Ludwik Hirszfeld Institute of Immunology and Experimental Thera
Ludwik
Hirszfeld Institute of Immunology and Experimental Therapy
Polish
Academy of Sciences
Rudolfa
Weigla 12, 53-114 Wrocław
Research
Report - 2000
DEPARTMENT OF IMMUNOCHEMISTRY
Head: Czesław Ługowski, Ph.D., Professor
Laboratory of Microbial
Immunochemistry and Vaccines
Head: Czesław Ługowski, Ph.D.,
Professor
Biochemical
characteristics of macromolecules involved in immunological
processes -
immunochemical studies
of bacterial endotoxins
The
aim of our studies in the year 2000 was the immunochemical
characterization of endotoxins, important virulence factors known
also as lipopolysaccharides, isolated from opportunistic pathogens
such as Plesiomonas shigelloides, Hafnia alvei, Proteus
mirabilis and Proteus vulgaris. All these bacteria cause
typical nosocomial infections, sometimes resulting in severe
complications, such as bacteraemia and endotoxic shock. The
carbohydrate parts of lipopolysaccharide (LPS), O-specific
polysaccharides (PS) and core oligosaccharides, are the targets of
specific antibodies induced by LPS in the host immune system. Most
O-antigens have a unique chemical structure, but some possess a
significant structural similarity to other polysaccharides, which
accounts for the serological cross-reactivity of strains.
Through
the use of NMR spectroscopy and matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry, in
conjunction with chemical and immunological methods, we have
established novel structures of bacterial O-specific polysaccharides
isolated from the Pl. shigelloides strain CNCTC 113/92, P.
mirabilis O48 and P. vulgaris O21 LPS. We concluded that
the O-specific polysaccharide of the Pl. shigelloides strain
is composed of a hexasaccharide repeating unit containing
D-glycero-b-D-manno-heptopyranose
and 6-deoxy-b-D-manno-heptopyranose,
sugars which are rarely present in O-specific PS. It was also
possible to perform full structural NMR studies using high-resolution
magic-angle spinning (HR-MAS) NMR on the LPS suspension and in
situ on bacteria. HR-MAS NMR spectra of intact bacteria showed
that the chemical shifts of reporter groups could be a good marker
for the main surface bacterial antigens (O-antigens), as well as
supply chemical information concerning the structure of this
substance. This technique provides the means of in situ
detection and characterization of large flexible molecules observed
in real time. This approach could be used in the fast fingerprinting
of bacterial and other cellular antigens. (This project in
collaboration with the Department of Chemistry, Swedish University of
Agricultural Sciences, Uppsala, Sweden).
We
have also determined the structure of O-specific polysaccharides
isolated from cross-reacting strains of P. vulgaris O21 and P.
mirabilis O48 lipopolysaccharides. Both polysaccharides have
similar backbones built up of tetrasacharide phosphate repeating
units and differ in the presence of an O-acetyl group and a glucose
residue. The O-specific polysaccharide of P. vulgaris O21 has
the same structure as that of Hafnia alvei 744 except for the
GlcN residue that carries an N-acetyl group rather than a
3-hydroxybutyryl group. Serological investigations confirmed a close
relatedness of the Proteus and Hafnia O-antigen
studied. (This project in collaboration with the Institute of
Microbiology and Immunology, University of Lodz, Poland)
Our recent studies on
Hafnia alvei core oligosaccharides have revealed a new
structure present in strains 1185 and 1204. Sugar, and methylation,
analyses, mass spectrometry and NMR spectroscopy proved that the core
oligosaccharide differs from the typical H. alvei core. The
difference refers to one sugar residue, i.e. the terminal galactose
in the cores of the two investigated strains replaces the glucose
residue present in the typical core. (In collaboration with the N.D.
Zielinsky Institute of Organic Chemistry, Russian Academy of
Sciences, Moscow, the Russian Federation)
Laboratory of Glycoconjugate
Immunochemistry
Head: Elwira Lisowska, Ph.D.,
Professor
Cell membrane glycoproteins and
characterization of antibodies recognizing their carbohydrate and
peptidic epitopes
Despite
long-lasting studies on glycophorins, not all of the minor structures
of their oligosaccharide chains have been elucidated. In our studies
(in cooperation with Dr. B. Nilsson, Sweden) the structures of
glycans containing epitopes of the ABO blood group system have been
determined. This year, O-glycans of glycophorin A (GPA) from blood
group O erythrocytes were isolated and the structures containing
blood group H determinants were identified by means of mass
spectrometric methods. These studies are important in view of the
emerging role of GPA as a marker of glycosylation changes under
pathological conditions.
A
subject of our interest has also been the carbohydrate antigens TF
and Tn, which are present in cryptic form in normal glycophorins but
are commonly exposed in cancer cells. In search of reagents for
testing cancer cells, four new monoclonal antibodies (anti-TF and
anti-Tn, obtained from Dr. D. Blanchard, France) were examined. Two
of them seemed to fulfill the required criteria, as they recognized
Tn and sialylTn epitopes present in various model glycoproteins.
However, the anti-Tn MAbs did not react with antigens of human colon
adenocarcinoma cell lines, despite the fact that these cells strongly
reacted with anti-Tn lectins (in collaboration with Dr. D. Duś). The
most likely explanation is that the cancer cells lack the clusters of
GalNAc-residues required for reaction with antibodies.
For the
identification of peptidic epitopes we introduced a method involving
peptides synthesized on plastic pins (Pepscan). The epitopes for
human anti-Mg antibodies were studied. (Mg is
a rare GPA variant; natural antibodies against it are present in 1-2%
of individuals.) Our initial results suggest a more homogeneous
subspecificity of human antibodies compared with the mouse monoclonal
anti-Mg studied earlier. The Pepscan method was also
applied for the identification of peptidic epitopes of the Duffy
antigen, which is interesting as a chemokine-binding protein. Only a
few anti-Duffy MAbs have been reported and we identified epitopes for
most of them. The latest stage of these studies has been an
identification of the epitope for the MAb anti-Fyb,
representing the sequence 41YDANLE46 of
the Duffy antigen. The identification of the Duffy epitopes is
important, because the antibodies studied by us inhibit
chemokine-binding to the Duffy antigen.
Our
earlier studies on the cloning, arrangement and sequencing of genes
for anti-glycopeptidic MAbs resulted in an offer of cooperation in
studies on the monoclonal antibody against protein G of the rabies
virus, obtained in the laboratory of Dr. W. Wunner (USA). The gene
segments encoding variable fragments of the antibody H and L chains
were identified and sequenced.
Other
studies dealt with antibodies which agglutinate erythrocytes of rare
NOR phenotype, in which we had earlier identified the presence of
unique glycosphingolipids. Recently, we determined the full structure
of one of the NOR glycolipids with the use of immunochemical methods
(reactions of the glycolipid and its enzymatic degradation products
with lectins and antibodies, examined on TLC plates) and mass
spectrometry techniques (in collaboration with Dr. V. Reinhold, USA).
Lastly,
we examined recombinant glycophorin C, the normal form and its Ge and
Yus variants expressed in COS7 cells, by means of lectins. The
results indicated the presence of N-glycans, but lacking
polylactosamine sequences.
Laboratory of General Immunochemistry
Head:
Maria Janusz, Ph.D., Associate Professor
Immunomodulatory
peptides and Fc receptors
It is
now accepted that active oxygen species (e.g. O2-)
and NO may play a role in the pathogenesis of Alzheimer’s disease
(AD). This prompted us to investigate whether proline-rich
polypeptide (PRP) tablets (ColostrininŇ)
can be used as a potential drug for the treatment of AD and whether
its active nonapeptide fragment (NP) may act as an inducer of the
secretion of NO and O2-. It was found that the
peptides, when applied intraperitoneally to mice, did not induce
production of NO. However, both PRP and NP induced production of O2-.
The effect was dose dependent and the amount of O2-
produced did not exceed a value twice that of the control
level. The increase in the O2- level might be
transient and is not toxic to the cells.
The
effect of PRP and NP on the secretion of NO induced in mice by LPS
was also studied. The results obtained showed that the peptides
inhibited (by 10 - 50%) the production of NO induced by LPS. The
effect is dose dependent (doses of PRP and NP were 0.1 - 100 mg
per mouse). The effect of the peptides on the production of O2-
in mice treated with LPS is currently under investigation.
The aim of other
studies was to establish whether nuclear proteins bind to the
promotor sequence of the hFcRn gene and to investigate whether the
DNA-protein interactions are specific. The results obtained showed
that there are functional binding sites for SP1or SP1-like factors,
AP1 or a related factor, and for additional unidentified proteins in
the promotor region. The presence of binding sites for several
transcription factors suggests that expression of the human FcRn gene
is controlled by a complex of proteins. The results obtained allow us
to understand the molecular mechanisms underlying the regulation of
the hFcRn gene expression and, thus, the mechanism of the biologic
functions of the hFcRn receptor.
Laboratory
of Glycobiology
Head: Maciej
Ugorski, Ph.D., Associate Professor
Structure and function of adhesion
molecules and their ligands associated with tumor progression
Sialylated Lewis
structures present on the surfaces of cancer cells are carried by the
carbohydrate chains of glycoproteins and glycolipids, and both
classes of glycoconjugates seem to be involved in binding to
endothelial E-selectin. In our current studies, employing inhibitors
of glycolipid and O-glycan biosynthesis, we have shown that
sialosyl-LeA-gangliosides are not involved in the adhesion of the
human colon cancer cells CX-1 to E-selectin-expressing CHO cells when
mucins are present on the cell surface. However, our data indicate
that sialosyl-LeA-gangliosides can be involved in the adhesion of
colon cancer cells when mucins are removed, e.g. with bacterial
O-sialoglycoprotease.
In the preliminary
studies on the adhesion of cancer cells in dynamic flow conditions,
the following parameters were analyzed: flow rate and number of cells
in suspension, number of acquired images per experiment and time of
image acquisition. The adhesion interactions of CX-1 and CX-1AS5
cells to E-selectin-expressing CHO and CEA-expressing CHO cells under
defined laminar flow were recorded.
To study whether the
adhesion and metastatic properties of human colon cancer cells can be
directly affected by changes in the expression level of
carcinoembryonic antigen (CEA), we created a specific
loss-of-function phenotype. Two stable sublines with low
(CX-1.1CEA-AS6Q) and barely detectable amounts (CX-1.1CEA.AS8Q) of
CEA were obtained after transfection of parental human colon
carcinoma CX-1.1 cells with an expression vector containing a
fragment of cDNA for CEA in anti-sense orientation. The inhibition of
CEA expression was analyzed on the level of (i) CEA synthesis by
Western blotting, (ii) mRNA expression by Northern blotting and (iii)
promotor activity by the measurement of luciferase activity as the
reporter gene. It was found that the specific lack of expression of
CEA on the surface of colon cancer cells completely abolished their
homotypic adhesion. Interestingly, transfected cells were
characterized by a slower growth rate in comparison with parental
cells.
In the research project
"Tumor-associated antigens, sialosyl-LewisA and CEA as targets
in gene anticancer therapy,” we propose a new approach to gene
therapy based on suicide gene strategy, in which tumor-associated
antigens are used as targets to achieve elevation of the therapy's
specificity and efficacy. This year, studies on the effectiveness of
an expression vector containing the cytosine deaminase (CD) gene
under the control of a CEA promotor in gene therapy of human colon
carcinoma were performed. In the preliminary experiments, the
specificity and activity of the CEA promotor were analyzed. Then the
sensitivity of human colon cancer CX-1 cells to 5-flurocytosine
(5-FCyt) was studied after their stable transfection with the
expression vector containing the CD gene. The estimated ID50
for 5FCyt was 3.5 mM. As liberated 5-fluorouracil (5FUra) kills
neighboring non-CD-expressing cancer cells, the bystander effect of
the drug was also studied. It was found that the presence of just 10%
CD-expressing cells in the culture killed all the tumor cells in the
presence of 5FUra.
DEPARTMENT
OF EXPERIMENTAL THERAPY
Head:
Michał Zimecki, Ph.D., Professor
Laboratory of Immunobiology
Head: Michał Zimecki, Ph.D.,
Professor
Synthetic and natural
immunomodulations of potential application in prevention and therapy
A
substantial part of our research program involved studying the
immunotropic properties of lactoferrin (LF). We found that human LF
regulated constitutive and lipopolysaccharide (LPS)-induced ICAM-1
molecule expression on human umbilical vein endothelial cells (HUVEC)
and mononuclear blood cells. These data, together with previous
results on LFA-1 expression regulation by LF, suggest a role for LF
in the modulation of cell migration through the endothelium in
inflammatory processes.
To further clarify the
mechanism of the protective effect of bovine LF in experimental
endotoxemia, we established that LF produced two kinds of effects in
a mouse model: i) a strong diminution of both pro-inflammatory and
anti-inflammatory cytokines in serum, or ii) an unbalanced,
predominant release of IL-6 and IL-10 with a minor component of
TNF-a.
The effects of
preoperative treatment of mice with bovine LF on their immune systems
were studied, anticipating future clinical application of LF. LF was
found to modify an early (1-3 day post-operational) decrease in the
mitogen-induced proliferation of T and B cells and the ability of
blood neutrophils to phagocytize Staphylococcus aureus.
The results of our first
clinical trial demonstrated an immunoregulatory role of bovine LF
administration, given orally to patients subjected to thyroid
surgery. Pretreatment of patients with LF resulted in regulation of
TNF-a and IL-6 production by the
patient’s blood cells and an upregulation of the proliferative
response to lymphocytes to PHA.
Our previous studies on
jaundiced rats revealed that the ability to produce LPS-induced TNF-a
by peritoneal exudate cells and splenocytes was strongly enhanced on
day 7 and depressed on day 14 following bile duct ligation.
Administration of a small dose of LPS, mimicking surgery, resulted in
an even deeper hyporeactivity of cytokine production by rat cells.
The results explain the very high mortality rates in jaundiced
patients subjected to surgery.
Recent
studies demonstrated that the normalization of cytokine production by
blood cells from patients subjected to phage therapy was associated
with recovery. Present data suggest that the regulation of other
parameters, such as blood picture, phagocytosis and serum LF levels,
also correlate with a positive outcome of therapy.
In a search for new
therapeutically applicable compounds, we studied two families of
compounds, derivatives of izoksazole, in mouse in vivo and in
vitro models, and established their structure/activity
relationships.
A part of our studies was
devoted to the effects of peptides on the immune system. They
included the C-terminal region of smallpox CIOL virus protein,
thymopentin-like fragments of MHC antigen fragments and ubiquitin,
and fragments of FAS/APOI and p55R proteins. The peptides were active
in several in vivo and in vitro mouse models.
Laboratory of Immunopharmacology
Head: Stanisław Szymaniec, M.D.,
Associate Professor
New synthetic and natural compounds
of potential anti-inflammatory and immunotropic activity
The cellular and
humoral immune responses in children with severe adverse reaction to
vaccination against Bordetella pertusis was investigated. It
was found that the sera from children receiving three doses of
vaccine contained antibody against LPS 186 and LPS 606. However, the
sera from four of these children contained antibody which only
reacted strongly with other Bordetella pertusis antigens.
In
previous studies we had examined an M II substance obtained by Prof.
Machoń (Dept. of Organic Chemistry, Medical Academy, Wrocław). Low
toxicity and strong anti-inflammatory activity were found.
Subsequently, we investigated the ulcerogenic activity of the M II
substance. Wistar rats were treated with 100 and 300 mg/kg body
weight p.o. of the M II substance for five days. Ibuprofen in
similar doses was used as a reference drug. The degree of gastric
mucosa damage (congestion, erosions, ulcers, bleeding) on a 0 - 5
scale was determined by two physician using a biomicroscope. No
ulcerogenic effect was seen in rats after treatment with 100 mg/kg of
the M II substance. After treatment with 300 mg/kg of the M II
substance, ulcerogenic activity was seen, but it was several times
lower than that caused by Ibuprofen.
The
aim of our next study was to use radiolabeled inhibitors of cystein
endopeptidase for the imaging of tongue and larynx cancer and
metastases. We found that I125 radiolabeled cystein
inhibitor derived from chicken egg had bound after incubation in
vitro to cancer cells. Cells preincubated with unlabeled
inhibitor reduced the binding of the I125 -inhibitor by
65%.
A new
test for measuring biological LPS activity was described. After a
single dose of 10 ng of LPS administered i.v., an increase of
the pulmonary retention of radiolabeled YAC cells, lymphocytes or
thymocytes, was observed. Rabbit anti-LPS antibody, mixed with LPS,
changed (decreased) the biological activity of LPS and the increase
in pulmonary cell retention was diminished.
Laboratory of Immunopathology
Head: Irena Frydecka, M.D., Professor
Immune deficiency in neoplastic
diseases
A part of our studies involved studying the
expression of the co-stimulatory CD28 and the down-regulatory CTLA-4
molecule on peripheral blood T lymphocytes derived from patients with
multiple myeloma (MM). The mean percentages of CD4+ and
CD8+ lymphocytes co-expressing the CD28 molecule was
statistically lower in MM patients than in controls. CTLA-4
expression was examined on unstimulated and on
anti-CD3+rIL-2-stimulated peripheral blood T lymphocytes stimulated
for 24, 48, 72 and 96 hours. We found a negligible percentage of
CTLA-4+/CD3+ cells before culture and after 96
hours of stimulation from MM patients as well as from controls. After
stimulation, the mean percentage of CTLA-4+/CD3+
cells from healthy subjects increased gradually, reaching a peak
after 72 hours of culture. In MM patients the mean proportion of
CD3/CTLA4+ cells was significantly higher compared with normal
subjects after 24, 48 and 72 hours of stimulation. Additionally, all
the MM patients studied exhibited different kinetics of CTLA-4
molecule expression, with a peak after 24 - 48 hours of stimulation.
Our data have shown an impaired expression of co-stimulatory CD28,
different kinetics and an increased expression of down-regulatory
CTLA-4 molecule, which, in consequence, may result in a
down-regulation of the immune response in these patients. Similar
results were obtained in patients with chronic lymphocytic leukemia,
Hodgkin's disease and cervical cancer.
Additionally, we have
studied the effect of human lactoferrin (HLF) on the expression of
the signal transduction CD3zeta chain on peripheral blood T cells
from patients with cervical cancer. HLF normalized the decreased
CD3zeta chain expression after 72 hours of culture. The results of
the study suggest the possibility of a clinical use of HLF in cancer
patients.
A part of our studies was devoted to the expression
of cell cycle regulatory proteins: cyclins D2, D3 and the inhibitor
of cyclin-dependent kinases p27 which are involved in the regulation
of G1 progression of the cell cycle. The study was performed in
patients with B-cell chronic lymphocytic leukemia (B-CLL). The
expressions of these proteins were estimated in leukemic lymphocytes
(CD19+/CD5+) and compared with normal lymphocytes. The proportion of
CD19+/CD5+ lymphocytes which synthesized cyclin D3 was statistically
lower, and of those producing cyclin D2 statistically higher, in
comparison with a control group. There was no correlation between the
increased proportion of CD19+/CD5+/D2+ cells and the proportion of
cells in the G2/M phase of the cell cycle. These observations suggest
that over-expression of cyclin D2 may block the cell cycle
progression of B-PBL cells and inhibit cyclin D3 synthesis.
The mean level of p27
expression in leukemic lymphocytes was statistically higher compared
with normal B cells. Our results revealed a severely impaired
production of cell cycle regulatory proteins in B-CLL.
DEPARTMENT OF MICROBIOLOGY
Head: Jolanta Zakrzewska-Czerwińska, Ph.D.,
Associate Professor
Laboratory
of the Molecular Biology of Microorganisms
Head: Jolanta Zakrzewska-Czerwińska,
Ph.D., Associate Professor
The molecular basis of replication
and gene expression and designing of compounds inhibiting these
processes
Modular
polyketide synthases (type I) catalyze the biosynthesis of
polyketides - natural products with structural complexity and
remarkable medicinal properties. The biological activity of a new
type I polyketide synthase from Streptomyces bacteria has been
studied. Western-blot analysis was performed using polyclonal
antibodies directed against a ketosynthase domain as well as a
thioesterase protein representing a putative, type I polyketide
synthase multi-enzyme complex from Streptomyces coelicolor
A3(2). Immunoblot analyses of protein extracts from S. coelicolor
A3(2) mycelium harvested at different times of cultivation revealed
that formation of high-molecular-weight protein is growth phase
dependent and remains at the exponential phase.
In
light of the recent discoveries that the Streptomyces
chromosome is linear and that the oriC shows a higher
complexity than the oriC regions of unicellular bacteria, it
is interesting to better understand the initiation of DNA replication
in these myceliar prokaryotes. The Streptomyces oriC region
contains two clusters of DnaA boxes (nineteen) separated by a
flexible spacer. The Streptomyces DnaA protein consists, like
all other DnaA proteins, of four domains: domain III and the
carboxyterminal part (domain IV) are responsible for the binding of
ATP and DNA, respectively. We suggest that the arrangement of the
DnaA boxes allows the DNA-bound DnaA protein to induce bending and
looping of the oriC region. Domains I and III are
independently involved in dimerization of the S. lividans DnaA
protein molecules. We postulate that domain I and domain III could be
involved in cooperativity at distant and at closely spaced DnaA
boxes, respectively. The long domain II extends the range over which
the N-termini (domain I) of DNA-bound DnaA protein can form dimers.
Thus, interactions between DnaA molecules may bring the two clusters
of DnaA boxes separated by the spacer into functional contact by loop
formation.
The
key elements of the initiation of Helicobacter pylori
chromosome replication, DnaA protein and the oriC region, have
been characterized. The gene arrangement in the H. pylori dnaA
region differs from that found in other eubacterial dnaA
regions (rnpA-rmpH-dnaA-dnaN-recF-gyrB). H. pylori dnaA is
flanked by two open reading frames with unknown function, while the
dnaN-gyrB and rnpA-rmpH loci are separated from
the dnaA gene by 600 kbp and 90 kbp, respectively. We showed
that the dnaA gene encoding the initiator protein DnaA is
expressed in H. pylori cells. The H. pylori DnaA
protein, like other DnaA proteins, could be divided into four
domains. We demonstrated that the C-terminal domain of H. pylori
DnaA protein is responsible for specific DNA binding. Using in
silico and in vitro studies, the oriC region has
been located upstream of the dnaA gene. DNase I and gel
retardation analyses show the presence of at least four DnaA binding
sites, DnaA boxes within the H. pylori oriC region.
Germline
mutations of the p53 tumor suppressor gene, like the inherited
alterations of other tumor suppressor genes, determine a high risk of
cancer disease. Data from molecular analysis of the p53 in cancer
families indicate that germline p53 mutations are extremely rare in
Poland. The complex cancer phenotype observed in carriers of germline
p53 mutations and the overlapping of cancer phenotypes associated
with p53, BRCA1, and other genes mean that molecular tests are
indispensable for an identification and diagnosis of at-risk
individuals.
Indolo[2,3-b]quinolines
are a new family of DNA intercalators showing significant
cytotoxicity. The mechanism of their action is based on the
inhibition of DNA topoisomerase II activity. This depends on their
ability to induce and stabilize drug-topoisomerase II-DNA cleavable
complexes. The rational structural modification of
indolo[2,3-b]quinolines led us to synthesize hybrid molecules
composed of a 6H-indoloquinoline core (DNA intercalator) and an
alkylaminoalkyl chain (DNA-minor groove binder). Site-specific
intercalation of 6,11-dimethyl-6H-indolo[2,3- b]quinoline was
analyzed in vitro by Dnase I footprinting and by molecular
modeling. The results of the study allow the following
generalizations to be made: (i) the intercalator was found to bind
preferentially to the pBR 322 DNA plasmid in the
5’-TGCTAACGC-3’
region between adjacent adenine bases, (ii) diethylaminoethyl
substituent introduced at the N-6 position of indolo[2,3-b] quinoline
fits best (of several analogues analyzed) to the DNA minor groove,
(iii) the selected compound, synthesized by a modified Graebe-Ullman
reaction, showed significant cytotoxic properties (ID50=1.8 mM),
inhibited the activity of topoisomerase II at 1.5 mM and interacted
with the cellular cycle (G2M inhibition at 5 mM).
Laboratory of Signaling Proteins
Head: Wojciech Gorczyca, Ph.D.,
Associate Professor
Function and physicochemical
properties of proteins involved in Ca2+ -dependent signal
transmission by cAMP and cGMP in cells of the immune system
Our
studies have focused on: 1) the determination of guanylyl cyclases
activity in different cells of the immune system, 2) analysis of
structural properties of calcium-binding proteins belonging to the
Neuronal Calcium Sensors (NCS) family, and 3) the
identification of autoantibodies recognizing proteins of neuronal
origin.
Cyclic
GMP (cGMP) is synthesized by two distinct forms of guanylyl cyclases
(GC), particulate (pGC) and soluble (sGC), which differ in structure,
subcellular localization, and mechanism of activation. Reports
concerning activity of both GC forms in cells of the immune systems
are contradictory. Thus, the aim of our studies was to determine
which form of GC is responsible for cGMP synthesis in macrophages,
neutrophils, as well as in T cells at different stages of maturation.
The intact cells were treated with ANP (atrial natriuretic peptide -
the particulate GC activator) or with SNP (sodium nitroprusside - the
soluble GC activator) and the cGMP formed was measured. Additional
experiments were performed on isolated cytosolic and membrane
fractions of macrophages and neutrophils. We found that, in rat
peritoneal macrophages (PM) and neutrophils, the main active form of
the enzyme is particulate GC, while in guinea pig PM, soluble GC is
responsible for cGMP synthesis. These observations suggest that the
mechanisms regulating cGMP formation might be species specific.
Experiments on isolated membrane fractions indicated that
dephosphorylated particulate cyclase loses its activity. We also
found that murine T cells are able to synthesize cGMP using both
forms of guanylyl cyclases; however, the pattern of GC activities
appeared to be dependent on the maturation stage of the cells. Since
the main enzyme regulated by cGMP is protein kinase PKG1, we have
also tested its expression in T cells. Our results indicate that PKG1
could be a target for cGMP in thymocytes but not in unstimulated
mature single positive cells, and we suggest that cGMP and PKG1 might
be involved in the development of T cells.
Recoverin
and guanylyl cyclase-activating proteins (GCAP1 and GCAP2) are
homologous proteins recently found in the retinas of vertebrates.
Both GCAPs have been shown to regulate the activity of retinal
guanylyl cyclase in a calcium-dependent manner. Recoverin has been
postulated to be a calcium-dependent regulator of rhodopsin kinase.
All three proteins belong to a larger family of neuronal
calcium-binding proteins, the so-called Neuronal Calcium Sensors
(NCS). Since recoverin binds to Phenyl-Sepharose in a
calcium-dependent manner, while GCAPs do not, we tried to answer the
question of how calcium ions influence the hydrophobicity of all
three proteins in solution. Using different approaches, we have found
that the binding of calcium by GCAPs apparently decreases their
hydrophobicity, what is opposite to the effect of calcium on
recoverin. These results indicate that recoverin and GCAPs, although
structurally related, differently change their conformations after
binding of calcium. Our data also support earlier reports concerning
the differential calcium-dependent binding of recoverin and GCAPs to
biological membranes. Structural 3D models that fit the above
observations indicate that GCAPs, unlike recoverin, rather remain in
the "open form” at low calcium concentration. We also obtained
rabbit sera against several NCS proteins and confirmed that
antibodies better recognize NCS proteins in their Ca2+-loaded
form.
Neurodegenerative
disorders can be associated with cancers which have not invaded the
nervous system. Such a "remote" effect of cancer is called
"paraneoplastic syndrome" and is believed to be based on an
autoimmune response to the expression of neuronal proteins by
neoplastic cells. The aim of our studies was to find if the sera of
patients with different cancers or neurological diseases contain
antibodies recognizing neuronal antigens. We have detected several
antibodies recognizing antigens of different molecular weight.
Identification of these antigens is in progress.
DEPARTMENT
OF CLINICAL IMMUNOLOGY
Head:
Andrzej Lange, M.D., Professor
Laboratory of
Immunogenetics
Head: Piotr
Kuśnierczyk, Ph.D., Associate Professor
The role of major histocompatibility
complex molecules in immune reactions and the factors affecting their
expression
A
model of mouse fibroblasts transfected with class II MHC molecule
covalently linked to antigenic peptide as well as mouse CD4+ T-cell
hybridomas specifically responding to this MHC/peptide complex was
used. The Interleukin-2 (IL-2) responses of the hybridomas to
fibroblasts presenting specific peptide alone or in the presence of
naturally bound peptides were compared. Surprisingly, hybridoma cells
responded stronger to the antigenic peptide when other peptides were
present on the same cells. This unexpected finding might result from
a cross-reactivity of some naturally bound peptide(s) with the T-cell
receptors of the hybridoma cells that was too weak to stimulate the
hybridoma on its own but acted synergistically with the specific
peptide.
In
earlier projects we showed that nonapeptide HIV-1 p24gag144-152
did not affect cell surface HLA-C expression, although octapeptide
p24gag145-152 did stabilize the HLA-C upon binding. We
found that a B-lymphoblastoid cell line HAJ generated in our
institute was extremely resistant to hygromycin B and gamma
irradiation. This cell line was found to be HLA class II negative and
class I low, similarly to bare lymphocyte syndrome cells. Interferon
gamma increased class I but did not restore class II expression,
although class II genes were shown earlier to be present in these
cells. The paper describing the association of C4A and C4B complement
component alleles with psoriasis vulgaris was submitted for
publication.
Laboratory
of Clinical Immunology
Head: Andrzej Lange,
M.D., Professor
The genetic basis and
pathomorphological verification of allogeneic reaction following
grafting of hematopoietic cells
In studies on genetic
markers used for recipient-donor matching and on the assessment of
the risk factors associated with transplant-related morbidity, we
revealed an association between the genotype of the TNFA and TNFB
genes and susceptibility to transplant-related toxicity. An influence
of IL-6 gene polymorphism on the potential to generate the cytokine
was shown, and the IFNg gene
genotype was found to be associated with the incidence of fatal GvHD
but not with toxicity.
Elutriation was employed
and the technique was optimised for the removal of T cells from G-CSF
mobilised peripheral blood cells used for allotransplantation and for
the discrimination of bcr/abl positive and negative cells from
non-mobilised newly diagnosed CML patients’ progenitor cells.
Peripheral blood
progenitor cells (PBPC) were characterised for the presence of CXCR4
and it was found that progenitors in the periphery differed from
those residing in the bone marrow with respect to the coexpression of
this receptor.
It was found that ovarian
carcinoma cells may exploit either resistance to apoptosis or
proliferative capacity to expand, and this was associated with a
different gene cluster expression (Ki67, p53, bcl-2 and IL-6 genes).
It was documented that
tumours IL-6 gene expression influenced CRP level and the
accumulation of CD45RO+ cells associated with the survival of ovarian
carcinoma patients.
It was shown that
HLA-DRB1 and DRB3 associated features independently influenced the
course of sarcoidosis.
The STR (short tandem
repeats) technique was employed for monitoring bone marrow
transplant patients for chimerism and the pace of chimerism post BMT
was described for patients receiving a more and a less aggressive
conditioning regimen.
Computer tools were
employed for donor-recipient matching and this documented the fact
that the progress in telematics and communications has improved the
efficacy of the donor search procedure.
It was discovered that
KIR (killer inhibitory receptors) influenced the proliferation
of NK cells in the MLC, depending on the HLA of the stimulators.
An
association between apoptosis and the accumulation of cyclins and
Ki67 antigen in epidermal cells in Graft-versus-Host-Disease was
discovered.
DEPARTMENT
OF MEDICAL IMMUNOLOGY
Head:
Andrzej Górski, M.D., Professor
Laboratory
of Bacteriophages
Acting head: Beata Weber-Dąbrowska,
Ph.D., Assistant Professor
The immunobiology of bacteriophages
and their application in the therapy of bacterial infections
During the last year we
started preparing a large amount of phage lysates of Staphylococcus
and Pseudomonas. Attempts were undertaken to obtain pure phage
preparations using a modified method of column chromatography.
Specific bacteriophages
were applied in the treatment of long term bacterial infections in 20
cancer patients. In all cases, the infections were caused by
multi-drug resistant bacteria and, as a result, antibiotic treatment
had failed. Phage therapy of the infections was effective in all the
patients.
In collaboration with the
Department of Immunology, Institute of Transplantology, Medical
Academy in Warsaw, preliminary results were obtained showing that
short incubation of human lymphocytes and monocytes with
bacteriophage lysates may induce intracellular cytokine synthesis.
Further studies are currently underway involving purified phages.
The Laboratory of
Bacteriophages started collaboration with the Agriculture Academy in
Wrocław. The bacterial flora from several infected boars was isolated
and identified. These studies are still underway.
Phage therapy was used
extensively in last year in the treatment of bacterial infections
caused by drug resistant bacteria of different origin. Therapy was
applied in 143 patients with infections of different organs and
tissues. Positive therapeutic effects were obtained in 83% of the
cases.
We have continued studies
on the immunoregulatory effect of phage therapy. It was found earlier
that effective phage therapy was associated with the normalization of
cytokine production (TNF-a and
IL-6) by blood cell cultures. In the last two years, other
parameters, such as blood picture, phagocytosis and serum lactoferrin
levels, were investigated. The results obtained indicate that
regulation of these parameters also correlates with positive effect
of phage therapy.
Trials were undertaken to
isolate and characterize a set of phages active against Enterococcus.
New phages will be useful in the treatment of persistent infection
caused by multi-drug resistant Enterococcus and in studies on
the immunoregulatory effect of phage therapy.
Laboratory of
Reproductive Immunology
Acting head: Małgorzata
Jerzak, M.D., Assistant Professor
Immunological aspects
of reproduction failures
b1
integrins are receptors responsible for the binding of extracellular
matrix proteins that mediate activation, adhesion, migration and
differentiation in different tissue compartments. It has recently
been demonstrated that integrins are distributed on the surfaces of
spermatozoa and on T cells, as well. It is also known that sperm
antibodies are found in 10- 15% of women with unexplained
infertility. The aim of the study was to establish some parameters of
the cellular and humoral immune responses in women with unexplained
infertility. We determined that sperm antibody-negative infertile
women have increased PHA-activated T-cell adhesion to collagen
(C-IV), elastin (E) and fibronectin (FN), and PMA-activated T-cell
adhesion to C-IV and E compared with normal healthy women (P<0.05).
Our data suggest the existence of disturbed T-cell:ECM interactions
in infertile women. Further studies are needed to determine the role
of those abnormalities in infertility.
Previous
studies revealed that 1,25-dihydroxyvitamin D3
(calcitriol)-induced differentiation of human promyelocytic leukemia
cells leads to an increased resistance of the cells to
apoptosis-inducing agents. Present results suggest that the acquired
resistance to pro-apoptotic agents in HL-60 cells is not mediated by
the CD95 receptor/ligand system, but by an increased sensitivity of
differentiated cells to survival factors. One of the survival factors
is insulin, which exerts its activity in a PI3 kinase-dependent way.
Apoptosis has been
proposed as a mechanism for maintaining tolerance in the immune
system. Expression of Fas ligand (FasL) by the human trophoblast has
recently been accepted as a mechanism providing protection against
the lytic action of decidual immune cells. Therefore, the purpose of
this study was to determine the role of T-cell apoptosis in pregnancy
maintenance. The apoptosis of T cells after culture with
extracellular matrix proteins, which are a part of the placental
structure, was studied. Preliminary data suggest that disturbances in
the programmed cell death of activated T cells in the human decidua
can be responsible for pregnancy loss. Therefore, the apoptosis of
activated cytotoxic T cells might be an interesting possible
explanation of successful pregnancy outcome.
Laboratory of Tissue Immunology
Acting head: Beata Nowakowska, Ph.D.,
Assistant Professor
HLA profiles
in the south-western Polish population
The
HLA class II DR, DQ and DP antigens are heterodimeric cell surface
glycoproteins that play a central role in the human response. They
are characterized by their extensive degree of allelic polymorphism
and the phenomenon of linkage disequilibrium. A knowledge of the
allelic frequency and linkage in haplotypes is important both for
general population genetics and for the selection of suitable
donor-recipient pairs for transplantation. Allele matching within
these loci significantly improves the outcome of transplantation,
especially that of bone marrow. The extensive polymorphism of these
genes is useful as a genetic marker for anthropological and disease
studies, as well.
The
aim of our first project was to establish the gene polymorphism of
the HLA-DPB1 locus and a linkage analysis of alleles from the HLA D
- region. The sample consisted of DNA from 41 unrelated individuals
(parents) from families originating from the south-west of Poland.
Family data are most informative for defining the haplotypic profile
and for assessing the pattern of linkage disequilibrium in a
population.
Population
frequencies of the alleles of the HLA DPB1 locus have not yet been
described in the Polish population. Among the 82 different DPB1
alleles, only 18 were represented in our material. The allele
DPB1*0401 had the highest frequency (33.9%), followed by DPB1* 0201
(12.9%), DPB1* 0402 (11.3%) and DPB1*0301 (8.1%). The rest of the
alleles were present with frequencies ranging between 1.0 - 4.0%. The
results obtained do not differ substantially from data published on
other European Caucasoid populations. The heterozygosity index (H)
for the DRB1 locus was high, at H= 0.8387. The DPB1 locus allelic
distribution was close to the neutrality expectation (the
Fisher-Wright neutral allele model) with a Watterson index of
homozygosity of F= 0.1594.
We investigated the linkage in the
haplotypes DRB1- DQA1- DQB1 and associations with DPB1 alleles. A
total of 82 haplotypes were included in the analysis. The frequent
associations DRB1* 1501- DQA1* 0102- DQB1*0602, DRB1*0701-
DQA1*0201- DQB1* 0201 and DRB1*0301- DQA1* 0501 - DQB1*0201 found in
our population have also been reported in many other Caucasoid
populations. The haplotype DRB1*0701- DQA1*0101 - DQB1* 0303 was more
frequent in our population than in others. We found only one frequent
association of the alleles loci DR, DQ and DP: DRB1*1501 -DQA1*0102 -
DQB1* 0602 and DPB1* 0401. It is interesting that one of the most
frequently cited examples of positive disequilibrium, the haplotype:
DRB1* 0301- DQA1*0501 -DQB1* 0201 and DPB1* 0101, was not present in
our material.
Laboratory of Virology
Acting head: Zofia Błach-Olszewska,
Ph.D., Professor
Innate immunity in viral infections
The local and systemic response of
nitric oxide in relation to the production of IL-6 during the course
of bronchial asthma was evaluated in the studies. The high production
of nitric oxide by bronchoalveolar and peripheral blood leukocytes,
irrespective of steroid treatment, airway infections or the
exacerbation or remission of asthma symptoms, suggests that NO may be
considered as a marker of chronic inflammation in asthmatics, whereas
IL-6 may only be implicated in the processes of the acute phase of
the disease.
In another study we revealed a
reduction of the innate antiviral immunity of BALB/c mice treated
intraperitoneally with cyclosporine A. The effect was dose-dependent
and statistically significant.
Interferon
levels in 249 sera specimens from HIV/AIDS patients were also
determined. All of the patients were on a combined antiretroviral
therapy and not treated with IFN. Pathologically enhanced IFN levels
(>20 U/ml) were found only in the sera taken before the beginning
of the therapy or during unsuccessful therapy. The interferon was
identified as acid labile IFN-a.
Ovine
colostrinine induced IFN-g and TNF
in whole-blood cultures of HIV- or HCV-infected patients. In
progressed HIV infection, a decreased ability of the cells to produce
cytokines was observed. A significant correlation between cytokine
levels after PHA or LPS stimulation and after colostrinine treatment
was found. The results suggest that colostrinine acts similarly to
the classical cytokine inducers in the cases of immune defects caused
by HIV infection.
Antiviral properties of 64 new
organoselenium compounds, analogs of ebselen, were measured. Ebselen
and many of the other compounds were shown to have anti-HSV-1 and
anti-EMCV activities. However, only a weak anti-VSV effect was
observed.
In addition, the antiviral activity of
photosensitisers was studied in culture medium and in the presence of
human blood. The enveloped viruses (HSV-1 and VSV) were
photoinactivated by the compounds. However, there was no activity
against EMCV, a non-enveloped virus. The presence of blood
considerably decreased the antiviral effects.
Laboratory of Cellular Interactions
Head: Danuta
Duś, Ph.D., Associate Professor
The interaction of tumor cells with
endothelium involving constituents of the immune system in the
neoplastic process
Extravasation of tumor cells is a prerequisite for distant tissue
colonization and further metastasis development. A necessary step in
the process is the adhesive interactions of endothelial cell adhesion
molecules with their ligands, presented by glycosylated tumor cell
surface molecules. The aim of the study was to search for new human
tumor markers engaged in the blood-borne metastatic spread of tumor
cells. This is one of the possible ways to learn about possible
further interference in the process of the mutual interactions of
tumor cells with endothelial cells at the site of tumor cell
extravasation. The studies were performed on two main subjects: tumor
cells and partner endothelial cells. The first was a continuation of
the search for a correlation between the expression of the tumor
markers chosen, i.e. tumor-associated antigens (TAA), and metastatic
potential in vivo, in immuno-incompetent mice for established
tumor cell lines of human colon carcinoma. In our previous study we
demonstrated that the acquisition of tissue-specific highly
metastatic phenotype by the selected variant LS-180 human colon
carcinoma cells was accompanied by an increased expression of Lewis
antigens, which correlated with their adhesion pattern to endothelial
cells. A recent study on these variant cells, done in cooperation
with Prof. H. Debray from the University of Lille, France, revealed
differential ?1-3/1,4 fucosyltransferases activities which catalyze,
among others, the biosynthesis of sialyl Lex and sialyl
dimeric Lex as well as Lea and
sialyl Lea. The results obtained indicate that colon
carcinoma variant cells differently synthesize glycoconjugate ligands
for endogenous lectins, which may be of potential use for them during
recruitment to a specific metastatic site (manuscript in
preparation).
In collaboration with Prof. Z. Kiliańska's group from the Department
of Cytobiochemistry of the University of Łódz, a new nuclear
protein, p36, which accompanies colon carcinoma, has been described.
A part of studies done in collaboration with dr. T. Kręcicki from
Department and Clinic of Otolaryngology, Medical University of
Wrocław, concerned the prognostic value of molecular markers in
laryngeal cancer. Our previous results proved the potential value of
nm23 gene product expression evaluation in predicting the
metastatic capacity of laryngeal tumors (manuscript in preparation).
Recently, the products of two oncogenes: c-myc and bcl-2XL
are under study.
The second subject was the further characterization of human
endothelial cells, which are the tumor cell interaction partners
during the process of metastasis. The vascular endothelium is a site
of inflammatory reactions. As a response to specific signals,
adhesion molecules are induced to recruit leukocytes as well as tumor
cells. By studying a panel of eight immortalised human endothelial
cell lines of distinct tissue origin (Patent No 99 16169, 21/12/99,
CNRS, France), their organ-specificity was confirmed by proof of the
differential display of endogenous lectins and cytokine receptors, in
addition to the tissue-specific sets of addressins and other adhesion
molecules.
We
described for the first time the ability of human endothelial cells
to be stimulated by IL-7 due to the presence of specific IL-7
functional receptor. We also found that the human endothelial cell
lines displayed selective reactions towards H2O2-mediated
oxidative stress, as well as low energy 808 nm laser stimulation,
which resulted in modulation of the NO level and the transient
expression of some adhesion molecules, respectively (manuscript
submitted).
In
studies performed in cooperation with Dr. J. Heimrath from the
Department of Reproduction and Obstetrics of the Medical University
of Wrocław, we followed the role of the presence of trophoblasts and
activated endothelium-secreted products VCAM-1 and endothelin in the
peripheral blood of pregnant women -1 in the pathogenesis of
pregnancy-induced hypertension (PIH).
DEPARTMENT
OF INFECTIOUS DISEASE MICROBIOLOGY
Head:
Andrzej Gamian, Ph.D., Associate Professor
Laboratory of Medical Microbiology
Head: Andrzej Gamian,
Ph.D., Associate Professor
The pathogenesis of some autoimmune
diseases of bacterial etiology: the role of sialic acid, glycolipids,
endotoxins and bacterial proteins
The general strategy for
the elaboration of the protective tools against invading bacteria
involves the determination of the structures of the molecules
involved in the infection and immune processes, their chemical and
genetic manipulations, as well as understanding their biological
activities. Sialic acid is one of the key molecules on the surface of
tissue cells participating in immunological functions. In some
bacteria, sialic acid may be a constituent of surface antigens, and
the occurrence of sialic acid is associated with an increased
pathogenicity of bacteria; it is particularly involved in autoimmune
processes. In the group of bacterial pathogens studied in our
laboratory, the function of sialic acid in endotoxins is not known.
In the course of our studies, the structure of the O-specific
polysaccharide repeating unit from Salmonella Toucra O48 has
been determined as:
®4)-a-Neup5NAc7,9OAc(2®3)-L-a-FucpNAc(1®3)-D-b-GlcpNAc(1®.
Sialic acid is also structurally similar to 3-deoxy-octulosonic acid,
an inherent component of endotoxin. Thus, the interference of these
bacterial components with functions of tissue sialic acid may
contribute to the mechanisms of pathogenicity. Due to the structural
mimicry of sialic acid-containing structures, care should be taken to
avoid the induction of autoantibodies when antibacterial vaccines are
constructed.
Most
of the research on sepsis in the last ten years has focused on
methods of blocking cytokine responses. The failure of this approach
has encouraged a return to basic principles, which means to enhance
resistance to infection and to characterize the inflammatory reaction
in the individual patient. Our studies are thus focused on the
developing methods of protection against infection and monitoring
sepsis and septic shock. Neutralization of endotoxin is still one of
the most effective and safe ways to protect against bacterial
infections. The monitoring of specific markers for sepsis and septic
shock could significantly facilitate the prognosis of these diseases
and their treatment. In a collaboration with the Clinic of
Anesthesiology and Intensive Care at the Medical Academy in Wrocław,
studies were carried out on some biochemical markers of septic shock.
Neopterin was found to be an early marker for the prognosis of the
development of septic complications, since patients who died from
septic shock or multiorgan failure had elevated concentrations of
this marker. In another study, we confirmed the value of neopterin
and procalcitonin measurements as diagnostic tools in monitoring the
clinical course of septic patients and those with postoperative
complications. Current work focuses on a determination of endotoxins
as markers in the monitoring of different stages of septic shock and
immune status during treatment.
DEPARTMENT
OF CANCER IMMUNOLOGY
Acting
head: Leon Strządała, Ph.D., Associate Professor
Laboratory of Tumor Immunology
Acting head: Adam Opolski, Ph.D.,
Assistant Professor
The mechanisms of the proliferation,
differentiation and apoptosis of tumor cells, metastasis and tumor
progression. Studies on the antitumoral effects of cytostatics
The
aim our investigation was to elaborate in vivo models to study
the pathogenesis of metastasis using human and murine tumor cell
lines. The antitumoral and antiangiogenic effects in vivo of
genistein applied alone or combined with cyclophosphamide was also
studied.
We
prepared a model to determine the amount of blood in the tumor
tissue, which should correspond to the degree of its vascularisation.
Using the B16F-10 melanoma model, no antiangiogenic effect of
genistein was detected. In contrast, a 44% tumor growth inhibition
and a 60% blood volume reduction by genistein was observed. These
results indicate a higher antiangiogenic than cytostatic effect of
genistein. A synergistic antiangiogenic effect of genistein combined
with cyclophosphamide was observed in mice bearing transplantable
Lewis lung cancer.
Hu
1703He cells express a high level of sialosyl LewisA
antigen on their surfaces, while HCV 29T cells are sialosyl
LewisA-negative. These cancer cells, in addition to their
tumorigenic and invasive properties, are highly metastatic when
inoculated into athymic nu/nu mice. The ability to form secondary
tumor foci in the lung and liver seems to be, in these uroepithelial
cells, independent of the expression of sialosyl LewisA
antigen.
Our
results also showed that pretreatment of HL-60 human promyelocytic
leukemia cells with calcitriol or its new analogues significantly
potentiates their sensitivity to the antiproliferative effect in
vitro of cisplatin, doxorubicin or genistein. The mechanism of
this potentiating effect remains obscure and is currently under our
investigation.
New
methotrexate-fibrinogen conjugates, obtained by Dr. J. Boratyński
according to his own original procedure, revealed a higher
antitumoral activity than free methotrexate. They were, however, more
toxic, and this problem is currently under our investigation. The
newly designed conjugates should be less toxic but still possess or
increase their antitumoral activity.
Studies of the NO
production in the tissue of syngeneic, transplantable mouse colon
cancer MC38 were undertaken. Initial injections of IL-2
gene-transfected allogeneic cells (X63-mIL-2) exerted an antitumoral
effect, but did not significantly change the level of NO production.
However, after 3 or 4 peritumoral injections an increase in the
nitric oxide level was observed.
Laboratory
of Cellular Immunology
Head: Leon Strządała, Ph.D.,
Associate Professor
Signaling disturbances in the
apoptotic cellular pathways in normal and malignant differentiation
Previous
studies have shown that up to 50% of mice expressing a transgenic TCR
specific for male (H-Y) antigen develop spontaneous thymic lymphomas.
Disturbance of homeostasis through the deregulation of proliferative
pathways and repression of cell death may be the main mechanisms of
tumorigenesis. In agreement with this possibility, we found that
over-expression of the Ras, Raf, and L-myc proteins, but not the
Bcl-2 family proteins, plays an important role in the basal oncogenic
potential of TCR transgens and resistance of the lymphomas to
TCR-induced apoptosis. Further, it was found that a defect of
apoptosis in these cells is located downstream of the Nur77 induction
and upstream from the execution phase of apoptosis.
The aim of the
studies on the role of Fas/FasL in the induction of apoptosis in
thymic lymphomas was to learn more about a defect in the
triggering of the execution phase of apoptosis and the resistance of
the lymphomas to Nur77-mediated apoptosis. Results show that a defect
in the signaling pathway leading to apoptosis in the lymphoma cells
involves regulation of Fas/FaL expression in two separate ways.
Mice
that are heterozygous for the APC gene mutation (MIN and APCD1638)
develop intestinal tumors within several weeks of life. We
established cell lines originating from normal epithelial cells of
the small intestine of the C57BL/6 mouse (B1V), normal and tumor
cells of the small intestine of the APCD1638+/-
mouse (AC1 and AN1, respectively) and tumor cells of the small
intestine of the MIN+/- mouse (MIN). The cell lines expressed high
levels of Ras and Raf. In cells originating from tumors (AN1, MIN),
we observed a higher level of expression of anti-apoptotic proteins
(Bcl-2, Bcl-xl) and pro-apoptotic Bax protein than in cell lines
established from normal tissue (AC1, B1V). In addition, we observed
that AN1 and MIN cells, in contrast to B1V cells, underwent apoptosis
after treatment with ionomycin. AN1 cells underwent apoptosis after
treatment with etoposide. None of the examined cell lines underwent
apoptosis after treatment with dexamethason. The examined cell lines
expressed Fas receptor on their surface, but did not express FasL.
B1V and MIN cells underwent apoptosis after stimulation of the Fas
receptor. We also examined the differences in gene expression between
normal tissue of the small intestine and tumor from the same mutant
MIN+/- mouse. Using cDNA-RDA and RT-PCR techniques, we identified a
group of 13 genes that are over-expressed in the tumor cells.
Stimulation
of PC12 cells with nerve growth factor (NGF) induces their neuronal
differentiation, while treatment with glucocorticoids (dexametasone)
promotes adrenergic differentiation toward neuroendocrine cells. Our
results show that etoposide induces caspase-dependent apoptosis of
PC12 cells., which is inhibited by ZVAD-fmk, and that dexamethasone
inhibits spontaneous apoptosis as well as etoposide-induced death of
PC12 cells.
|
