Cellular & Molecular Biology Letters

International Scientific Journal
Established 1996

Volume 12 (2007) No. 2

DOI: 10.2478/s11658-006-0061-z Volume 12 (2007) pp 149 - 161
Title	THE IMMUNOREGULATORY EFFECTS OF EDEINE ANALOGUES IN MICE
Authors	Zbigniew Czajgucki¹, Michał Zimecki²* and Ryszard Andruszkiewicz¹
Abstract	The edeines analogs were tested in several in vitro and in vivo assays using the mouse model, with edeine B (peptide W1) and cyclosporine A as reference compounds. The peptides displayed moderate, stimulatory effects on concanavalin A-induced (ConA-induced) splenocyte proliferation, whereas their effects on pokeweed mitogen-induced (PWM-induced) splenocyte proliferation were inhibitory. The peptides inhibited lipopolysacharide-induced (LPSinduced) tumor necrosis factor alpha production but had little effect on interleukin 6 production. In the model of the humoral immune response in vitro to sheep red blood cells, peptide 1 was distinctly stimulatory in the investigated concentrations (1-100 μg/ml), whereas peptides 3 and 4 only stimulated the number of antibody-forming cells at the highest concentration (100 μg/ml). In the model of the delayed type hypersensitivity in vivo to ovalbumin, the peptides were moderately suppressive (3 being the most active). The reference peptide W1 stimulated ConA-induced cell proliferation at 1-10 μg/ml but was inhibitory at 100 μg/ml. It also inhibited PWM-induced cell proliferation in a dosedependent manner. This peptide had no effect on the humoral immune response in vitro or on cytokine production, but inhibited DTH reaction in vivo. The relationship between structure and activity, and a possible mode of action of the peptides, is discussed in this paper.
Keywords	Edeine, Immune response, Mice
Adress and Contact Informations	¹Department of Pharmaceutical Technology and Biochemistry, University of Technology, 80-952 Gdańsk, Poland, ²Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, 53-114 Wrocław, Poland *Author for correspondence; e-mail: zimecki@iitd.pan.wroc.pl, phone: +48-71-370- 99-53, fax: +48-71-337-13-82

DOI: 10.2478/s11658-006-0062-y Volume 12 (2007) pp 162 - 175
Title	N-TERMINAL BRAIN NATRIURETIC PROPEPTIDE LEVELS CORRELATE WITH PROCALCITONIN AND C-REACTIVE PROTEIN LEVELS IN SEPTIC PATIENTS
Authors	Mariusz Piechota¹, Maciej Banach²*, Robert Irzmański³, Małgorzata Misztal⁴, Jacek Rysz⁵, Marcin Barylski³, Magdalena Piechota-Urbańska⁶, Jan Kowalski³ and Lucjan Pawlicki³
Abstract	The aim of this study was to find the relationship between N-terminal brain natriuretic propeptide (NT-proBNP), procalcitonin (PCT) and C-reactive protein (CRP) plasma concentrations in septic patients. This was a prospective study, performed at Medical University Hospital No. 5 in Łódź. Twenty patients with sepsis and severe sepsis were included in the study. N-terminal brain natriuretic propeptide, procalcitonin and C-reactive protein concentrations, and survival were evaluated. In the whole studied group (128 measurements), the mean NT-proBNP, procalcitonin and C-reactive protein concentrations were, respectively: 140.80±84.65 pg/ml, 22.32±97.41 ng/ml, 128.51±79.05 mg/l. The correlations for the NT-proBNP level and procalcitonin and C-reactive protein levels were 0.3273 (p<0.001) and 0.4134 (p<0.001), respectively. NT-proBNP levels correlate with PCT and CRP levels in septic patients. In the survivor subgroup, the mean NT-proBNP plasma concentrations were significantly lower than in the non-survivor subgroup.
Keywords	N-terminal brain natriuretic propeptide, Procalcitonin, C-reactive protein, Sepsis, Severe sepsis
Adress and Contact Informations	¹Department of Anaesthesiology and Intensive Care Unit, Bolesław Szarecki University Hospital No. 5 in Łódź, Medical University of Łódź, Poland, ²1st Department of Cardiology and Cardiac Surgery, University Hospital No. 3 in Łódź, Medical University of Łódź, ul. Sterlinga 1/3, 91-425 Łódź, Poland, ³Department of Internal Diseases and Cardiological Rehabilitation, Bolesław Szarecki University Hospital No. 5 in Łódź, Medical University of Łódź, Poland, ⁴Department of Statistical Methods, University of Łódź, Poland; ⁵2nd Department of Family Medicine, University Hospital No. 2 in Łódź, Medical University of Łódź, Poland; ⁶Department of Pharmacy, Medical University of Łódź, Poland *Author for correspondence; e-mail: m.banach@termedia.pl, tel./fax: (+48) 42 633 1558

DOI:10.2478/s11658-007-0003-4 Volume 12 (2007) pp 176 - 191
Title	MICROARRAY ANALYSIS REVEALS THE ROLE OF MATRIX METALLOPROTEINASES IN MOUSE EXPERIMENTAL AUTOIMMUNE MYOCARDITIS INDUCED BY CARDIAC MYOSIN PEPTIDES
Authors	Qizhu Tang¹*, Ji Huang¹, Haiyan Qian², Ran Xiong¹, Difei Shen¹, Hui Wu¹, Zhouyan Bian¹ and Xiaohong Wei¹
Abstract	Autoimmune myocarditis develops after the presentation of heartspecific antigens to autoaggressive CD⁴⁺ T cells and after inflammation has infiltrated the tissues. To shed light on global changes in the gene expression of autoimmune myocarditis and to gain further insight into the molecular mechanisms underlying the genesis of myocarditis, we conducted a comprehensive microarray analysis of mRNA using an experimental mouse autoimmune myocarditis model via immunization with α-myosin heavy chainderived peptides. Of over 39,000 transcripts on a high density oligonucleotide microarray, 466 were under-expressed and 241 over-expressed by ≥ 1.5-fold compared with the controls in BALB/C mouse with autoimmune myocarditis. In this paper, we list the top 50 up-regulated genes related to the immune response. These altered genes encode for leukocyte-specific markers and receptors, the histocompatibility complex, cytokines/receptors, chemokines/receptors, adhesion molecules, components of the complement cascade, and signal transduction-related molecules. Interestingly, matrix metalloproteinases (MMPs) such as MMP-3 and MMP-9 were up-regulated, as further revealed by the reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry assays. This indicates that MMPs may act as major regulators of the cytokine profile. Together, these findings provide new insight into the molecular events associated with the mechanism of the autoimmune genesis of myocarditis.
Keywords	Autoimmune, Matrix metalloproteinases, Microarray, Myocarditis
Adress and Contact Informations	¹Department of Cardiology, Renmin Hospital of Wuhan University, 430060 Wuhan, P.R. China, ²Department of Cardiology, Fuwai Hospital and Cardiovascular Institute, Chinese Academy of Medical Science and Peking Union Medical College, 100037 Beijing, P.R. China *Author for correspondence; e-mail address: qzhtang@yahoo.com, fax: (86)-27-88083385

DOI: 10.2478/s11658-006-0063-x Volume 12 (2007) pp 192 - 205
Title	*GENETIC INSTABILITY IN THE RAD51 AND BRCA1* REGIONS IN BREAST CANCER**
Authors	Maria Nowacka-Zawisza¹, Magdalena Bryś¹, Hanna Romanowicz-Makowska², Andrzej Kulig² and Wanda M. Krajewska¹*
Abstract	Breast cancer is the most prevalent cancer type in women. Accumulating evidence indicates that the fidelity of double-strand break repair in response to DNA damage is an important step in mammary neoplasias. The RAD51 and BRCA1 proteins are involved in the repair of double-strand DNA breaks by homologous recombination. In this study, we evaluated loss of heterozygosity (LOH) in the RAD51 and BRCA1 regions, and their association with breast cancer. The polymorphic markers D15S118, D15S214 and D15S1006 were the focus for RAD51, and D17S855 and D17S1323 for BRCA1. Genomic deletion detected by allelic loss varied according to the regions tested, and ranged from 29 to 46% of informative cases for the RAD51 region and from 38 to 42% of informative cases for the BRCA1 region. 25% of breast cancer cases displayed LOH for at least one studied marker in the RAD51 region exclusively. On the other hand, 31% of breast cancer cases manifested LOH for at least one microsatellite marker concomitantly in the RAD51 and BRCA1 regions. LOH in the RAD51 region, similarly as in the BRCA1 region, appeared to correlate with steroid receptor status. The obtained results indicate that alteration in the RAD51 region may contribute to the disturbances of DNA repair involving RAD51 and BRCA1 and thus enhance the risk of breast cancer development.
Keywords	RAD51, BRCA1, Loss of heterozygosity (LOH), Breast cancer
Adress and Contact Informations	¹Department of Cytobiochemistry, University of Łódź, Banacha 12/16, 90-237 Łódź, Poland, ²Department of Clinical Pathomorphology, Polish Mother’s Memorial Hospital, Research Institute, Rzgowska 281/289, 93-338 Łódź, Poland *Author for correspondence: e-mail: wmkraj@biol.uni.lodz.pl, tel.: +48-42-6354487, fax: +48-42-6354484

DOI: 10.2478/s11658-006-0064-9 Volume 12 (2007) pp 206 - 219
Title	*DIPLOID POTATO (Solanum tuberosum L.) AS A MODEL CROP TO STUDY TRANSGENE EXPRESSION*
Authors	Anna Nadolska-Orczyk*, Aleksandra Pietrusinska, Agnieszka Binka-Wyrwa, Dominik Kuc and Wacław Orczyk
Abstract	This paper presents a method of Agrobacterium-mediated transformation for two diploid breeding lines of potato, and gives a detailed analysis of reporter gene expression. In our lab, these lines were also used to obtain tetraploid somatic hybrids. We tested four newly prepared constructs based on the pGreen vector system containing the selection gene nptII or bar under the 35S or nos promoter. All these vectors carried gus under 35S. We also tested the pDM805 vector, with the bar and gus genes respectively under the Ubi1 and Act1 promoters, which are strong for monocots. The selection efficiency (about 17%) was highest in the stem and leaf explants after transformation with pGreen where nptII was under 35S. About half of the selected plants were confirmed via PCR and Southern blot analysis to be transgenic and, depending on the combination, 0 to 100% showed GUS expression. GUS expression was strongest in multi-copy transgenic plants where gus was under Act1. The same potato lines carrying multi-copy bar under Ubi1 were also highly resistant to the herbicide Basta. The suggestion of using Agrobacterium-mediated transformation of diploid lines of potato as a model crop is discussed herein.
Keywords	Binary vectors, Gene expression, Genetic transformation, Polyploid
Adress and Contact Informations	Plant Transformation and Cell Engineering Department, Plant Breeding and Acclimatization Institute, Radzików, 05-870 Błonie, Poland *Author for correspondence: e-mail: a.orczyk@ihar.edu.pl

DOI: 10.2478/s11658-006-0067-6 Volume 12 (2007) pp 220 - 230
Title	THE INFLUENCE OF PROTONS AND ZINC IONS ON THE STEADYSTATE INACTIVATION OF Kv1.3 POTASSIUM CHANNELS
Authors	Andrzej Teisseyre* and Jerzy W. Mozrzymas
Abstract	Using the whole-cell patch-clamp technique, we investigated the influence of extracellular pH and zinc ions (Zn²⁺) on the steady-state inactivation of Kv1.3 channels expressed in human lymphocytes. The obtained data showed that lowering the extracellular pH from 7.35 to 6.8 shifted the inactivation midpoint (Vi) by 17.4 ± 1.12 mV (n = 6) towards positive membrane potentials. This shift was statistically significant (p < 0.05). Applying 100 μM Zn2+ at pH 6.8 further shifted the Vi value by 16.55 ± 1.80 mV (n = 6) towards positive membrane potentials. This shift was also statistically significant (p < 0.05). The total shift of the Vi by protons and Zn²⁺ was 33.95 ± 1.90 mV (n = 6), which was significantly higher (p < 0.05) than the shift caused by Zn²⁺ alone. The Zn2+-induced shift of the Vi at pH 6.8 was almost identical to the shift at pH = 7.35. Thus, the proton- and Zn²⁺-induced shifts of the Vi value were additive. The steady-state inactivation curves as a function of membrane voltage were compared with the functions of the steady-state activation. The total shift of the steady-state inactivation was almost identical to the total shift of the steady-state activation (32.01 ± 2.10 mV, n = 10). As a result, the “windows” of membrane potentials in which the channels can be active under physiological conditions were also markedly shifted towards positive membrane potentials. The values of membrane voltage and the normalised chord conductance corresponding to the points of intersection of the curves of steady-state activation and inactivation were also calculated. The possible physiological significance of the observed modulatory effects is discussed herein.
Keywords	Zinc, Lymphocyte, Potassium channels, Patch-clamp, pH, Neuronal excitability
Adress and Contact Informations	Department of Biophysics, Wrocław Medical University, ul. Chałubińskiego 10, 50-368 Wrocław, Poland *Author for correspondence: ateiss@biofiz.am.wroc.pl

DOI: 10.2478/s11658-006-0066-7 Volume 12 (2007) pp 231 - 239
Title	*SNPs IN THE PORCINE PPARGC1A* GENE: INTERBREED DIFFERENCES AND THEIR PHENOTYPIC EFFECTS**
Authors	Monika Stachowiak, Maciej Szydlowski, Jakub Cieslak and Marek Switonski
Abstract	Due to its function, the peroxisome proliferative activated receptor-γ, coactivator-1α (PPARGC1A) gene is a candidate in the search for genes that may affect production traits in the pig. The purpose of this study was to screen for new SNPs in exon 8 of the porcine PPARGC1A gene and to test their possible association with production traits. Altogether 736 pigs representing five breeds (Polish Landrace, n=242; Polish Large White, n=192; Hampshire, n=27; Duroc, n=21; Pietrain, n=12) and synthetic line 990 (n=242) were scanned via SSCP assay. Four SNPs were found; two new ones: C/G (His338Gln) and G/A (Thr359Thr), and two previously reported ones: C/A (Arg369Arg) and T/A (Cys430Ser). The missense T/A and C/G SNPs demonstrated pronounced interbreed variability in terms of allele frequencies, including the exclusive presence of the C/G substitution in the Hampshire breed. The tested SNPs occurred in five putative haplotypes, and their frequency also differed substantially between breeds. The association of the SNPs with production traits was tested for G/A (Thr359Thr), C/A (Arg369Arg) and T/A (Cys430Ser) substitutions in Polish Large White, Polish Landrace and line 990. The analysis revealed only breed-specific associations. The T/A (Cys430Ser) SNP was related to the feed conversion ratio in the Polish Large White (P=0.02), and the silent G/A and C/A substitutions were respectively associated with abdominal fat in line 990 and backfat thickness in Polish Landrace (P=0.04). The combined effects of the substitutions were estimated as haplotype effects. Three significant contrasts between haplotypes were calculated, but the observed associations were again only breed-specific.
Keywords	Pig, PPARGC1A gene, Quantitative trait loci, SNP
Adress and Contact Informations	Department of Genetics and Animal Breeding, Agricultural University of Poznan, 60-637 Poznan, Poland *Author for correspondence: e-m ail: switonsk@jay.au.poznan.pl, tel./fax: +48-61-8487246

DOI: 10.2478/s11658-006-0069-4 Volume 12 (2007) pp 240 - 252
Title	*THE MOLECULAR DIVERSITY OF DIFFERENT ISOLATES OF Beauveria bassiana* (BALS.) VUILL. AS ASSESSED USING INTERMICROSATELLITES (ISSRS)**
Authors	M. Elena Estrada^1,2, Manuel V. Camacho¹and Cesar Benito¹*
Abstract	Inter-microsatellite PCR (ISSR-PCR) markers were used to identify and to examine the genetic diversity of eleven Beauveria bassiana isolates with different geographic origins. The variability and the phylogenetic relationships between the eleven strains were analyzed using 172 ISSR-PCR markers. A high level of polymorphism (near 80%) was found using these molecular markers. Seven different isolates showed exclusive bands, and ISSR primer 873 was able to distinguish between all the strains. The dendrogram obtained with these markers is robust and in agreement with the geographical origins of the strains. All the isolates from the Caribbean region were grouped together in a cluster, while the other isolates grouped in the other cluster. The similarity exhibited between the two clusters was less than 50%. This value of homology shows the high genetic variability detected between the isolates from the Caribbean region and the other isolates. ISSR-PCR markers provide a quick, reliable and highly informative system for DNA fingerprinting, and allowed the identification of the different B. bassiana isolates studied.
Keywords	Beauveria bassiana, Strain identification, Inter-microsatellite, Phylogenetic relationships
Adress and Contact Informations	¹Departamento de Genética, Facultad de Biología, Universidad Complutense, 28040-Madrid, Spain, ²Instituto Nacional de Investigaciones de la Cańa de Azúcar (INICA), Programa de Fitomejoramiento, La Habana, Cuba. *Author for correspondence; e-mail: cebe8183@bio.ucm.es, tel: 34-913944860, fax: 34- 913944844

DOI: 10.2478/s11658-006-0068-5 Volume 12 (2007) pp 253 - 267
Title	*TETRAPLOID SOMATIC HYBRIDS OF POTATO (Solanum tuberosum L.) OBTAINED FROM DIPLOID BREEDING LINES*
Authors	Jarosław Przetakiewicz, Anna Nadolska-Orczyk, Dominik Kuć and Wacław Orczyk*
Abstract	Intraspecific somatic hybrids between 16 different diploid breeding lines of Solanum tuberosum L. were produced by PEG-induced fusion. Manually selected heterokaryons were cultured in a Millicells-CM using a postfusion protoplast mixture. Plants were regenerated from calli derived from heterokaryons obtained from 10 out of 38 combinations of diploid lines. Of the tested putative somatic hybrids, 14.2% were diploid, 72.8% were tetraploid and 13% pentaploid. The DNA amplification pattern obtained with RAPD or semirandom primers confirmed that 6 fusion combinations were hybrids. In most cases, the morphological traits were intermediate to those of the diploid fusion partners. About 23.0% of the tested somatic hybrids showed variation in their morphology. Of the tested somatic hybrids, 78.0% flowered and 86.0% tuberized. The cytoplasm of 9 diploid lines and 6 somatic hybrid combinations was analysed. Two of the diploid lines had W/S chloroplasts and α or ε mitochondria; the remainder contained T chloroplasts and β mitochondria. All the analysed somatic hybrids carried T chloroplasts and β mitochondria.
Keywords	Cytoplasmic hybrids, Heterokaryons, Molecular markers, Protoplast fusion
Adress and Contact Informations	Plant Transformation and Cell Engineering Department, Plant Breeding and Acclimatization Institute, Radzików, 05-870 Błonie, Poland *Author for correspondence; e-mail: w.orczyk@ihar.edu.pl, tel: +48 22 7253717, fax: +48 22 7254714

DOI: 10.2478/s11658-007-0001-6 Volume 12 (2007) pp 268 - 279
Title	INCREASED EXPRESSION OF HSP70 BY COLON CANCER CELLS IS NOT ALWAYS ASSOCIATED WITH ACCESS TO THE DENDRITIC CELL CROSS-PRESENTATION PATHWAY
Authors	Lina Matera¹*, Sarah Forno¹, Alessandra Galetto^1I, Francesco Moro², Stefano Garetto¹ and Antonio Mussa³
Abstract	Dendritic cells (DCs) are highly specialized antigen-presenting cells endowed with the unique ability to not only present exogenous antigens upon exposure to MHC II, but also to cross-present these upon exposure to MHC I. This property was exploited to generate the tumor-specific CD8 cytotoxic lymphocyte (CTL) response in DCs-based cancer vaccine protocols. In this context, the source of tumor antigens remains a critical challenge. A crude tumor in the context of danger signals is believed to represent an efficient source of tumor antigens (TAs) for DCs loading. In our previous work, increased DCs cross-presentation of antigens from necrotic gastric carcinoma cells paralleled up-regulation of the heat shock protein hsp70. We studied the expression of hsp70 on primary colon carcinoma cells and its relevance in the cross-priming of anti-tumor CTL by tumor-loaded DCs. Hsp70 was expressed on all three of the tumors studied, but was never detected in the peritumoral normal mucosa (NM). The uptake of the tumor induced a trend towards down-modulation of the monocyte-specific marker CD14, but had no effect on the chemokine receptors CCR4 and CCR7. The IFN-γ enzyme-linked immunospot assay (ELIspot) showed cross-priming of CTL by tumor-loaded but not NM-loaded DCs in four of the six cases studied. The CTL response generated in DC+tumor cultures was directed towards the tumor, but not towards NM, and it was characterized by refractoriness to polyclonal (Ca ionophores, PKC activators) stimuli. Of the three CTL-generating tumors, only one expressed hsp70. This data indicates a tumor-specific expression of hsp70, but does not support its relevance in the DC cross-presentation of TAs.
Keywords	Dendritic cells, Cytotoxic T lymphocyte, Colon cancer, Necrosis, Heat shock proteins
Adress and Contact Informations	¹Deptartment of Internal Medicine, University of Turin, Italy, ²Unit of Liver Transplantation, Molinette Hospital, Turin, Italy, ³S.C.D.U. of Surgical Oncology, University of Turin, Italy *Author for correspondence: e-mail: lina.matera@unito.it, tel: +390116961813, fax: +390116334146 ^I Present address: University of East Piedmont “Amedeo Avogadro”, Novara, Italy

DOI: 10.2478/s11658-007-0002-5 Volume 12 (2007) pp 280 - 293
Title	PHENOL RED IN THE CULTURE MEDIUM STRONGLY AFFECTS THE SUSCEPTIBILITY OF HUMAN MCF-7 CELLS TO ROSCOVITINE
Authors	Józefa Węsierska-Gądek*, Tanja Schreiner, Margarita Maurer, Astrid Waringer and Carmen Ranftler
Abstract	Estrogens play an important role in the growth and terminal differentiation of the mammary gland. Prolonged exposure to estrogens seems to predispose women to breast cancer. It recently became evident that not only the intrinsic hormonal status but also external factors such as the occurrence of pharmaceuticals and chemicals with hormone activity in the environment may put women at greater risk of developing breast cancer. We focused on the interference of endocrine disruptors in breast cancer therapy. We observed that phenol red added to the culture medium strongly promoted the cell proliferation and cell cycle progression of human cells expressing the estrogen receptor, and affected their susceptibility to chemotherapy.
Keywords	Endocrine disrupters, Apoptosis, Cell cycle arrest, Cyclindependent inhibitors
Adress and Contact Informations	Cell Cycle Regulation Group, Division: Institute of Cancer Research, Department of Medicine I, Vienna Medical University, Borschkegasse 8 a, A-1090 Vienna, Austria *Author for correspondence: e-mail: Jozefa.Gadek-Wesierski@meduniwien.ac.at, tel: 43-1-4277 ext. 65247, fax: 43-1-4277 ext. 65194

DOI: 10.2478/s11658-006-0065-8 Volume 12 (2007) pp 294 - 307
Title	PARTIAL REVERSAL OF TRANSFORMED FUSIFORM PHENOTYPE BY OVEREXPRESSION OF CALRETICULIN
Authors	Michal Opas* and Marc P. Fadel
Abstract	Calreticulin, a Ca²⁺-storage and chaperone protein of the ER, has also been shown to affect cell adhesiveness. To examine the effects of differential expression of calreticulin on cellular adhesiveness, we used L fibroblast cell lines stably expressing either elevated or reduced amounts of full length, ER-targeted calreticulin. Overexpression of calreticulin correlates with an increase in adhesiveness of L fibroblasts such that these transformed cells acquire epithelioid morphology and form an epithelial-cell sheet when crowded. Functionally, the “reversal” of transformed phenotype in L fibroblasts differentially overexpressing calreticulin can be accounted for by changes in levels of expression of N-cadherin and vinculin. Structurally, however, although the form and extent of cell-cell contacts in L fibroblasts overexpressing calreticulin mimicked those in normal epithelia, electron microscopical examination revealed that cell-cell junctions formed by these transformed cells bore only superficial resemblance to those of normal epithelia in culture. Our data imply that overexpression of calreticulin, while partially reverses fusiform transformed phenotype is in itself insufficient to re-establish bona fide zonulae adherens in transformed fibroblasts.
Keywords	Calreticulin, Adhesion, Vinculin, N-Cadherin
Adress and Contact Informations	Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King’s College Circle, Medical Sciences Building, Toronto, Ontario, Canada M5S 1A8 *Author for correspondence; e-mail: m.opas@utoronto.ca, tel: (416) 971-2140, fax: (416) 978-5959

DOI: 10.2478/s11658-007-0004-3 Volume 12 (2007) pp 308 - 316
Title	DETECTION OF THE STATUS OF TUMOUR-INFILTRATING CD4+ T-CELL SUBPOPULATIONS IN SQUAMOUS CELL CARCINOMA OF THE HEAD AND NECK (HNSCC) USING REAL-TIME RT-PCR DETECTION OF CD4 mRNA
Authors	Sava Smerkolj, Metka Volavsek and Damjan Glavac
Abstract	Several studies have reported tumour infiltrating CD4+ T cells as a favourable prognostic factor in some types of cancer. We investigated 37 head and neck squamous cell carcinomas (HNSCC) at different stages, using immunohistochemical staining for CD4+ infiltrates and real-time reverse transcription polymerase chain reaction (RT-PCR) detection of CD4 mRNA. The CD4+ infiltrates were evaluated and expressed as a percentage according to the ratio of CD4+ T cells to epithelial cells in the cancer cell nests and to the overall inflammatory cell infiltrate in the tumor stroma. The CD4 mRNA expression level strongly correlated with the CD4+ infiltration score in the cancer epithelium (rs = 0.858, P < 0.001) and in the cancer stroma (rs = 0.797, P < 0.001). These results indicate that the real-time RT-PCR assay is a sensitive and reliable method for the detection of CD4 mRNA, and that it could be used to reassess CD4+ infiltration status in resected specimens from patients with HNSCC.
Keywords	Tumour infiltrating CD4⁺ T cells, RT-PCR CD4 mRNA detection, HNSCC
Adress and Contact Informations	Department of Molecular Genetics, Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia *Author for correspondence: damjan.glavac@mf.uni-lj.si

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