Quantification of chemokine and cytokine levels after standardized in vitro stimulation of peripheral blood mononuclear cells from patients with haematological malignancies

Ref ID: 19409

Author:

J. U. Springer,1 C. Lass-Fl€orl,2 K. Huenniger,3 M. Lackner,2
O. Kurzai,3 W. Heinz,4 A. J. Ullmann,4 H. Einsele4 and J. Loeffler4

Author address:

1University of W€urzburg Medical Center, W€urzburg, Germany;
2Department of Hygiene and Social Medicine, University of
Innsbruck, Austria; 3Septomics Research Centre, Jena, Germany
and 4Universit€at W€urzburg, Medizinische Klinik und Poliklinik II

Full conference title:

6th Trends in Medical Mycology 2013

Date: 11 October 2014

Abstract:

Objectives Invasive aspergilllosis (IA) remains to be a major compli-
cation of patients with haematological malignancies (HM) and after
allogeneic stem cell transplantation (SCT). In these patients, IA is the
most common cause of mortality due to infection. Earlier diagnosis of
Aspergillus infections would facilitate more effective management and
prevent progression to invasive disease which typically has a poor response to antifungal drugs. Easily detectable biomarkers for early
detection or confirmation of IA are heavily needed.
Therefore, we have consecutively isolated peripheral blood mono-
nuclear cells (PBMC) from patients with haematological malignan-
cies. PBMCs were incubated with 2 different fungal stimuli,
respectively and chemokine and cytokine levels were quantified in
comparison to healthy controls.
Methods Since 2011, 1 ml of peripheral blood was consecutively
collected 3 times following a positive galactomannan result (GM,
Platelia, Bio-Rad). As controls blood from healthy volunteers and
from HM patients without positive GM assay was achieved. All
patients underwent chemotherapy or allogeneic SCT and were moni-
tored for invasive fungal diseases (IFD) using the current consensus
criteria of the European Organization for Research and Treatment of
Cancer / Mycoses Study Group. Blood samples were drawn bedside
into TruCulture tubes (TCT, Myriad RBM) and incubated at 37°C for
24 h. TCT stabilizes immune cells under standardized conditions,
thereby facilitating interaction and stimulation of PBMCs in vitro.
Zymosan, a fungal cell wall component, and inactivated Aspergillus
fumigatus germ tubes were used as a stimulus. Culture supernatants
were harvested and analyzed in a multiplex ELISA assay (Bio-Rad)
quantifying n = 27 different human chemokines and cytokines.
Results PBMCs were obtained from 4 healthy volunteers, 5 unclassi-
fied patients (controls, 20 samples), and 1 possible and 6 probable IA
patients (cases, 19 samples). All blood specimens were incubated in
TCT with either zymosan or germ tubes or left untreated. Analysis of
the supernatants by multiplex ELISA revealed that 10 / 27 chemo- and
cytokines were not induced by either stimulus (IL-5, IL-7, IL-12p70,
IL-13, IL-15, Eotaxin, IP-10, PDGF-bb, RANTES and VEGF). In con-
trast, 15 / 27 analytes (IL-1b, IL-1ra, IL-4, IL-6, IL-8, IL-9, IL-17, basic
FGF, G-CSF, GM-CSF, IFN-g, MCP-1, MIP-1a, MIP-1b, TNF-a) were
induced by both stimuli, while IL-2 and IL-10 were induced by zymo-
san only. In general, stimulation with zymosan led to higher levels of
cytokine and chemokines, with IL-1b (37000-fold), TNF-a (7400-fold)
and MIP-1a (5500-fold change) showing maximal induction.
Conclusion In this pilot study, we were able to demonstrate that
prospectively collected human PBMCs showed cytokine and chemoki-
ne release after stimulation with zymosan and A. fumigatus germ
tubes in vitro. Although studies with higher number of patients are
necessary, we were able to demonstrate specific release of chemokin-
es and cytokines after PBMC stimulation. These markers might
become a potential tool for early and confirmative biomarker of IA.

Abstract Number: p161

Conference Year: 2013

Link to conference website: NULL

New link: NULL

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