progressão da doença. Estas vacinas oferecem uma série de
vantagens quando comparadas às vacinas clássicas, em termos económicos e técnicos. O
controlo de qualidade é mais fácil e a comercialização não necessita de uma rede de
refrigeração, pois estas vacinas são estáveis à temperatura ambiente e podem ser liofilizadas.
Além disso, as vacinas de DNA induzem uma resposta imunitária celular através da produção
de linfócitos T auxiliares (CD4+) e linfócitos T citotóxicos (CD8+) e também uma resposta
humoral por produção de linfócitos B.
O pDNA usado na produção de vacinas deve conter vários elementos funcionais para garantir a
sua propagação (origem de replicação procariótica) e seleção em microrganismo bacterianos (marcador de seleção, como por exemplo a resistência a um antibiótico). Adicionalmente, deve
apresentar elementos responsáveis por uma elevada expressão nos hospedeiros eucarióticos
(promotor de expressão eurariótica, sinal de poliadenilação, sítio de múltipla clonagem) e
ativação do sistema imunitário (gene de interesse). O pDNA para fins terapêuticos deve estar
em conformidade com as especificações das agências reguladoras, seguindo rigorosos critérios
de qualidade em termos de uso de reagentes tóxicos ou de origem animal, bem como de pureza
relativamente aos restantes constituintes dos lisados celulares. Contudo, as semelhanças
existentes entre o pDNA e os seus contaminantes comuns, como proteínas, endotoxinas, DNA
genómico e RNA, podem complicar a sua separação, pelo que o processo de purificação deverá
ser extremamente eficiente. Portanto a preparação destas vacinas de DNA requer o
desenvolvimento de processos de produção e purificação que permitam obter grandes
quantidades de plasmídeo na sua forma mais compacta e biologicamente ativa (isoforma
superenrolada (sc)).
Hoje em dia qualquer processo para obtenção de vacinas de DNA requer a utilização de métodos
cromatográficos para obtenção do plasmídeo sc na sua forma mais pura. A cromatografia é uma
técnica quantitativa que tem por finalidade geral a identificação e a separação dos
componentes de uma mistura. Os compostos presentes são distribuídos entre uma fase
estacionária e uma fase móvel e a separação ocorre porque os compostos têm diferentes
afinidades com a fase estacionária e com a fase móvel, portanto deslocam-se com diferentes
velocidades. Os métodos cromatográficos exploram propriedades do pDNA como tamanho,
hidrofobicidade, carga e a afinidade das suas bases para os ligandos, de modo a promover a
separação das impurezas com a maior eficiência possível. A flexibilidade deste tipo de processo
advém da grande variedade de suportes e ligandos que podem ser usados, tendo em conta as
características da molécula a separar.
Em relação aos suportes cromatográficas existentes, alguns problemas ainda precisam de ser
ultrapassados, como a baixa capacidade de ligação dos suportes, o tempo do processo de
purificação das biomoléculas e os rendimentos de recuperação. Apesar dos bons resultados
obtidos com as matrizes convencionais há necessidade de utilizar novas matrizes com melhor
performance. Os monolitos poderão ser uma alternativas às matrizes convencionais uma vez
que mostram várias vantagens em relação a estas, nomeadamente: elevada capacidade de
ligação devido às excelentes propriedades de transferência de massa; separação mais rápida o
que leva a uma baixa degradação; resolução independente do caudal; fácil manuseamento e
elevada reprodutibilidade. Desta forma, o trabalho apresentado nesta tese consistiu na
utilização de colunas monolíticas com a finalidade de purificar a isoforma sc da vacina de DNA
NTC 7482-41H-VA2 HA com 6.471kbp que contém o codão de iniciação e sequência necessária
para expressar a HA do vírus influenza.
Numa fase inicial, foram realizados vários ensaios com plasmídeos modelo de diferentes
tamanhos (até 14 kbp) utilizando o monolito CarbonylDiImidazole (CDI) não derivatizado. Este
ligando foi escolhido pelas suas semelhanças com a coluna histidina agarose previamente
utilizada com sucesso na purificação de um plasmideo modelo. Plasmídeos maiores necessitaram de menor concentração de sal (sulfato de amónio) para ligar ao monolito devido
ao maior número de interações entre estas biomoléculas e o monolito. A caracterização do
suporte foi possível através da determinação da capacidade dinámica de ligação (DBC) por
variação de parâmetros como o tamanho do plasmídeo, força iónica, caudal e o pH da fase
móvel. Verificou-se que quanto maior concentração de sal usada, menor a repulsão
electrostática dos grupos fosfato no pDNA, o que torna estas moléculas mais compactas
aumentando a DBC. Por outro lado, observou-se que para o mesmo caudal, plasmídeos maiores
possuem menor DBC, uma vez que ocupam maior área de superfície dentro dos canais do
monolito. Por fim, constatou-se que a diminuição do pH também tem uma influência positiva
na DBC.
No seguimento do trabalho, foi também testado um supporte monolítico imobilizado com
agmatina para a purificação da vacina de pDNA contra o vírus influenza. A agmatina é
naturalmente sintetizada a partir do aminoácido arginina na forma descarboxilada, e é
conhecida pela sua intervenção em inúmeros processos biológicos. As semelhanças estruturais
da agmatina com o seu precursor arginina, previamente utilizado como ligando numa coluna de
agarose para purificar sc pDNA, foram a razão de escolha para o propósito aqui referido. A
agmatina foi pela primeira vez usada como ligando em processos cromatográficos, verificandose
que possui a particularidade de funcionar sob dois modos de interação, o que permitiu a
aplicação de duas estratégias de purificação: gradiente por diminuição da concentração de
sulfato de amónio ou por aumento da concentração de cloreto de sódio. Os melhores resultados
foram obtidos com a estratégia que usa sulfato de amónio, atingindo-se um grau de pureza de
98 % e um rendimento de 51,8 %. Adicionalmente, o método apresentou uma elevada DBC (5.656
mg/mL) e uma redução significativa das impurezas do hospedeiro quando comparadas com o
lisado injectado no monolito. A eficiência de transfeção nos fibroblastos transfectados com o
pDNA sc purificado com esta estratégia foi de 73 %.
Por último, a implementação de uma abordagem baseada em cromatografia de troca iónica
utilizando o monolito com o ligando etilenodiamina (EDA) para a purificação do pDNA sc com
expressão do gene HA do vírus influenza foi também bem sucedida. Este último ligando funciona
por troca aniónica e possui semelhanças estruturais com o ligando DEAE, previamente utilizado
como método analítico para quantificação de impurezas e da isoforma sc em amostras de pDNA.
O processo permitiu uma redução significativa das impurezas provenientes do hospedeiro E.
coli obtendo-se um grau de pureza de 97,1% e um redimento de 47%. Após a verificação dos
padrões de qualidade do pDNA sc purificado, foram realizados ensaios in vitro com 2 tipos de
células (A549 e CHO). A expressão da HA determinada por imunofluorescência permitiu verificar
uma maior eficiência de transfecção nas células CHO com cerca de 70,6 %, enquanto as células
A549 apenas registaram uma eficiência de transfecção de 61,4 %. De entre todos os métodos
cromatográficos utilizados, o EDA permitiu obter um processo mais rápido e com menor impacto
económico.
No global, este projeto de doutoramento demonstrou que os monolitos possuem um grande
potencial de aplicação em processos cromatográficos para a purificação de vacinas de DNA obtidas a partir de lisados complexos de E. coli seguindo as exigências das agências reguladoras.
Apesar de os monolitos utilizados com os ligandos acima referidos apresentarem valores de
rendimento e purificação relativamente semelhantes às matrizes particuladas convencionais, a
elevada capacidade dos monolitos permite uma maior rapidez e eficiência do processo de
purificação.Influenza viruses (of the Orthomyx-oviridae family) are enveloped, negative-stranded, RNA
viruses with segmented genomes responsible for a significant human respiratory disease named
flu. Researchers have made efforts to fight this contagious disease that still shows high levels
of morbidity and mortality. The best option for reducing the impact of this viral infection is
through vaccination. Even though traditional influenza vaccines are safe and usually effective,
they only provide protection against the dominant strains of a given year, and thus need to be
annually updated. This limitation, together with the use of embryonated chicken eggs as the
substrate for vaccine production, is time-consuming and could involve potential biohazards in
the growth of new strains of the virus. In the last years, the expansion of efficient plasmid DNA
purification processes has fostered new therapeutic applications, concretely gene therapy and
DNA vaccination. The latter is a promising alternative to conventional vaccination since it
induces all three arms of adaptative immunity (antibodies, helper T cells, cytolytic Tlymphocytes),
providing cross-strain protection. Moreover, DNA vaccines need to be obtained
in high quantities, can be easily stored and the production process is generic, in contrast to the
complicated process needed for conventional vaccines. However, there are still bottlenecks in
the large scale manufacturing of this and other DNA pharmaceuticals, mainly at downstream
processing level. It is known that a large quantity of the highly pure biologically active
supercoiled (sc) plasmid DNA (pDNA) with pharmaceutical grade is necessary to implement this
technology. For that purpose, the application of chromatographic operations has demonstrated
good results since there are simple, robust, versatile and high reproducible. However, there
are still some bottlenecks associated with conventional matrices namely their low binding
capacity and diffusivity for pDNA samples. Owing to these limitations, monolithic supports have
emerged as interesting alternatives due to the versatility of their structural characteristics,
high binding capacity and the excellent transfer mass properties. Thus, in the present project
it is proposed the production and purification of pDNA NTC 7482-41H-VA2 HA with 6.471kbp
expressing the influenza virus protein hemagglutinin (HA) with new and more efficient
processes based on monolithic supports.
With these in mind, the non-grafted CarbonylDiImidazole (CDI) monolithic column was explored
in order to study the interaction behavior of different plasmids with different sizes. The
biorecognition of the intact and undamaged plasmid form, the supercoiled (sc) isoform, was
studied on each plasmid with different sizes (at least up to 14 kbp). The characterization of
the monolithic support was also evaluated by dynamic binding capacity (DBC) manipulating
different salt concentration, flow rates and pH values. The strategy applied in this work showed
that the isoforms of plasmids with different sizes can be separated using the CDI monolithic
disk. These results showed that it was possible to have a selective separation of the sc isoforms
of different plasmids. Higher size plasmids needed a lower ammonium sulphate concentration
to bind to the monolith. On the other hand, capacity studies proved that at the same flow rate, the largest plasmid seems to have a lower capacity value whereas the smallest plasmid showed
the best capacity value. In addition, high salt concentrations increase the DBC. Finally, the
effect of diminishing the pH also had a positive consequence in the breakthrough experiments.
These results were useful for the implementation of a new chromatographic strategy based on
monolithic supports.
An agmatine monolithic disk was also tested in the purification of the sc isoform of a pDNAbased
vaccine against influenza. This was the first application of agmatine (decarboxylated
arginine) as ligand in a chromatographic process. Due to the role of this molecule in biological
processes different interactions between the ligand and other biomolecules of interest can be
exploited. Accordingly, two different purification strategies were used by applying either a
descending ammonium sulphate or an ascending sodium chloride elution gradient. The best
strategy was obtained using ammonium sulphate with a purification degree over 98 % and a
recovery yield of 51.8 %. Moreover, this method presented a high binding capacity (5.656
mg/mL) and all the host impurities were significantly reduced or undetectable when compared
with the injected lysate. Furthermore, the transfection efficiency of fibroblast cells using the
sc pDNA purified with this strategy was also high, reaching an efficiency of 73%. All these results
proved that this agmatine-functionalized monolith is a versatile column for pDNA purification.
Additionally, an ion exchange interaction chromatography approach using an ethylenediamine
(EDA) monolith for the purification of a sc pDNA expressing the influenza virus HA protein was
successful. The applied process exhibited a significant reduction of the E. coli host impurities
while achieving a sc pDNA purity degree of 97.1 % and a step yield of 47 %. Finally in vitro
experiments using A549 and CHO cells were performed. A strong intracellular fluorescence was
observed in the transfected CHO cells with the purified sc isoform, presenting 70.6% of
transfection while A549 cells showed a weaker signal for a transfection efficiency of 61.4%. The
HA expression was recognized by a mouse monoclonal antibody directed to the HA protein with
high viability and high transfection efficiency.
Overall, this doctoral research work revealed that monoliths have the potential to be further
applied in chromatographic processes for purification of a pDNA influenza vaccine from complex
lysates under the requirements of the regulatory agencies
THE MECHANISM OF CYTOKINE SYNERGY INDUCED BY COMBINATORIAL TLR ACTIVATION
Ph.DDOCTOR OF PHILOSOPH
- …