The pathogenesis of dengue virus infection in patients with sickle cell disease

Annually an estimated number of 50 - 100 million dengue virus (DENV) infections
occur in tropical and subtropical countries around the world. DENV-infection
usually results in a subclinical or self-limiting febrile disease but may also
lead to severe disease, previously known as Dengue haemorrhagic fever (DHF) and
Dengue shock syndrome (DSS). Severe disease is characterised by
thrombocytopenia, haemorrhagic manifestations, liver disturbances and a sudden
onset of vascular permeability, believed to be caused by a cytokine storm.

Important clinical symptoms of severe DENV infection are plasma leakage and
bleeding. It is believed that both symptoms are partially caused by activation
of endothelial cells. However, the primary target cells of DENV infection are
monocytes. It has been shown that endothelial cells can be infected with DENV
in vitro, but whether viral replication in endothelial cells really happens in
vivo is still a matter of debate.

A fatal case of a DENV infected patient with sickle cell disease celled to the
hypothesis that sickle anaemia may worsen the clinical course of a DENV
infection. During an outbreak in Cuba, a relationship between sickle cell
anaemia and severe DENV infections has been described. It was hypothesized that
the monocytes of patients with sickle cells disease have a lower inflammatory
set-point, which leads to activation of endothelial cells. Since endothelial
cells in patients with sickle cell disorder are activated without any
concurrent infection, it is conceivable that hyper-reactivity of macrophages
during DENV infection in these patients amplifies the activational status of
endothelial cells, leading to severe plasma leakage and bleeding.

To elucidate the pathogenetic mechanism of endothelial cell activation in DENV
infected patients with sickle cell disorder.

Experimental in vitro study with human-derived material

Patients visiting the out-patient clinic of the department of hematology (only
adults) of internal medicine will be asked to participate in this study. After
informed consent is obtained blood will be drawn via venipuncture.

The following specimens will be collected:
EDTA blood (20cc) in order to isolate PBMCs

Materials/methods
Peripheral blood will be drawn in 2 X 10 mL EDTA tubes. Samples will be kept at
40C and be processed immediately. The PBMCs will be separated from the other
blood fraction with a Ficoll layer. To set a baseline the PBMCs of patients
with HbSS and of healthy controls will be stimulated with LPS and conA to
investigate whether the inflammatory setpoint of PBMCs from patients with
sickle cell disease is indeed increased. Afterwards the PBMCs will be infected
with dengue virus and incubated with Human Umbilical Vein Endothelial Cells for
approximately 5-7 days. Every day samples from the supernatant will be taken
and the virus titre and certain coagulation (TF, sTM) and inflammatory
parameters (TNF-α, IL-1β) will be measured.

There may be a risk for pain and/or a hematoma at the venipuncture site.