Ceramide galactosyltransferase (UGT8) is a molecular marker of breast cancer malignancy and lung metastases.

BACKGROUND: It was shown recently on the level of gene expression that UGT8, coding UDP-galactose:ceramide galactosyltransferase, is one of six genes whose elevated expression correlated with a significantly increased the risk of lung metastases in breast cancer patients. In this study primary tumours and their lung metastases as well as breast cancer cell lines were analysed for UGT8 expression at the protein level. METHODS: Expression of UGT8 in breast cancer tissue specimens and breast cancer cell lines was analysed using IHC, real-time PCR and Western blotting. RESULTS: Comparison of the average values of the reaction intensities (IRS scale) showed a significant difference in UGT8 expression between (1) primary and metastatic tumours (Mann-Whitney U, P<0.05), (2) tumours of malignancy grades G3 and G2 (Mann-Whitney U, P<0.01) as well as G3 and G1 (Mann-Whitney U, P<0.001) and (3) node-positive and node-negative tumours (Mann-Whitney U, P<0.001). The predictive ability of increased expression of UGT8 was validated at the mRNA level in three independent cohorts of breast cancer patients (721). Similarly, breast cancer cell lines with the 'luminal epithelial-like' phenotype did not express or weakly expressed UGT8, in contrast to malignant, 'mesenchymal-like,' cells forming metastases in nude mice. CONCLUSION: Our data suggest that UGT8 is a significant index of tumour aggressiveness and a potential marker for the prognostic evaluation of lung metastases in breast cancer.

Transcriptomic "portraits" of canine mammary cancer cell lines with various phenotypes.

In light of the high incidence of mammary cancer in dogs and completion of the canine genome sequencing, the new possibilities of gene profiling by using DNA microarrays give hope to veterinary oncology. The cell lines isolated from mammary tumors are a valuable tool in developing and testing new pathway-specific cancer therapeutics. Differential cytometric analysis of 6 canine mammary cancer cell lines was performed. We divided cell lines into 3 groups based on their phenotype: 2 lines with high proliferative potential, 2 lines with high antiapoptotic potential, and 2 lines with high metastatic potential. DNA microarray analysis revealed common genes for cell lines of each group. We found that genes encoding the receptors for growth hormone and ghrelin are related to high proliferation rate, while ABR (active BCR-related) and TMD1 (TM2 domain containing 1) genes are related to a high antiapoptotic potential of the cancer cells. Metastatic properties of mammary cancer cells seem to be associated with elevated expression of PGP (P glycoprotein), SEMA3B (semaphorin 3B), and STIM1 (stromal interaction molecule 1).

Transcriptomic signature of cell lines isolated from canine mammary adenocarcinoma metastases to lungs.

Metastasis is a final step in the progression of mammary gland cancer, usually leading to death. Potentially, a molecular signature of metastasis can be defined via comparison of primary tumors with their metastases. Currently, there is no data in the literature regarding the molecular portrait of metastases in dogs and only few reports regarding human cancer. This is the first report describing the transcriptomic signature of canine cancer metastatic cells. Two adenocarcinoma cell lines isolated from the canine mammary gland (CMT-W1 and CMT-W2) were compared with cell lines isolated from their lung metastases (CMT-W1M and CMT-W2M) with regards to the following cytometric parameters: cell cycle, ploidy, Bcl-2 expression, susceptibility to induced apoptosis, and transcriptomic profile. Cytometric analyses revealed significant differences in cell cycle and antiapoptotic potential between the examined cells. Using oligonucleotide microarrays, we found 104 up-regulated genes in the metastatic cell line CMT-W1M and 21 up-regulated genes in the primary CMT-W1 cell line. We also found 83 up-regulated genes in the CMT-W2M cell line and only 21 up-regulated genes in the CMT-W2 cell line. Among the up-regulated genes in both metastatic cell lines, we found 15 common genes. These differently expressed genes are involved mainly in signal transduction, cell structure and motility, nucleic acid metabolism, developmental processing, and apoptosis (GHSR, RASSF1, ARF1GAP, WDR74, SMOC2, SFRP4, DIAPH1, FSCN1, ALX4, SNX15, PLD2, WNT7B, POU6F2, NKG7, and POLR2F). Seven of them are involved in a cellular pathway dependent on ghrelin via growth hormone secretagogue receptor (GHSR). Our results suggest that this pathway may be essential for mammary cancer cells to have a metastatic potential.

Isolation of Yersinia enterocolitica from aborted fetuses and sows in pig farms with reproductive disturbances.

The purpose of the study was to determine the occurrence of Yersinia enterocolitica in tissues of aborted fetuses, placentas, vaginal and rectal swabs of aborting sows from pig farms where reproductive disturbances were found and to determine and analyze the biotype and serotype affinity of the strains isolated. Altogether 97 fetuses aborted in various stages of pregnancy, 25 placentas and swabs from 231 sows were taken. All sows originated from farms where reproductive disorders appeared. In general, 1069 samples were collected. Two enrichment methods were used in this study; fast enrichment techniques in ITC broth, then plating onto CIN agar (ITC/CIN), and cold enrichment in phosphate buffered saline followed by plating onto CIN agar (PBS/CIN). From all samples examined, 96 Y. enterocolitica strains were isolated including 57 (59.4%) from rectal swabs of sows, followed by 6 (6.3%) from vaginal swabs and 2 (2.1%) from placentas. The bacteria were isolated from tissues of 18 out of 97 aborted fetuses. A total of 60 strains were selected for further examination--29 strains originated from aborting sows and 31 from aborted fetuses. Among strains examined 54 isolates (90%) belonged to the biotype 1A of Y. enterocolitica and to the different serotypes O:3, O:5, O:6, O:7/13, O:8 and NT (not typable). Only 6 strains belonged to serotype O:3, biotype 4 Y. enterocolitica. Our study has revealed the possibility of Y enterocolitica isolation from internal organs of aborted swine fetuses and sows from farms with reproductive disturbances. The results suggest the connection between fetal death, pregnancy course disorders and Y. enterocolitica infection.

Genotypic characterization of hospital Enterococcus faecalis strains using multiple-locus variable-number tandem-repeat analysis.

AIMS: The level of genetic diversity and relationships between the specific genotypes and the distribution of virulence determinants among Enterococcus faecalis strains isolated from patients hospitalized in different wards of two hospitals were investigated. METHODS AND RESULTS: Fifty-six clinical strains of E. faecalis, isolated from patients hospitalized in the period of 1999-2004 in several wards in Wroclaw (Poland), were analysed by multiple-locus variable-number tandem-repeat analysis (MLVA). Analysis of seven genomic loci identified 40 novel genotypes among the analysed E. faecalis strains, with two major genomic groups, designated I and II, distinguished at a cut-off of 35%. With a similarity cut-off of 85.7%, the genotypes could be combined into 12 clusters (C1-C12), containing at least two isolates. The remaining 18 MLVA types were represented by a single isolate. CONCLUSIONS: Based on the data obtained by MLVA, it was found that (i) many E. faecalis isolates recovered from patients from the wards whose location allowed the potential transmission of micro-organisms, belonged to closely related MLVA types and (ii) possible relationships between specific E. faecalis genotype and the virulence factors lipase, haemolysin and esp gene can exist. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study confirms that MLVA is a suitable method for the epidemiological study of E. faecalis and for the first time shows possible relationships between specific genotypes and such virulence determinants, i.e. lipase, haemolysin and esp gene.

Tumor-associated carbohydrate antigens: Sialyl Lea and T/Tn antigens in canine mammary tumors.

Twenty-eight canine mammary tubulopapillary carcinomas and 14 simple adenomas were studied by immunohistochemistry for the expressions of the tumor-associated carbohydrate antigens. Sialyl Le(a) was detected in 71.42% of the malignant and 92.84% of the benign tumors. Staining with anti-T and anti-Tn monoclonal antibodies revealed that 85.70% of the tubulopapillary carcinomas expressed T and Tn antigens. In contrast, 50% of the adenomas did not express T antigen, and 42.85% of them were only weakly stained for this carbohydrate structure. In the case of Tn antigen, the majority (57.14%) of samples was weakly stained, and no binding was observed in 35.71% of the analyzed specimens. Comparison of average values of reaction intensity (IRS) scale for malignant versus benign tumors by the Mann-Whitney U-test revealed a significant relationship between T and Tn antigens expression and type (malignant vs. benign) mammary tumors. Based on the results obtained, it is suggested that each of the studied antigens can be treated as a tumor-associated antigen of canine mammary tumors. However, only the T and Tn antigens seem to be associated with malignant transformation of mammary gland cells and to be of potential value as diagnostic markers.

Analysis of the presence of ail, ystA and ystB genes in Yersinia enterocolitica strains isolated from aborting sows and aborted fetuses.

Forty-five Yersinia enterocolitica strains isolated from aborted fetuses and placentas and from vaginal and rectal swabs of aborting sows were subjected to serotyping, biochemical typing and polymerase chain reaction multiplex analyses to detect the presence of the ail, yst A and ystB genes. The isolates were recovered from the internal organs (tonsil, lung, liver, spleen, kidney, mesentheric lymph nodes, small intestine and rectal intestine) of 18 (18.6%) of 97 aborted fetuses examined, two (8%) of 25 aborted placentas and 27 (15.8%) of 172 examined aborting sows. Serotyping of Y. enterocolitica revealed that only six (13.3%) of the examined isolates belonged to serotype O:3, with a considerable number of isolates (31.1%) having serotype O:5, while biochemical studies showed that as many as 40 of the 45 strains belonged to biotype 1A. As expected, the Y. enterocolitica strains of bioserotype 4/O:3 contained ail and ystA genes, while strains of biotype 1A contained only the ystB gene.

Differential effect of IFN-tau on proliferation and distribution of lymphocyte subsets in one-way mixed lymphocyte reaction in cows and heifers.

IFN-tau is a signaling protein secreted by the bovine conceptus during the peri-implantation period and responsible for pregnancy recognition. Its main role is the prevention of pulsatile release of luteolytic PGF2alpha, but it also exerts immunomodulatory activities characteristic for other type I interferons. The aim of the study was to examine the effect of IFN-tau on the proliferation and distribution of peripheral blood lymphocyte subsets during one-way mixed lymphocyte reaction (MLR) in cows and heifers. IFN-tau inhibited the proliferative response of lymphocytes in MLR both in cows and heifers in a dose-dependent manner, but cow lymphocytes were less susceptible than those ones from heifers. It was also showed that IFN-tau differentially changed lymphocyte subsets distribution in MLR in cows and heifers. In cows, the relative percentage of CD8(+) cells after MRL in the presence of IFN-tau was significantly lower than in heifers. Differential effect of rIFN-tau on proliferation and lymphocyte subsets distribution in a one-way MRL in cows and heifers indicated that the age of the mother is an important factor in immunomodulatory effect towards developing bovine embryo.

Differentiation of Salmonella Gallinarum biovar Gallinarum from Salmonella Gallinarum biovar Pullorum by PCR-RFLP of the fimH gene.

In our studies on FimH adhesins expressed by different Salmonella serovars, we cloned and sequenced the fimH genes from Salmonella enterica ssp. Enterica ser. Gallinarum biovar Gallinarum and S. enterica ssp. Enterica ser. Gallinarum biovar Pullorum. Comparison of the nucleotide sequences revealed the presence of a single-nucleotide polymorphism (SNP) at position 544 bp from the A of the start codon of the fimH open reading frame (ORF). Further analysis of the restriction enzyme sites in fimH gene showed that the SNP at this position is responsible for a sequence specifically recognized by SacI in S. Gallinarum biovar Gallinarum only, making it possible to differentiate both biovars with the use of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Digestion of PCR amplicons of the fimH gene from S. Gallinarum biovar Gallinarum strains with SacI gave two DNA fragments of 554 and 472 bp and only one fragment of 1026 bp for S. Gallinarum biovar Pullorum. This allows a clear differentiation between these two biovars.

Genotype analysis of Campylobacter jejuni isolated from broilers in Poland.

Twenty-six Campylobacter jejuni strains isolated from poultry were analyzed by genotypic typing including ITS-profiling, REP- and ERIC-PCR. ITS-profiling revealed the presence of 8 different genotypes. Amplification of REP sequences by PCR gave similar results with 10 different genotypes. ERIC-PCR was found to be the most discriminatory for typing C. jejuni. As many as 13 different DNA patterns were obtained with this technique. Based on data obtained it was found that C. jejuni isolates recovered from broilers at the slaughterhouses in southwest Poland are characterized by a high degree of genetic heterogeneity.

Combined effect of surface electrostatic charge and poly(ethyl glycol) on the association of liposomes with colon carcinoma cells.

Particulate drug formulations are considered to be a means that may improve the pharmacokinetics and biodistribution of active compounds. By using them, drug distribution is determined solely by the properties of the carrier. The surface properties of such supramolecular aggregates determine how they will interact with various biological structures. Among others, surface electrostatic charge and surface grafted polymers are considered to be among the major factors affecting its interaction with proteins and cells. In this article, we present experimental evidence that properly selected surface electrostatic charge and grafted polymers can alter the association of liposomes with colon cancer cells. The dependence of the adsorption of liposomes onto the cell surface on the quantity and length of surface grafted polymers for a certain surface charge density exhibits a distinct maximum. For example, when liposomes were formed with 20 mol% of DOTAP, PE-PEG350 increased liposome adsorption by up to 6 mol%. This adsorption maximum depends on both polymer length and charge type. Results presented in this article show that the interaction of liposomes with colon cancer cells can be tuned by a proper combination of liposome surface electrostatics and surface grafted polymers.

Typing of Yersinia Enterocolitica Isolates by ITS profiling, REP- and ERIC-PCR.

Thirty-five Yersinia enterocolitica strains isolated from humans, pigs and foxes were analysed by genotyping including intergenic transcribed sequence (ITS) profiling, REP- and ERIC-PCR. ERIC-PCR revealed the presence of seven different genotypes. Amplification of the 16S-23S rDNA spacer region by ITS profiling gave similar results with nine different genotypes. REP-PCR was found to be more discriminatory for typing of Y. enterocolitica than ERIC-PCR and ITS profiling. Fifteen different DNA patterns were obtained by this technique. Based on data obtained by three methods it was found that: (i) Y. enterocolitica strains belonging to the same serotype can represent different genotypes and vice versa; (ii) isolates recovered from humans, pigs and foxes exhibit limited heterogeneity and, independent of the origin, one or two prevailing genotypes were always observed; and (iii) many human Y. enterocolitica isolates shared common genotypes with porcine isolates.

Complex nature of the human antisperm antibody response in SCID mice.

Human peripheral blood mononuclear (PBMs) cells were introduced into the peritoneal cavity of severely-combined immunodeficient (SCID) mice in concentrations of 2.5-4.0 x 10(7) cells per mouse. Whole mononuclear cell suspensions were used either unstimulated or following primary in vitro culture with human spermatozoa. In some experiments, immunodepletion of CD8(+) cells was carried out prior to grafting. Lymphocytes were obtained from nonsensitized (to antigen) human subjects or from individuals who were primed in vivo (vasectomized individuals in case of sperm antigens). An enzyme-linked immunosorbent assay was employed to assess total human immunoglobulin (G or M) levels as well as the specificity of the antibodies generated. We have been successful by generating primary and secondary immune responses with 'naïve' human lymphocytes, challenged with chlamydia or ovalbumin but without adjuvant or CD8(+) immunodepletion; however, we were unable to induce specific antibodies to spermatozoa under this regime in SCID male mice. We then employed female SCID mice, treated with sperm antigen extracts (glycosylated or deglycosylated) encapsulated in liposomes and human lymphocytes obtained from 'naïve' or pre-sensitized in vivo subjects. It was found that the most pronounced humoral response to sperm antigens was obtained with deglycosylated antigens and PBMs from vasectomized (in vivo pre-primed to spermatozoa) individuals. A presented SCID mice model can be helpful at understanding of antisperm antibody development and the molecular nature of generated antibodies to modified sperm antigenic entities.

Comparison of ITS profiling, REP- and ERIC-PCR of Salmonella Enteritidis isolates from Poland.

Thirty-one Salmonella Enteritidis strains isolated from chickens, broilers and hens were analysed by genotypic typing including REP-PCR. ERIC-PCR and ITS profiling (PCR-ribotyping). Analysis of DNA banding patterns generated by REP-PCR revealed the presence of 22 different genotypes, which were grouped by dendrogram analysis into three distinct lineages (maximum similarity approx. 50%). Each isolate of S. Enteritidis analysed by ERIC-PCR generated an individual DNA pattern. Again, these isolates could be divided into three distinct genomic groups (maximum similarity approx. 60%) by their ERIC-PCR fingerprints. REP- and ERIC-PCR were found to be more discriminatory for typing of S. Enteritidis than ITS profiling. Amplification of the 16S-23S rDNA spacer region gave nine different profiles of DNA, subdivided into two closely related groups by dendrogram analysis. In summary, data obtained by genotyping methods for S. Enteritidis isolates from regions located in the south-west and the central parts of Poland revealed an enormous heterogeneity among analysed samples, and proved that REP- and ERIC-PCR are highly discriminatory techniques, which can be used, in addition to conventional methods, in epidemiological studies of S. Enteritidis infections.

Surface charge and the association of liposomes with colon carcinoma cells.

Interaction between the plasma membrane and aggregate lipid surface determines how efficiently the encapsulated drug will be delivered into the cell. Electrostatic interactions are one of the main forces affecting liposome and aggregate association with the charged cell surface. In this study, the effect of surface charge on the association of liposomes with human colon CX-1.1 cancer cells was studied. When phosphatidylserine was incorporated into a lipid bilayer, the amount of liposomes associated with cells tended to increase along with the amount of negatively charged lipid present in the liposomal lipid bilayer. When the cationic lipid dioleoyl-1,2-diacyl-3-trimethylammonium-propane (DOTAP) was included into the liposome formula, their uptake by the cells was also increased. Maximum binding occurred when the amount of positively charged lipids in liposomes was about 10 mol% of lipids.

Sialosyl Le(a)-carrying gangliosides present on the surface of colon carcinoma cells are not directly involved in adhesion to E-selectin.

We have shown previously that human colon cancer CX-1 cells contain lipid- and protein-bound sialosyl Lewis(a) structures that support the adhesion of these cells to E-selectin. Treatment of cancer cells with O-sialoglycoprotease did not decrease either the binding of anti-sialosyl Le(a) antibodies or binding to E-selectin-expressing CHO cells. This suggested that cleavage of sialomucins uncovered cryptic sialosyl Le(a) gangliosides that support such interactions. In the present study, inhibitors of glycolipid and O-glycan biosynthesis, d,l-threo-PPPP and GalNAc-alpha-O-benzyl, respectively, were used to study whether the binding of anti-sialosyl Le(a) antibody and adhesion of CX-1 cells to E-selectin can be mediated by sialosyl Le(a) gangliosides. Treatment of cancer cells with each of the inhibitors decreased the expression of the respective glycoconjugates as shown by TLC-binding assay and immunoblotting with anti-sialosyl Le(a) antibody. However, only slight differences in binding of antisialosyl Le(a) antibody to the surfaces of control and inhibitor-treated CX-1 cells were found by flow cytometry, as well no differences were observed in binding of control and inhibitor-treated CX-1 cells to E-selectin-expressing CHO cells, supporting the earlier hypothesis on the involvement of gangliosides in binding of anti-sialosyl Lewis(a) in the partial absence of mucin O-glycans. This hypothesis was further proven by electron microscopy data. Both native CX-1 and d,l-threo-PPPP-treated cells were labelled with anti-sialosyl Lewis(a) antibody mostly at a distance 70-90 nm from cell surface, suggesting interaction with protein-bound carbohydrate structures only. In contrast, the cancer cells treated with GalNAc-alpha-O-benzyl showed most of the staining around 20 nm distance from the plasmalemma, implying that the antibody interacts with lipid-bound sialosyl Lewis(a) instead. The electron microscopy data in conjunction with other results described in this report strongly support the hypothesis that sialosyl Lea gangliosides are not involved in the adhesion of CX-1 cells to E-selectin when mucins are present on the cell surface, but they may be involved in binding to E-selectin in their absence.

[REP and ERIC repetitive DNA sequences in bacteria--diagnostic significance]. / Powtórzone sekwencje DNA typu REP i ERIC u bakterii--znaczenie diagnostyczne.

Main part of eukaryotic genomes is build of unique sequences coding proteins and RNAs, but they contain as well numerous repeats interspersed with single-copy fragments. Existence of repetitive sequences were also demonstrated in prokaryotic genomes. They are found in different species of Gram-negative and Gram-positive bacteria. Interspersed repetitive sequence elements called REP and ERIC sequences are present in different species of Enterobacteriaceae family, including Escherichia coli and Salmonella typhimurium. Their functions are not completely clear, probably they play important role in regulation of gene expression. Nevertheless, REP and ERIC elements are widely use in identification and genetic analysis of bacteria. For example, using rep-PCR technique it is possible to discriminate between closely related serovars of the same species, which enables to analyze phylogenetic and epidemiological relations among them.

Metastatic potential of human uroepithelial cancer cells is not dependent on their adhesion to E-selectin.

In our previous studies we have found that human uroepithelial cell lines differ in their expression of sialosyl LewisA antigen. We have also shown that among the studied cell lines, only Hu 1703He cells with the highest expression of this tetrasaccharide bind to E-selectin-expressing cells. In the present study we put forward the question, of whether sialosyl LewisA-mediated adhesion of uroepithelial cells to E-selectin is important in the formation of metastases. The HCV 29T and Hu 1703He cells, representing two uroepithelial cell lines, were transplanted into NCr nu/nu mice. Hu 1703He cells express on their surface a high level of sialosyl LeA antigen, while HCV 29T cells are sialosyl LewisA-negative. We have shown that human uroepithelial 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 tumour foci in lung and liver seems to be independent of the expression of sialosyl LewisA antigen.

Study on ganglioside composition in brain tumours supra- and infratentorial.

The composition of gangliosides in primary tumors depends on their histological origin and differentiation grade (biological malignancy). The aim of the present study was the comparison of gangliosides in various types of brain tumours. The studies were performed on specimens from 20 high grade gliomas, 5 low grade gliomas, 8 meningiomas and 9 metastatic tumours. The isolated gangliosides were separated on silica 60 HPTLC plates and their mobilities were compared with glycolipids standards. The following observations were made: 1. high and low grade gliomas had similar ganglioside profiles comprising 7 different species; 2. the profiles of gangliosides isolated from metastatic neoplasms differ considerably from those of gliomas as well as menigiomas.