Institutionen för laboratoriemedicin / Department of Laboratory Medicine
Abstract
Acute graft-versus-host disease (GVHD) remains a major barrier to the
wider application of allogeneic stem cell transplantation (SCT) for a
variety of diseases. GVHD occurs when the transplanted donor Tlymphocytes
react to host antigens on antigen-presenting cells and attack host
tissues. The donor lymphocytes are introduced into a milieu that promotes
their direct attack on target cells, causing tissue damage through
perform, granzyme B, and Fas/Fas ligand (FasL) interactions. In addition,
the dysregulated production of inflammatory cytokines, such as TNF-alpha,
IFN-gamma, IL-1, and others, may cause direct tissue damage. The
immunobiology of acute GVHD is complex and the precise mechanisms by
which host tissues are damaged remain unclear. Despite progress in
understanding the mediators involved in acute GVHD, treatment has
remained frustrating; most patients who develop the severe manifestations
of GVHD succumb to it or to complications of its treatment. Today, the
only available method of diagnosing acute GVHD relies on clinical
observations and clinical judgments. Since plenty of other problems can
influence the symptoms seen in patients after SCT and further confuse the
picture of the patient's disease, diagnosing acute GVHD can be rather
difficult. It is well known that predicting the risk of acute GVHD before
its clinical manifestation and early administration of additional therapy
may result in less incidence of severe GVHD. Hence, new methods to
diagnose acute GVHD are desired. Therefore, the major aim of this thesis
has been to find new reliable molecular methods to diagnose acute GVHD
after allogeneic SCT.
The gene expression of T-cell effector molecules, granzym B, perform,
FasL, and TNF-alpha, was evaluated as diagnostic markers for acute GVHD
after SCT. Peripheral blood samples from patients were analysed by
competitive or real-time polymerase chain reaction (PCR). An
up-regulation of the gene expression of was seen in association with
acute GVHD diagnosis in 23 of 27, 26 of 27, and 24 of 27 patients
diagnosed with acute GVHD for granzyme B, perform and FasL, respectively.
The gene expression of TNF-alpha did not show the same pattern.
Interestingly, we also found that patients with increasing levels during
steroid treatment, 2 of 3 in Paper I and 10 of 10 in Paper II, showed
persistent or deteriorating acute GVHD.
Using VNTR analysis, chimerism patterns were investigated in 34 patients
during the early posttransplantation period. The difference in the
clearance rate of host T-cells between day 7 and day 10 was compared. In
this study, we found that there was a significantly higher risk for
patients with complete donor chimerism on day 7, together with patients
with an increase of more than 50% in the donor CD4+ T-cell population
between day 7 and 10, to develop grades II-IV acute GVHD. Our data
indicate that early monitoring of T-cell chimerism after SCT may be of
use for early identification of patients at risk of developing moderate
to severe acute GVHD after SCT.
We hypothesized that the gene expression of chemokine receptors in
peripheral blood from patients after SCT would reflect the development of
acute GVHD since these molecules are involved in the recruitment of
activated T-lymphocytes during acute GVHD. Quantitative real-time PCR was
used to assess the gene expression of the chemokine receptors CCR5,
CXCR3, CCR1, and CCR2. In this study we found that increasing levels of
these receptors were associated with acute GVHD. We found increasing
levels in connection to acute GVHD diagnosis of all four markers in 35,
33, 35, and 35 of 46 occasions of acute GVHD for CCR5, CXCR3, CCR1, and
CCR2, respectively. Another important finding was that the gene
expression levels increased before acute GVHD was diagnosed clinically
with the median number of days before diagnosis ranging from 3 to 5 days.
In conclusion, although the pathophysiology of acute GVHD still remains
complex, this study shows that different molecular markers involved in
this complicated disorder may be used to diagnose and predict acute GVHD
and to monitor steroid treatment. This may prevent life-threatening
complications and improve the outcome for patients after allogeneic SCT