Volume 50 (2004) No. 6

Volume 50 (2004) No. 6
Original Articles
Allogeneic Gene-Modified Tumour Cells in Metastatic Kidney Cancer. Report II
G. PIZZA1, C. DE VINCI1, G. LO CONTE1, A. MAZZUCA1,  V. DI MAIO1, S. RATINI1, G. SEVERINI1, L. BUSUTTI2,
A. P. PALARETI3, A. GULINO4, A. VACCA4, L. MELCHIORRI5, M. FERRARI6, L. GIACOMELLI7,
O. R. BARICORDI5, S. FORZINI8, R. CAPANNA8.........................................................................................175
1Immunotherapy Module, Operative Unit (O. U.) of Urology, Department of Urology and Nephrology, and 2O.U. of Radiotherapy,
Department of Oncology, S. Orsola-Malpighi Hospital, Via P. Palagi, Bologna, Italy
3Department of Computer Science, University of Bologna, Italy
4Experimental Medicine, University “La Sapienza”, Rome, Italy
5Department of Experimental and Diagnostic Medicine, Section of Medical Genetics, University of Ferrara, Ferrara, Italy
6Experimental Institute of Zooprophylaxis, Emilia-Romagna and Lombardia Regions, Brescia, Italy
7Department of Surgical Science University of Rome "La Sapienza", Rome, Italy
82nd Division of Orthopaedics and Reconstructive Surgery Centre, Florence, Italy
Corresponding author: Giancarlo Pizza, Immunotherapy Module, Operative Unit of Urology, Department of Urology and Nephrology,
S. Orsola-Malpighi Hospital, Via P. Palagi, 9, 40138 Bologna, Italy.   Tel.: +39-051-6362478;    Fax: +39-051-6362476; e-mail: gpizza@med.unibo.it. 
Abstract.
Full text. 175-183

Immunogenicity of Dendritic Cell-Based HPV16 E6/E7 Peptide Vaccines: CTL Activation and Protective Effects
M. INDROVÁ*, M. REINIŠ*, J. BUBENÍK, T. JANDLOVÁ, J. BIEBLOVÁ, V. VONKA1, J. VELEK2..........184
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague 6, Czech Republic
1Institute of Haematology and Blood Transfusion, Prague 2, Czech Republic
2Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech   Republic, Prague 6, Czech Republic
Corresponding author: Marie Indrová, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic,
Flemingovo nám. 2, 166 37 Prague 6, Czech Republic.
Abstract.
Full text. 184-193
Aneuploidy  in  the  Transgenic  Rabbit
V.  PARKÁNYI1,  P.  CHRENEK1,   J.  RAFAY1,  K.  SUVEGOVÁ1,  R.  JURČÍK1, A. V. MAKAREVICH1,
J.  PIVKO1,  L.  HETÉNYI1, R. K. PALEYANDA2.....................................................................................194
1Research Institute of Animal Production  (RIAP),  Nitra, Slovak  Republic
2American Integrated Biologics, Inc., E. Woodstock,  USA
Corresponding author: Vladimír  Parkányi, Research Institute of Animal Production, Hlohovská 2, 949 92  Nitra, Slovak Republic.
Tel.: (+421) 37 6546 276; fax: (+421) 37 6546 480; e-mail: parkanyi@vuzv.sk
Abstract.
Full text. 194-199
Short Communications
The Role of NK1.1+ Cells in the Protection against MHC Class I+ HPV16-Associated Tumours
J. ŠÍMOVÁ, J. BUBENÍK, J. BIEBLOVÁ, T. JANDLOVÁ...............................200
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Corresponding author:
Jana Šímová, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic,
Flemingovo nám. 2, 166 37 Praha 6, Czech Republic.
Abstract.
Full text. 200-202
The -455G/A Polymorphism of the β Fibrinogen Gene and the Bgl II Polymorphism of the α2β1 Integrin Gene and
Myocardial Infarction in Patients with Type 2 Diabetes
G. POGLAJEN1, J. KIRBIŠ2, A. MILUTINOVIĆ3...............................................203
1Medical Center Medicor Izola
2Department of Cardiovascular Surgery, Medical Centre Ljubljana
3Institute of Histology and Embryology, Medical Faculty, University of Ljubljana
Corresponding author: Aleksandra Milutinović, Institute of Histology and Embryology, Medical Faculty, University of Ljubljana,
 Korytkova 2, 1105 Ljubljana, Slovenia. Tel +386 1 543 7382; Fax +386 1 543 7361; E-mail: sandramilutinovic@yahoo.com
Abstract.
Full text. 203-204
Original Articles
Allogeneic Gene-Modified Tumour Cells in Metastatic Kidney Cancer. Report II
G. PIZZA, C. DE VINCI, G. LO CONTE, A. MAZZUCA,  V. DI MAIO, S. RATINI, G. SEVERINI, L. BUSUTTI,
A. P. PALARETI, A. GULINO, A. VACCA, L. MELCHIORRI, M. FERRARI, L. GIACOMELLI,
O. R. BARICORDI, S. FORZINI, R. CAPANNA

In a limited study, comprising only ten patients, we have previously reported that allogeneic irradiated RCC-cell-line cells,
engineered to produce IL-2 (ACHN-IL-2), admixed with autologous metastatic formalin-treated tumour cells were used to vaccinate
MRCC patients in progression of disease and also receiving IL-2 immunotherapy. The cells, admixed to autologous TC, were administered subcutaneously. We now report an extended study on thirty patients and one hundred thirty-one controls. Patients received 4–20 injections (mean 10 ± 4), containing an average of 92 x 106 ± 45 x 106 ACHN-IL-2 transfected cells (a minimum of 25 x 106, and a maximum
of 200 x 106). Autologous TC, admixed to allogeneic, were also administered by 4–16 s.c. injections (mean 7 ± 3) i.e., a total  of
12 x 106–160 x 106  cells. Vaccination was administered during 73–1451 (307 ± 316) days, and the follow-up continued for
1122 ± 1240 days (106–5137). Throughout this period, the patients continued receiving the previously set immunotherapy treatment.
No adverse side effects related to the treatment were noticed. One complete and four partial tumour responses were observed, as well
as nine cases of stable disease. Thirteen patients died in the treated group (43%) and 63 (44%) in the control group. Responding patients resumed progression in 4–11 months and died 18 and 36 months after beginning the vaccine therapy. The Gehan Wilcoxon’s test showed a
significantly (P < 0.01) better survival in the vaccinated patients compared to that of the controls. Thus, we confirm, in an increased number
of patients and an extensive follow-up, that our vaccination protocol is safe, devoid of adverse side effects, and promising.
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Immunogenicity of Dendritic Cell-Based HPV16 E6/E7 Peptide Vaccines: CTL Activation and Protective Effects
M. INDROVÁ, M. REINIŠ, J. BUBENÍK, T. JANDLOVÁ, J. BIEBLOVÁ, V. VONKA, J. VELEK

We have investigated the capacity of cellular vaccines based on dendritic cells loaded with human HPV16 E6/E7 oncoprotein-derived
peptides to induce immune responses in vitro and to elicit protective immunity in a murine experimental model mimicking human HPV16-associated carcinomas. Dendritic cells loaded with the HPV16 E6/E7 peptides exhibiting cytotoxic T lymphocyte (CTL) or Th
epitopes (E641-50, E681-90, E698-107, E6130-137, E749-57, and E744-62) were able to stimulate in vitro DNA synthesis in  syngeneic  H-2b
spleen cells. The priming capacity of peptide-loaded BMDC and peptide-loaded dendritic cell lines DC2.4 and JAWS II was
compared.  It has been found that both, peptide-loaded BMDC and established dendritic cell lines can activate the syngeneic responder cells,
 but the priming capacity of BMDC was substantially higher. In the second set of experiments, we have examined the cytolytic activity of syngeneic spleen cells after repeated activation in vitro with BMDC loaded with HPV16 synthetic peptides containing CTL epitopes.
Significant cytotoxic responses against HPV16 E6/E7 antigen-expressing TC-1 targets have been found after repeated in vitro activation
with all peptides containing the CTL epitopes. In contrast, peptide E744–62 harbouring both Th and CTL epitopes induced significant
cytotoxic responses already after single in vitro activation. This cytotoxic effect could be enhanced with admixture of the E749-57 peptide. Experiments with MHC class I proficient (TC-1, MK16-IFNγ) and deficient  (MK16) target cells revealed that the dendritic cells loaded
with the E6/E7 HPV16 peptides activated CTLs in vitro, but not the other cytolytic effector mechanisms. The effectiveness of the
peptide-loaded BMDC-based cellular vaccines was also investigated in vivo. C57BL/6  (H-2b) mice were immunized with various peptide-loaded BMDC and subsequently challenged with TC-1 cells. The strongest protective effect was achieved with the BMDC
loaded with the peptide E744–62 harbouring both CTL and Th epitopes.  Mice immunized with the E744–62 peptide remained
tumour-free after s.c. transplantation of the TC-1 cells and exhibited long-lasting protective immunity, whereas the mice immunized
with E681-90 and E749-57 peptides did not remain tumour-free and exhibited only partial inhibition of tumour growth detectable as
depression of the tumour growth curves.
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Aneuploidy  in  the  Transgenic  Rabbit
V.  PARKÁNYI,  P.  CHRENEK,   J.  RAFAY,  K.  SUVEGOVÁ,  R.  JURČÍK, A. V. MAKAREVICH,
J.  PIVKO,  L.  HETÉNYI, R. K. PALEYANDA

The aim of this study was to determine whether there are differences in the karyotypes between transgenic and non-transgenic
or control rabbits. New Zealand White transgenic rabbits (F1 generation) were obtained after breeding of transgenic founder
rabbits that were derived from single – SM – or double microinjection – DM – with a WAP-hFVIII transgene. C-metaphase plates
were obtained from short-time culture of peripheral blood lymphocytes synchronized by the addition of colcemide. A significantly higher
rate of aneuploidy was observed in c-metaphase spreads of transgenic (56–66%) rabbits, as compared to non-transgenic ones (28–38%)
(P  < 0.05; P < 0.01). The patterns of chromosome banding were identical in both groups of rabbits. No structural aberrations were
revealed in either group. These findings demonstrate that transgenic rabbits have a higher frequency of numerical chromosomal aberrations
 in their peripheral blood lymphocytes than normal rabbits, but without apparent deleterious effects on health or reproduction.
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Short Communications
The Role of NK1.1+ Cells in the Protection against MHC Class I+ HPV16-Associated Tumours
J. ŠÍMOVÁ, J. BUBENÍK, J. BIEBLOVÁ, T. JANDLOVÁ

Depletion of NK1.1+ cells by repeated i.p. injections of PK136 antibody significantly enhanced growth of MHC class I+ tumours in
syngeneic mice. Depletion starting before tumour transplantation or on the day of transplantation substantially accelerated tumour growth; depletion starting on day 7 or 14 after tumour transplantation was without any effect. These results indicate that the NK1.1+ cells play an important inhibitory role during the early phase of the growth of some MHC class I+ tumours. Since the relevant target for NK cells is a
 "missing self" signal, absence of the MHC class I molecules, the NK cells cannot be expected to directly inhibit the growth of the
MHC class I+ tumours. The results indicate that the effects of non-NK cells or indirect effects mediated by NK cell interactions and
 release of cytokines were responsible for the results.
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The -455G/A Polymorphism of the β Fibrinogen Gene and the Bgl II Polymorphism of the α2β1 Integrin Gene and
Myocardial Infarction in Patients with Type 2 Diabetes
G. POGLAJEN, J. KIRBIŠ, A. MILUTINOVIĆ

Platelets and fibrinogen might be involved in the pathogenesis of thrombus formation and MI. The Bgl II gene polymorphism of the α2β1
 integrin, which is a platelet collagen receptor, and the -455G/A polymorphism in the β fibrinogen gene have been suggested as genetic risk factors for MI. The aim of this study was to look for a relationship between the -455G/A polymorphism in the β fibrinogen gene and the development of MI in Caucasians with type 2 diabetes. One hundred and forty-two subjects with type 2 diabetes and MI were compared
 to 234 diabetic subjects with no history of coronary artery disease. There were no significant differences in the frequency of the Bgl II gene polymorphism or of the -455G/A polymorphism in the β fibrinogen gene in the patients with MI compared to the patients without MI:
Bgl II (+/+) genotype was found in 19.7% of patients with MI and 15.4% of controls and -455GG genotype was found in 58.4% of patients with MI and 57.7% of controls. The present study demonstrates that neither the Bgl II gene polymorphism nor -455G/A polymorphism
in the β fibrinogen gene is a genetic marker for MI in Slovene population (Caucasians) with type 2 diabetes.
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