Researchers involved
V. Ramakrishnaiah
(Vedashree),
PhD student
W. Farid
(Waqar),
PhD student
P. de Ruiter
(Petra),
technician
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Researchers involved |
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V. Ramakrishnaiah (Vedashree), PhD student |
W. Farid (Waqar), PhD student |
P. de Ruiter (Petra), technician |
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Background:
The research line of dr. van der Laan and prof. dr. Tilanus focuses on immunological
and virological aspects of liver transplantation. The research is translational
of character and aims to provide basic and applied knowledge to improve the
outcome of Liver transplantation in patients. Work is preformed in close collaboration
with dr. Kazemier and dr. de Jonge of the department of Surgery and dr. Kwekkeboom,
prof. dr. Janssen , prof. dr. Peppelenbosch and prof. dr. Metselaar of the department
of Gastroenterology and Hepatology.
Most of the patients experience infectious complication after transplantation.
Clinical signs are non-specific and in many instances cannot be differentiated
from rejection. Therefore we need to increase our knowledge on the distinct
immunological aspects of acute rejection and opportunistic infections. Improving
monitoring and treatment of hepatitis C virus re-infection after transplantation.
Of particular importance for this study is the use of fine needle aspiration
biopsy which allows direct monitoring of intrahepatic immune events. This method
is safe, non-traumatic and can be used frequently. The FNAB technique already
proved to be a useful tool for the diagnosis of rejection and HBV infection.
The role of Treg in immunological tolerance to the allograft is studied in a
cohort of liver transplant patents. Treg subsets are characterized by phenotypic
markers (CD4+CD25+) and tested functionally for allo-specific suppressive activity.
The overall aim is to identify on basis of Treg, patients who are effectively
tolerant to the graft and can be safely taken off immunosuppressive medication.
We are currently developing a gene therapy approach to prevent HCV infection
of the liver graft using RNA interference to inhibit HCV replication. Conditions
to achieve effective lentiviral transduction during the perfusion of the liver
graft were being determined. Most recent research endeavors aim to better understand
liver regeneration after liver resection for living donor liver transplantation.
The role of growth factors, cytokines and mesenchymal stem cells is investigated.
The summary of the current research focus is shown in the graph bellow.
Innovative therapy for hepatitis C recurrence.
Chronic viral infections have a tremendous impact on global health and therefore
new therapeutic options should be urgently explored. RNA interference (RNAi)
represents a promising new approach to combat viral infections and recent developments
in the field of gene therapy have increased the feasibility of clinical applications.
We aim to explore the utility of RNAi for the treatment of the ultimately life-threatening
liver disease caused by the hepatitis C virus (HCV), affecting approx. 170 million
people worldwide. RNAi technologies have fuelled a rapid progress in the basic
understanding of the HCV biology and revealed numerous new viral and host cell
factors as potential targets for therapy. Together with the improvement of gene
delivery technology and the discovery of the critical role of miRNA in HCV infection,
RNAi and miRNA-based antiviral strategies hold great promise for the future.
The current standard interferon-alpha (IFN- )-based therapy for chronic HCV
infection is only effective in approximately half of the patients, prompting
the need for alternative treatments. RNA interference (RNAi) represents novel
approach to combat HCV by sequence-specific targeting of viral or host factors
involved in infection. Monotherapy of RNAi, however, may lead to therapeutic
resistance by mutational escape of the virus. Here we proposed that combining
lentiviral vector mediated RNAi and IFN- could be more effective and avoid therapeutic
resistance. We found that IFN- treatment did not interfere with RNAi mediated
gene silencing. RNAi and IFN- act independently on HCV replication showing combined
antiviral activity when used simultaneously or sequentially. Transduction of
mouse hepatocytes in vivo and in vitro was not effected by IFN- treatment. In
conclusion, RNAi and IFN- can be effectively combined without cross-interference
and may represent a promising combinational strategy for the treatment of hepatitis
C. Effects of immunosuppressive medication on HCV. Immune suppression considerably
affects hepatitis C virus (HCV) recurrence and outcome of antiviral treatment
after liver transplantation. Previously we have reported that mycophenolic acid
(MPA of MMF) has direct antiviral activity on HCV replication and we are currently
extending these findings using new in vitro and in vivo models. Furthermore,
recent findings suggest that the calcineurin inhibitor FK506 (tacrolimus, Tac)
but not cyclosporine A (CsA) interfere with the antiviral activity of interferon-
(IFN- ) in vitro. The aim of our study was to more extensively investigate the
effects of calcineurin inhibitors on IFN- signaling and antiviral activity in
subgenomic and infectious HCV models. Treatment with both Tac and CsA did not
affect Huh7 cell proliferation at 10 to 500 ng/ml doses, however, completely
inhibited T cell proliferation. In contrast to previous reports, Tac had no
effect on IFN- stimulated reporter gene expression, even at 5 g/ml dose. Furthermore,
in Huh7 subgenomic HCV replicon cells, treatment with Tac had no significant
effect on the suppression of viral replication by IFN- . In the infectious HCV
model, treatment with IFN- effectively inhibited both viral RNA replication
and de novo production of virus particles and both were not attenuated at any
concentration of Tac. CsA had no significant effect on IFN- stimulated reporter
gene expression, however as shown previously, a combination of CsA (at 500 ng/ml
and higher) and IFN- resulted in an enhanced inhibition of viral replication
both in the subgenomic and infectious HCV models. In conclusion, our study shows
no evidence that Tac or CsA interfere with IFN- -mediated inhibition of HCV
replication and virion production in vitro. Therefore, no further mechanistic
arguments are found to break the clinical controversy about choice of calcineurin
inhibitor during post-transplantation antiviral therapy.
Optimizing immune suppression for transplantation tolerance.
CD4+Foxp3+ regulatory T cells (Treg) depend on IL-2 for their function and survival.
By interfering with the IL-2 production, calcineurin inhibitors (CNI) may negatively
affect Treg. We have investigated the effects of conversion from CNI to mycophenolate
mofetil (MMF) on renal function, Treg frequency and phenotype in liver transplant
recipients. Patients with renal impairment on CNI were converted to MMF and
received a single dose of IL-2-receptor blocking antibody (Daclizumab). Control
patients continued CNI treatment. Six months after conversion to MMF the percentage
of CD4+Foxp3+ and CD4+CD25bright T cells increased both by 1.2 fold. FOXP3 mRNA
analysis of mononuclear cells confirmed the enrichment of Foxp3 in peripheral
blood. CD25 expression on CD4+Foxp3+, but not CD4+Foxp3-, cells significantly
increased at six months compared to pre-conversion. Daclizumab treatment resulted
in a 75 percent blocking of CD25 at one month but did not affect Foxp3+ Treg
levels. After conversion renal function rapidly and significantly improved and
acute rejection occurred in two patients (12%). In conclusion, conversion to
MMF increases the percentage and CD25 expression of CD4+Foxp3+ cells indicating
that MMF therapy can overturn the repressive effect of CNI on circulating Treg
and therefore may promote Treg mediated tolerance.
Liver graft injury and repair.
Bone marrow (BM) mesenchymal stem cells (MSC) have self-renewal and pluripotent
differentiation capacities. MSC are mobilized from the BM compartment in response
to systemic injury were they migrate and contribute to tissue regeneration.
Recent evidence suggests that trophic factors produced by MSC play an important
role in these regenerative responses. For instance, treatment with MSC conditioned
medium can protect rats against lethal hepatic failure. Our group has recently
identified a resident population of MSC in human liver, which are highly comparable
to BM MSC regarding surface marker expression, lineage differentiation capacity,
Wnt-stimulated proliferative responses and genome-wide gene expression profiles
(only two-fold different expression found in 0.25% of genes). We demonstrated
that liver MSC can differentiate towards hepatocyte-lineage both in vitro and
in vivo with a persistent capacity to self-renew in mice. Moreover we found
that like BM MSC also liver MSC secrete factors that promote liver regeneration
in mice after partial hepatectomy and in the context of immunosuppression. Treatment
with concentrated MSC-conditioned medium stimulated hepatoctyte proliferation
and significantly up regulated cytokine gene expression (TNF and HGF). Recent
evidence suggests that MSC-derived trophic factors do not only include cytokines
and extracellular matrix modulating factors but also secrete microvesicles/exosomes
containing mRNA. Microvesicle-mediated transfer of genetic information between
stem cells and injured tissue as a paracrine mechanism shuttling defined patterns
of mRNA and microRNA between cells. Conversely, release of microvesicles from
injured cells may reprogram the phenotype of stem cells to acquire specific
features and may induce dedifferentiation. Finally, we newly discovered that
MSC have antiviral properties and can inhibit virus replication (including of
hepatitis viruses) by secretion of soluble factors with distinct antiviral properties
from interferons.
Selected publications