IITDLogo
Research Report - 2000

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.