997 research outputs found
Auto-fluorescent intracellular sink - A novel Inherent biomarker for drug discovery in pancreatic cancer stem cells
Tesis doctoral inédita, leída en Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 20/09/2013.Pancreatic
adenocarcinoma
(PDAC),
the
fourth
leading
cause
of
cancer-‐
related
death
world-‐wide,
is
a
malignant
neoplasm
of
the
exocrine
pancreas.
It
has
been
hypothesized
that
a
subset
of
tumor
cells
with
stem-‐like
properties,
termed
cancer
stem
cells
(CSCs),
drives
pancreatic
tumor
growth,
metastasis,
and
chemoresistance.
While
multiple
surface
markers
such
as
CD133
and
CD44
have
been
successfully
used
to
isolate
and
characterize
CSCs,
their
expression
are
not
exclusively
linked
to
a
CSC
functional
phenotype.
Therefore,
since
isolating
and
characterizing
CSCs
is
of
paramount
importance
for
understanding
pancreatic
cancer,
we
sought
to
identify
new
and
novel
markers
that
functionally
enrich
for
CSCs
using
primary
human
pancreatic
cancer
cells.
While
our
classic
approaches,
such
as
cell
surface
expression
of
known
CSC
markers
or
side
population
were
either
prone
to
alterations
by
the
tissue
culture
environment
or
did
not
enrich
for
CSCs,
we
inadvertently
identified
a
distinct
population
of
cells
characterized
by
a
subcellular
compartment
exhibiting
strong
autofluorescence,
which
did
exhibit
defined
CSC
characteristics.
Specifically,
these
autofluorescent
cells
were
markedly
enriched
both
in
sphere
culture
and
during
chemotherapy,
strongly
expressed
pluripotency-‐associated
genes,
and
were
highly
invasive
both
in
vitro
and
in
vivo.
Most
importantly,
they
were
exclusively
tumorigenic
in
vivo
at
the
single
cell
level
and
were
capable
of
recapitulating
the
heterogeneity
of
the
parental
tumor.
Autofluorescence
was
determined
to
be
due
to
an
accumulation
of
riboflavin
in
membrane-‐restricted
cytoplasmic
structures
by
means
of
the
ATP-‐dependent
ABCG2
transporter,
which
did
not
overlap
with
cells
defined
as
the
side
population,
but
could
be
selectively
eliminated
with
the
ABCG2
inhibitor
Fumitremorgin
C.
In
addition,
we
show
that
autofluorescence
is
not
restricted
to
PDAC,
as
other
solid
tumors
similarly
contain
autofluorescent
cells.
Thus,
to
take
advantage
of
this
broad
phenotype,
we
developed
a
low
throughput
screening
platform,
and
show
that
autofluorescent
cells
are
highly
amendable
to
anti-‐cancer
compound
screening.
Thus,
unbiased
and
label-‐free
tracking
of
autofluorescent
cells
in
cancers
such
as
PDAC
represents
a
promising
new
technological
advancement,
which
can
be
used
not
only
for
isolating
and
studying
CSC,
but
can
be
additionally
exploited
for
low
throughput
screening
of
compounds
with
anti-‐cancer
activity
in
a
clinical
setting.El
adenocarcinoma
pancreático
es
una
neoplasia
maligna
del
páncreas
exocrino,
siendo
la
cuarta
causa
principal
de
muerte
relacionada
con
cáncer
en
todo
el
mundo.
Se
ha
planteado
la
hipótesis
de
que
un
subconjunto
de
células
tumorales
con
propiedades
troncales,
denominadas
células
madre
de
cáncer,
impulsan
el
crecimiento
del
tumor
de
páncreas,
metástasis,
y
la
quimiorresistencia.
Mientras
que
varios
marcadores
de
superficie
tales
como
CD133
y
CD44
se
han
utilizado
con
éxito
para
aislar
y
caracterizar
este
tipo
de
células,
su
expresión
no
está
vinculada
exclusivamente
a
un
fenotipo
funcional
de
células
madre
de
cáncer.
Por
tanto,
ya
que
el
aislamiento
y
caracterización
de
este
tipo
de
células
es
de
vital
importancia,
hemos
tratado
de
identificar
marcadores
novedosos
que
de
manera
funcional,
enriquezan
en
este
tipo
celular
utilizando
células
primarias
de
cáncer
de
páncreas
de
humanos.
Los
resultados
premilinares
de
la
búsqueda
de
un
buen
marcador
de
células
madre
de
cáncer,
tales
como
la
expresión
de
marcadores
de
superficie
o
las
células
Side-‐Population
(SP),
observamos
que
podían
estar
sujetos
a
alteraciones
por
el
cultivo
de
tejidos
o
que
no
enriquencían
de
manera
especifíca
en
células
madre.
Esto
nos
forzó
a
la
búsqueda
de
otro
marcador
que
no
fuse
sensible
a
este
tipo
de
alteraciones
en
su
expresión,
siendo
éste
nuestro
principial
objetivo
de
este
trabajo.
Partiendo
de
cultivos
primarios
de
xenoinjertos
humanos,
identificamos
una
población
de
células
que
se
caracteriza
por
un
compartimento
subcelular
exhibiendo
una
fuerte
autofluorescencia.
Específicamente,
estas
células
autofluorescentes
fueron
enriquecidas
tanto
en
cultivo
de
esferas
como
durante
la
quimioterapia,
sobre-‐expresan
los
genes
de
pluripotencia-‐asociados
y
mostraron
ser
altamente
invasivas,
tanto
in
vitro
como
in
vivo.
Además,
estas
celulas
son
exclusivamente
tumorigénicas
in
vivo,
siendo
capaces
de
formar
un
tumor
desde
tan
sólo
una
célula.
Se
determinó
que
la
autofluorescencia
era
una
acumulación
de
riboflavina
en
la
estructura
autofluorescente
y
estaba
mediado
por
el
transportador
ABCG2.
Debido
a
que
se
expresan
en
una
gran
parte
de
tumores
primarios
de
páncreas
y
su
expresión
se
mantiene
constante
bajo
diferentes
condiciones,
desarrollamos
una
plataforma
de
screening
donde
podemos
investigar
el
efecto
de
distintos
compuestos
anticancerígenos,
así
como
obtener
un
diagnóstico
clinico
para
medicina
personalizada
Development of a purification process for HIV-1 VLPs, from supernatant to lyophilization
HIV-1-based virus-like particles (VLPs) have high potential as scaffold for the development of chimeric or multivalent vaccines by functionalizing them with specific antigens [1], [2]. The obtention of vaccine formulations independent of cold chain is desirable to facilitate transportation and administration worldwide [3]. Recently, efforts are being made to develop cost-effective scalable processes to obtain these particles. The present study aims to compare some of the most used downstream processing (DSP) technologies for capture and purification of VLPs, taking especial consideration in using technologies enabling operation at large scale [4]. First, suspension adapted HEK 293 cells cultured in chemically defined cell culture media were used to produce the Gag-eGFP VLPs. Then, some steps of the purification process were studied, including a primary and secondary clarification by depth filtration and filtration respectively, an intermediate step by tangential flow filtration (TFF) or multimodal chromatography (MC), a capture step by ion exchange (IEC), heparin affinity (AC) and hydrophobic interaction chromatography (HIC), a polishing step by size exclusion chromatography (SEC) and a finally lyophilization step by freeze-drying process. Different operation units were tested for each step. Finally, a complete DSP train was implemented using the best results obtained in each stage. A concentration of 2.2 ± 0.8·109 VLPs/mL in the lyophilized samples was obtained after its storage at room temperature for 2 months. The morphology and structural integrity were further assessed by cryo-TEM. These first results in enveloped VLP lyophilization offer great promise to overcome the difficulty to distribute vaccines in poorly served remote rural areas and increase vaccine stability until their administration. Likewise, the purification methodologies proposed here could be easily scaled up and applied to purify similar enveloped viruses and vesicles.
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The miR-17-92 cluster counteracts quiescence and chemoresistance in a distinct subpopulation of pancreatic cancer stem cells
Objective Cancer stem cells (CSCs) represent the root of many solid cancers including pancreatic ductal adenocarcinoma, are highly chemoresistant and represent the cellular source for disease relapse. However the mechanisms involved in these processes still need to be fully elucidated. Understanding the mechanisms implicated in chemoresistance and metastasis of pancreatic cancer is critical to improving patient outcomes. Design Micro-RNA (miRNA) expression analyses were performed to identify functionally defining epigenetic signatures in pancreatic CSC-enriched sphere-derived cells and gemcitabine-resistant pancreatic CSCs. Results We found the miR-17-92 cluster to be downregulated in chemoresistant CSCs versus non-CSCs and demonstrate its crucial relevance for CSC biology. In particular, overexpression of miR-17-92 reduced CSC self-renewal capacity, in vivo tumourigenicity and chemoresistance by targeting multiple NODAL/ACTIVIN/TGF-beta 1 signalling cascade members as well as directly inhibiting the downstream targets p21, p57 and TBX3. Overexpression of miR-17-92 translated into increased CSC proliferation and their eventual exhaustion via downregulation of p21 and p57. Finally, the translational impact of our findings could be confirmed in preclinical models for pancreatic cancer. Conclusions Our findings therefore identify the miR-17-92 cluster as a functionally determining family of miRNAs in CSCs, and highlight the putative potential of developing modulators of this cluster to overcome drug resistance in pancreatic CSCs.CH: ERC Advanced Investigator Grant (Pa-CSC 233460), European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement No 256974 (EPC-TM-NET) and No 602783 (CAM-PaC), the Subdireccion General de Evaluacion y Fomento de la Investigacion, Fondo de Investigacion Sanitaria (PS09/02129 \& PI12/02643), and the Programa Nacional de Internacionalizacion de la I+D, Subprogramma: FCCI 2009 (PLE2009-0105; Ministerio de Economia y Competitividad, Spain). MC: La Caixa Predoctoral Fellowship.S
Intracellular autofluorescence as a new biomarker for cancer stem cells in glioblastoma
The identification of cancer stem cells (CSCs), which are implicated in tumor initiation, progression, therapy resistance, and relapse, is of great biological and clinical relevance. In glioblastoma (GBM), this is still a challenge, as no single marker is able to universally identify populations of GBM cancer stem cells (GSCs). Indeed, there is still controversy on whether biomarker-expressing cells fulfill the functional criteria of bona fide GSCs, despite being widely used. Here, we describe a novel subpopulation of autofluorescent (Fluo+) cells in GBM that bear all the functional characteristics of GSCs, including higher capacity to grow as neurospheres, long-term self-renewal ability, increased expression of stem cell markers, and enhanced in vivo tumorigenicity. Mechanistically, the autofluorescent phenotype is largely due to the intracellular accumulation of riboflavin, mediated by the ABC transporter ABCG2. In summary, our work identifies an intrinsic cellular autofluorescent phenotype enriched in GBM cells with functional stem cells features that can be used as a novel, simple and reliable biomarker to target these highly malignant tumors, with implications for GBM biological and clinical research.This research was funded by FEDER funds through the Operational Programme Competitiveness Factors–COMPETE and National Funds through FCT under the projects UIDB/50026/2020,
UIDP/50026/2020, and POCI-01-0145-FEDER-007038; by the project NORTE-01-0145-FEDER-000013,
NORTE-01-0246-FEDER-000012, and NORTE-01-0145-FEDER-000023, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership
Agreement, through the European Regional Development Fund (ERDF). J.V.d.C., C.S.G., E.P.M.,
and B.M.C. was funded by FCT-Foundation for Science and Technology (SFRH/BD/88121/2012
to J.V.d.C.; SFRH/BD/92786/2013 to C.S.G.; PD/BDE/143154/2019 to E.P.M.; and PTDC/SAUGMG/113795/2009, IF/00601/2012 and CEECIND/00072/2018 to B.M.C.). B.M.C. was also funded
by Fundação Calouste Gulbenkian and Liga Portuguesa Contra o Cancro
Microenvironmental hCAP-18/LL-37 promotes pancreatic ductal adenocarcinoma by activating its cancer stem cell compartment
This is the peer reviewed version of the following article: Microenvironmental hCAP-18/LL-37 promotes pancreatic ductal adenocarcinoma by activating its cancer stem cell compartment. Gut 64.12 (2015): 1921-1935 and which has been published in final form at http://dx.doi.org/10.1136/gutjnl-2014-308935OBJECTIVES:
The tumour stroma/microenvironment not only provides structural support for tumour development, but more importantly it provides cues to cancer stem cells (CSCs) that regulate their self-renewal and metastatic potential. This is certainly true for pancreatic ductal adenocarcinomas (PDAC), where tumour-associated fibroblasts, pancreatic stellate cells and immune cells create an abundant paracrine niche for CSCs via microenvironment-secreted factors. Thus understanding the role that tumour stroma cells play in PDAC development and CSC biology is of utmost importance.
DESIGN:
Microarray analyses, tumour microarray immunohistochemical assays, in vitro co-culture experiments, recombinant protein treatment approaches and in vivo intervention studies were performed to understand the role that the immunomodulatory cationic antimicrobial peptide 18/LL-37 (hCAP-18/LL-37) plays in PDAC biology.
RESULTS:
We found that hCAP-18/LL-37 was strongly expressed in the stroma of advanced primary and secondary PDAC tumours and is secreted by immune cells of the stroma (eg, tumour-associated macrophages) in response to tumour growth factor-β1 and particularly CSC-secreted Nodal/ActivinA. Treatment of pancreatic CSCs with recombinant LL-37 increased pluripotency-associated gene expression, self-renewal, invasion and tumourigenicity via formyl peptide receptor 2 (FPR2)- and P2X purinoceptor 7 receptor (P2X7R)-dependent mechanisms, which could be reversed by inhibiting these receptors. Importantly, in a genetically engineered mouse model of K-Ras-driven pancreatic tumourigenesis, we also showed that tumour formation was inhibited by either reconstituting these mice with bone marrow from cathelicidin-related antimicrobial peptide (ie, murine homologue of hCAP-18/LL-37) knockout mice or by pharmacologically inhibiting FPR2 and P2X7R.
CONCLUSIONS:
Thus, hCAP-18/LL-37 represents a previously unrecognised PDAC microenvironment factor that plays a critical role in pancreatic CSC-mediated tumourigenesis.CH: ERC Advanced Investigator Grant (Pa-CSC 233460), European Community's Seventh
Framework Programme (FP7/2007-2013) under grant agreement n° 256974 (EPC-TM-NET) and n°
602783 (CAM-PaC), the Subdirección General de Evaluación y Fomento de la Investigación, Fondo de
Investigación Sanitaria (PS09/02129 & PI12/02643) and the Programa Nacional de Internacionalización
de la I+D, Subprogramma: FCCI 2009 (PLE2009-0105; both Ministerio de Economía y Competitividad
(es), Spain), BSJr: Rámon y Cajal Merit Award from the Ministerio de Economía y Competitividad,
Spain and Clinic and Laboratory Integration Program (CLIP) grant from the Cancer Research Institute,
NY, NY. MC: La Caixa Predoctoral Fellowshi
Osteoprotegerin in Exosome-Like Vesicles from Human Cultured Tubular Cells and Urine
Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and receptors may be
present in exosomes and are expressed by proximal tubular cells. We have now studied the expression of selected TNF
superfamily proteins in exosome-like vesicles from cultured human proximal tubular cells and human urine and have
identified additional proteins in these vesicles by LC-MS/MS proteomics. Human proximal tubular cells constitutively
released exosome-like vesicles that did not contain the TNF superfamily cytokines TRAIL or TWEAK. However, exosome-like
vesicles contained osteoprotegerin (OPG), a TNF receptor superfamily protein, as assessed by Western blot, ELISA or
selected reaction monitoring by nLC-(QQQ)MS/MS. Twenty-one additional proteins were identified in tubular cell exosomelike
vesicles, including one (vitamin D binding protein) that had not been previously reported in exosome-like vesicles.
Twelve were extracellular matrix proteins, including the basement membrane proteins type IV collagen, nidogen-1, agrin
and fibulin-1. Urine from chronic kidney disease patients contained a higher amount of exosomal protein and exosomal
OPG than urine from healthy volunteers. Specifically OPG was increased in autosomal dominant polycystic kidney disease
urinary exosome-like vesicles and expressed by cystic epithelium in vivo. In conclusion, OPG is present in exosome-like
vesicles secreted by proximal tubular epithelial cells and isolated from Chronic Kidney Disease urine.This work was supported by grants from the Instituto de Salud Carlos III (ISCIIIRETIC REDINREN RD06/0016, RD12/0021, PI11/01854, PI10/00072 PI09/
00641 and PS09/00447); Comunidad de Madrid (Fibroteam S2010/BMD-2321, S2010/BMD-2378); Sociedad Española de NefrologÍa; European Network (HEALTH
F2-2008-200647); DIALOK European project LSHB-CT-2007-036644; Fundacion Lilly and IRSIN/FRIAT to JE; Programa Intensificación Actividad Investigadora (ISCIII/
Agencia Laín-Entralgo/CM) to AO; Instituto de Salud Carlos III (FIS PI11/01401, CP09/00229); and Fundación Conchita Rábago de Jiménez DÍaz to GAL. The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip
Famílies botàniques de plantes medicinals
Facultat de Farmàcia, Universitat de Barcelona. Ensenyament: Grau de Farmàcia, Assignatura: Botànica Farmacèutica, Curs: 2013-2014, Coordinadors: Joan Simon, Cèsar Blanché i
Maria Bosch.Els materials que aquí es presenten són els recull de 175 treballs d’una família botànica d’interès medicinal realitzats de manera individual. Els treballs han estat realitzat
per la totalitat dels estudiants dels grups M-2 i M-3 de l’assignatura Botànica Farmacèutica
durant els mesos d’abril i maig del curs 2013-14. Tots els treballs s’han dut a terme a través de la plataforma de GoogleDocs i han estat tutoritzats pel professor de l’assignatura i revisats i finalment co-avaluats entre els propis estudiants. L’objectiu principal de l’activitat ha estat fomentar l’aprenentatge autònom i col·laboratiu en Botànica farmacèutica
Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2
The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality
Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension
OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo
Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab
The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension
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