مجال
التميز
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تميز
دراسي وبحثي
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البحوث المنشورة
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البحث (1):
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فصل في
كتاب
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عنوان الفصل:
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Factors
in Homo and Heterotypic Aggregate Formation in Sepsis
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رابط إلى الفصل:
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Click Here
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تاريخ النشر:
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20/06/2016
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موجز عن الفصل:
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Sepsis, a severe systemic inflammatory
response to an infection that can be bacterial, viral or fungal in origin,
remains a serious condition with high mortality. The dynamics in the immune
response (immune activation, over activation and exhaustion) during
development and progression of sepsis pose a problem in the design of new
treatment approaches. This review focuses on the understanding of molecular
interactions that lead to the formation of cellular aggregates in sepsis and
puts novel treatment targets in the context of these interactions.
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البحث (2):
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عنوان البحث:
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Formation
Of Inflammatory Platelet-Leucocyte Aggregates In Vitro And Their Adhesion To
Inflamed Endothelial Cells
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رابط إلى البحث:
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Click
Here
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تاريخ النشر:
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14/04/2018
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موجز عن البحث:
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In sepsis,
there is extensive formation of inflammatory platelet leucocyte aggregates
(PLAs) that circulate and adhere to inflamed vascular endothelium. PLAs
correlate with severity of disease. Whole blood stimulation assays and flow cytometry
are widely used to study blood PLAs formed in vitro in response to pathogen
associated molecular patterns (PAMPs, most commonly LPS). However, this
approach lacks robust methodology. For example, the extent of spontaneous formation
of PLAs ex vivo is often unclear. Thus, a reliable in vitro model to
investigate sepsis relevant formation of PLAs and their adhesion to inflamed
endothelial cells is required.
We
aimed to develop a model of the acute inflammatory reaction to common
bacterial PAMPs, on which to test therapeutic agents. Whole blood from
healthy volunteers was drawn and immediately immunolabelled with anti
CD66b-APC and anti CD42b-PE to assess the extent of spontaneous PLA. Further,
whole blood samples were incubated with either endotoxin (E. coli 0111: B4 or
Salmonella enteritidis, Sigma), as single PAMP, or heat killed bacteria
(Klebsiella pneumoniae or Staphylococcus aureus, departmental archive), for
1h at 37_C.
PLAs
were identified by flow cytometry as CD66b and CD42b positive events.
Ultrastructural analysis of these aggregates was performed by scanning
electron microscopy (Scanning EM). Whole blood was stimulated with LPS (1000
ng/ml), heat-killed K. pneumoniae or heat killed S. aureus (106 CFU/ ml) or
left unstimulated (control), then co-incubated with TNFa stimulated EAhy.926,
to investigate PLA adherence to inflamed endothelial cells by transmission
electron microscopy (TEM) and light microscopy. Secreted IL-8 was measured by
ELISA. Data were analysed using the Kruskal-Wallis test, followed by Dunn’s
test for multiple comparisons between groups.
Twelve
individual samples were tested from eight volunteers.
There
was significant spontaneous aggregation of platelets and leucocytes, which
was not enhanced by LPS from E. coli or S. enteritidis. Scanning EM revealed
similar PLA surface area between unstimulated and endotoxinstimulated samples
but a significant increase after whole blood stimulation with heat killed
bacteria (p<0.0001). Light microscopy and TEM (Fig. 1) showed adherence of
more complexed cellular aggregates on the endothelial layer after stimulation
with heat killed bacteria, in conjunction with a significant increase in
IL-8.
Unexpectedly,
LPS was not a suitable stimulus to develop an in vitro model of acute
cellular interactions between PLAs and inflamed endothelium. Rather, the
combination of heat killed bacteria and Scanning EM successfully modelled
formation of complex PLAs and their adhesion to endothelial.
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المؤتمرات العلمية:
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المؤتمر (1):
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عنوان المؤتمر:
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9th Saudi
Students’ Conference
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تاريخ الإنعقاد:
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13/02/2016
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مكان
الإنعقاد:
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Birmingham,
UK
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طبيعة المشاركة:
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Poster
presentation
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عنوان المشاركة:
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Functional Assessment Of Regulatory
Motives Of Pneumococcal Transcriptional Regulators Involved In Biology
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ملخص المشاركة:
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Streptococcus
pneumonia is a leading cause of morbidity
and mortality among the young children especially under 5 years and elderly
(Varon et al., 2010). It is reported to cause death of around one
million children under the age of five annually ((Normark and Tuomanen,
2015).The significant increase in antibiotic resistant strains worldwide and
the limitations of the vaccines require a deeper understanding of
pneumococcal biology to find targets to develop antiinfectives. Recent data
show that there is a link between microbial adaptation and virulence.
Transcriptional
regulators (TR) allow S. pneumonia to live in diverse environments. The aim
of this study was to determine the functioning of selected TR (SPD_0144,
SPD_0939 and SPD_0447) known to be important for pneumococcal metabolism at
molecular level. Site directed deletions of putative -35 and -10 regions
believed to be promoters for selected transcriptional regulators were done by
overlap extension PCR using specifically modified primers(Horton et al.,
1990).By Using standered DNA cloning protocol, the mutated fragment of each
target was inserted into appropriate vector (pPP1or pPP2) and transformed
into E.coli(Fanglian, 2011). The recombinant plasmid of each target,
pPP2Pshp144Δ-35,pPP2Pshp939Δ-35 and pPP1Pspd0447Δ-10, was transformed into
D39 S. pneumonia (Lanelli and Pozzi, 2004). After that, the
pneumococcal mutants were assessed by β-galactosidase assay.
Expectedly,
pneumococcal mutants with mutation in Pshp939 and Pspd0447 exhibited marked
decrease in β-galactosidase activity compared to that of Pshp939-WT and
Pspd0447-WT, respectively. These findings could be contributed to the mutation
and indicate that the deleted sequences of Pshp939 and Pspd0447, including
-35 and -10 regions, are most likely to be part of promoter that up regulate
the expression of shp939 and spd0447 genes, respectively. However,
pneumococcal mutants with mutation in Pshp144 showed a significant increase
in β-galactosidase activity in comparison to that of Pshp144-WT, indicating
that the deleted sequence of Pshp144, involving -35 and -10 region is likely
to be a repressor.
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المؤتمر (2):
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عنوان المؤتمر:
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Joint
Respiratory Research Day
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تاريخ الإنعقاد:
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03/05/2017
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مكان
الإنعقاد:
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Nottingham,
UK
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طبيعة المشاركة:
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Poster
presentation
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عنوان المشاركة:
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Comparative Analysis Of Sepsis Relevant
Stimuli On Platelet Leukocyte Aggregate Formation
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ملخص المشاركة:
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Acute
respiratory distress syndrome (ARDS) is one of the life threatening
complication of severe sepsis in which platelet leukocyte aggregate formed in
sepsis play a critical role in its pathogenesis. Whole blood stimulation
assays are widely used to study the formation of aggregates involving
platelets and granulocytes as an in
vitro approach to understand the acute inflammatory reaction in the
bloodstream to the presence of pathogen associated molecular patterns (PAMP)
during septicaemia. Platelet leukocyte aggregates form in response to LPS.
However, the extent of spontaneous aggregate formation is unclear in most
assays. In this work, we expand our understanding of this phenomenon by
investigating the effect of different inflammatory stimuli on aggregate
formation: endotoxin as well as heat-killed Klebsiella pneumoniae (HKK).
To
analyse the spontaneous PGA at time point zero, the whole blood sample was
immune labelled immediately for flow cytometry. Meanwhile, to analyse the
effect of the stimulation, whole blood was immediately incubated with
endotoxin, as a single PAMP, or HKK, for 1h at 370C. The formation
of aggregates involving platelets and granulocyte was measured by flow
cytometry. Ultrastructural analysis of these aggregates was performed by means
of scanning electron microscopy.
There
was significant aggregation of platelets and granulocytes, which endotoxin
from different sources (E.coli and Salmonella enteritidis) did not
enhance significantly. By contrast, HKK stimulation increased the number of
platelet granulocyte aggregates significantly over the spontaneous
aggregation observed at the same time of incubation without HKK. Scanning EM
showed a significant increase in aggregate surface area after HKK stimulation
compared to endotoxin which was similar to that of the unstimulated sample.
We
conclude that endotoxin is not a suitable stimulus to provoke sepsis relevant
platelet granulocyte aggregates in
vitro.
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المؤتمر (3):
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عنوان المؤتمر:
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BJA
Research Forum
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تاريخ الإنعقاد:
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23-24/11/2017
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مكان
الإنعقاد:
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Leicester,
UK
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طبيعة المشاركة:
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Paper
presentation
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عنوان المشاركة:
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Formation Of Inflammatory
Plateletleucocyte Aggregates In Vitro And Their Adhesion To Inflamed
Endothelial Cells
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ملخص المشاركة:
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In
sepsis, there is extensive formation of inflammatory platelet leucocyte
aggregates (PLAs) that circulate and adhere to inflamed vascular endothelium.
PLAs correlate with severity of disease. Whole blood stimulation assays and
flow cytometry are widely used to study blood PLAs formed in vitro in
response to pathogen associated molecular patterns (PAMPs, most commonly
LPS). However, this approach lacks robust methodology. For example, the
extent of spontaneous formation of PLAs ex vivo is often unclear. Thus, a
reliable in vitro model to investigate sepsis relevant formation of PLAs and
their adhesion to inflamed endothelial cells is required.
We
aimed to develop a model of the acute inflammatory reaction to common
bacterial PAMPs, on which to test therapeutic agents. Whole blood from
healthy volunteers was drawn and immediately immunolabelled with anti
CD66b-APC and anti CD42b-PE to assess the extent of spontaneous PLA. Further,
whole blood samples were incubated with either endotoxin (E. coli 0111: B4 or
Salmonella enteritidis, Sigma), as single PAMP, or heat killed bacteria
(Klebsiella pneumoniae or Staphylococcus aureus, departmental archive), for
1h at 37_C.PLAs were identified by flow cytometry as CD66b and CD42b positive
events. Ultrastructural analysis of these aggregates was performed by
scanning electron microscopy (ScanningEM). Whole blood was stimulated with
LPS (1000 ng/ml),heat-killed K. pneumoniae or heat killed S. aureus (106
CFU/ml) or left unstimulated (control), then co-incubated with TNFa
stimulated EAhy.926, to investigate PLA adherence to inflamed endothelial
cells by transmission electron microscopy(TEM) and light microscopy. Secreted
IL-8 was measured by ELISA. Data were analysed using the Kruskal-Wallis test,
followed by Dunn’s test for multiple comparisons between groups.Twelve
individual samples were tested from eight volunteers.
There
was significant spontaneous aggregation of platelets and leucocytes, which
was not enhanced by LPS from E. coli or S. enteritidis. Scanning EM revealed
similar PLA surface area between unstimulated and endotoxinstimulated samples
but a significant increase after whole blood stimulation with heat killed
bacteria (p<0.0001). Light microscopy and TEM (Fig. 1) showed adherence of
more complexed cellular aggregates on the endothelial layer after stimulation
with heat killed bacteria, in conjunction with a significant increase in
IL-8.
Unexpectedly,
LPS was not a suitable stimulus to develop an in vitro model of acute
cellular interactions between PLAs and inflamed endothelium. Rather, the
combination of heat killed bacteria and Scanning EM successfully modelled
formation of complex PLAs and their adhesion to endothelial.
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