Galactosylceramide Upregulates the Expression of the BCL2 Gene and Downregulates the Expression of TNFRSF1B and TNFRSF9 Genes, Acting as an Anti-Apoptotic Molecule in Breast Cancer Cells.

Galactosylceramide (GalCer) increases the resistance of breast cancer cells to doxorubicin, paclitaxel, and cisplatin by acting as an anti-apoptotic molecule. GalCer was found to specifically downregulate the levels of the pro-apoptotic TNFRSF1B and TNFRSF9 genes and upregulate the levels of the anti-apoptotic BCL2 gene, suggesting that this glycosphingolipid regulates their expression at the transcriptional level. Consistent with this hypothesis, MDA-MB-231 and MCF7 breast cancer cells with high levels of GalCer showed lower activity of the TNFRSF1B and TNFRSF9 promoters than cells lacking GalCer. In contrast, the activity of the BCL2 promoter was higher in MCF7 cells overproducing GalCer than in MCF7 cells without GalCer. However, no difference in BCL2 promoter activity was observed between MDA-MB-231 cells with high and no GalCer content. Instead, we found that high levels of GalCer increased the stability of Bcl-2 mRNA. Subsequent studies showed that breast cancer cells with high levels of GalCer are characterized by significantly lower expression of P53. Importantly, inhibition of P53 expression by siRNA in MCF7 and MDA-MB-231 cells lacking GalCer resulted in decreased expression and promoter activity of the TNFRS1B and TNFRSF9 genes. On the other hand, increased expression and promoter activity of the BCL2 gene was found in such MCF7 cells, and increased stability of Bcl-2 transcripts was observed in such MDA-MB-231 cells. Taken together, these data strongly suggest that the regulatory protein that simultaneously increases the expression of the TNFRSF1B and TNFRSF9 genes and decreases the expression of the BCL2 gene and the stability of Bcl-2 transcripts is most likely P53, the expression of which is GalCer dependent.

Targeting SOX18 Transcription Factor Activity by Small-Molecule Inhibitor Sm4 in Non-Small Lung Cancer Cell Lines.

The transcription factor SOX18 has been shown to play a crucial role in lung cancer progression and metastasis. In this study, we investigated the effect of Sm4, a SOX18 inhibitor, on cell cycle regulation in non-small cell lung cancer (NSCLC) cell lines LXF-289 and SK-MES-1, as well as normal human lung fibroblast cell line IMR-90. Our results demonstrated that Sm4 treatment induced cytotoxic effects on all three cell lines, with a greater effect observed in NSCLC adenocarcinoma cells. Sm4 treatment led to S-phase cell accumulation and upregulation of p21, a key regulator of the S-to-G2/M phase transition. While no significant changes in SOX7 or SOX17 protein expression were observed, Sm4 treatment resulted in a significant upregulation of SOX17 gene expression. Furthermore, our findings suggest a complex interplay between SOX18 and p21 in the context of lung cancer, with a positive correlation observed between SOX18 expression and p21 nuclear presence in clinical tissue samples obtained from lung cancer patients. These results suggest that Sm4 has the potential to disrupt the cell cycle and target cancer cell growth by modulating SOX18 activity and p21 expression. Further investigation is necessary to fully understand the relationship between SOX18 and p21 in lung cancer and to explore the therapeutic potential of SOX18 inhibition in lung cancer.

Unraveling the role of type 1 fimbriae in Salmonella pathogenesis: insights from a comparative analysis of Salmonella Enteritidis and Salmonella Gallinarum.

Significant differences in pathogenicity between Salmonella Enteritidis and Salmonella Gallinarum exist despite the fact that S. Gallinarum is a direct descendant of S. Enteritidis. It was hypothesized that such various properties may be in part the result of differences in structure and functions of type 1 fimbriae (T1Fs). In S. Enteritidis, T1Fs bind to oligomannosidic structures carried by host cell glycoproteins and are called mannose-sensitive T1Fs (MST1F). In S. Gallinarum, T1Fs lost ability to bind such carbohydrate chains, and were named mannose-resistant MRT1Fs (MRT1F). Therefore, the present study was undertaken to evaluate the role of MST1Fs and MRT1Fs in the adhesion, invasion, intracellular survival and cytotoxicity of S. Enteritidis and S. Gallinarum toward chicken intestinal CHIC8-E11cells and macrophage-like HD11 cells. Using mutant strains: S. Enteritidis fimH::kan and S. Gallinarum fimH::kan devoid of T1Fs and in vitro assays the following observations were made. MST1Fs have a significant impact on the chicken cell invasion by S. Enteritidis as MST1F-mediated adhesion facilitates direct and stable contact of bacteria with host cells, in contrast to MRT1Fs expressed by S. Gallinarum. MST1Fs as well as MRT1Fs did not affected intracellular viability of S. Enteritidis and S. Gallinarum. However, absolute numbers of intracellular viable wild-type S. Enteritidis were significantly higher than S. Enteritidis fimH::kan mutant and wild-type S. Gallinarum and S. Gallinarum fimH::kan mutant. These differences, reflecting the numbers of adherent and invading bacteria, underline the importance of MST1Fs in the pathogenicity of S. Enteritidis infections. The cytotoxicity of wild-type S. Enteritidis and its mutant devoid of MST1Fs to HD11 cells was essentially the same, despite the fact that the number of viable intracellular bacteria was significantly lower in the mutated strain. Using HD11 cells with similar number of intracellular wild-type S. Enteritidis and S. Enteritidis fimH::kan mutant, it was found that the lack of MST1Fs did not affect directly the cytotoxicity, suggesting that the increase in cytotoxicity of S. Enteritidis devoid of MST1Fs may be associated with crosstalk between T1Fs and other virulence factors.

Prolactin-induced protein (PIP) increases the sensitivity of breast cancer cells to drug-induced apoptosis.

We have previously shown that high expression of prolactin-induced protein (PIP) correlates with the response of breast cancer (BC) patients to standard adjuvant chemotherapy (doxorubicin and cyclophosphamide), which suggests that the absence of this glycoprotein is associated with resistance of tumor cells to chemotherapy. Therefore, in the present study, we analyzed the impact of PIP expression on resistance of BC cells to anti-cancer drugs and its biological role in BC progression. Expression of PIP and apoptotic genes in BC cell lines was analyzed using real-time PCR and Western blotting. PIP was detected in BC tissue specimens using immunohistochemistry. The tumorigenicity of cancer cells was analyzed by the in vivo tumor growth assay. Apoptotic cells were detected based on caspase-3 activation, Annexin V binding and TUNEL assay. The interaction of PIP with BC cells was analyzed using flow cytometry. Using two cellular models of BC (i.e. T47D cells with the knockdown of the PIP gene and MDA-MB-231 cells overexpressing PIP), we found that high expression of PIP resulted in (1) increased sensitivity of BC cells to apoptosis induced by doxorubicin (DOX), 4-hydroperoxycyclophosphamide (4-HC), and paclitaxel (PAX), and (2) improved efficacy of anti-cancer therapy with DOX in the xenograft mice model. Accordingly, a clinical study revealed that BC patients with higher PIP expression were characterized by longer 5-year overall survival and disease-free survival. Subsequent studies showed that PIP up-regulated the expression of the following pro-apoptotic genes: CRADD, DAPK1, FASLG, CD40 and BNIP2. This pro-apoptotic activity is mediated by secreted PIP and most probably involves the specific surface receptor. This study demonstrates that a high expression level of PIP sensitizes BC cells to anti-cancer drugs. Increased sensitivity to chemotherapy is the result of pro-apoptotic activity of PIP, which is evidenced by up-regulation of specific pro-apoptotic genes. As high expression of PIP significantly correlated with a better response of patients to anti-cancer drugs, this glycoprotein can be a marker for the prognostic evaluation of adjuvant chemotherapy.

Expression of the Heterotrimeric GP2/GP3/GP4 Spike of an Arterivirus in Mammalian Cells.

Equine arteritis virus (EAV), an enveloped positive-strand RNA virus, is an important pathogen of horses and the prototype member of the Arteiviridae family. Unlike many other enveloped viruses, which possess homotrimeric spikes, the spike responsible for cellular tropism in Arteriviruses is a heterotrimer composed of 3 glycoproteins: GP2, GP3, and GP4. Together with the hydrophobic protein E they are the minor components of virus particles. We describe the expression of all 3 minor glycoproteins, each equipped with a different tag, from a multi-cassette system in mammalian BHK-21 cells. Coprecipitation studies suggest that a rather small faction of GP2, GP3, and GP4 form dimeric or trimeric complexes. GP2, GP3, and GP4 co-localize with each other and also, albeit weaker, with the E-protein. The co-localization of GP3-HA and GP2-myc was tested with markers for ER, ERGIC, and cis-Golgi. The co-localization of GP3-HA was the same regardless of whether it was expressed alone or as a complex, whereas the transport of GP2-myc to cis-Golgi was higher when this protein was expressed as a complex. The glycosylation pattern was also independent of whether the proteins were expressed alone or together. The recombinant spike might be a tool for basic research but might also be used as a subunit vaccine for horses.

Genomic and functional characterization of five novel Salmonella-targeting bacteriophages.

BACKGROUND: The host-unrestricted, non-typhoidal Salmonella enterica serovar Enteritidis (S. Enteritidis) and the serovar Typhimurium (S. Typhimurium) are major causative agents of food-borne gastroenteritis, and the host-restricted Salmonella enterica serovar Gallinarum (S. Gallinarum) is responsible for fowl typhoid. Increasing drug resistance in Salmonella contributes to the reduction of effective therapeutic and/or preventive options. Bacteriophages appear to be promising antibacterial tools, able to combat infectious diseases caused by a wide range of Salmonella strains belonging to both host-unrestricted and host-restricted Salmonella serovars. METHODS: In this study, five novel lytic Salmonella phages, named UPWr_S1-5, were isolated and characterized, including host range determination by plaque formation, morphology visualization with transmission electron microscopy, and establishment of physiological parameters. Moreover, phage genomes were sequenced, annotated and analyzed, and their genomes were compared with reference Salmonella phages by use of average nucleotide identity, phylogeny, dot plot, single nucleotide variation and protein function analysis. RESULTS: It was found that UPWr_S1-5 phages belong to the genus Jerseyvirus within the Siphoviridae family. All UPWr_S phages were found to efficiently infect various Salmonella serovars. Host range determination revealed differences in host infection profiles and exhibited ability to infect Salmonella enterica serovars such as Enteritidis, Gallinarum, Senftenberg, Stanley and Chester. The lytic life cycle of UPWr_S phages was confirmed using the mitomycin C test assay. Genomic analysis revealed that genomes of UPWr_S phages are composed of 51 core and 19 accessory genes, with 33 of all predicted genes having assigned functions. UPWr_S genome organization comparison revealed 3 kinds of genomes and mosaic structure. UPWr_S phages showed very high sequence similarity to each other, with more than 95% average nucleotide identity. CONCLUSIONS: Five novel UPWr_S1-5 bacteriophages were isolated and characterized. They exhibit host lysis range within 5 different serovars and are efficient in lysis of both host-unrestricted and host-restricted Salmonella serovars. Therefore, because of their ability to infect various Salmonella serovars and lytic life cycle, UPWr_S1-5 phages can be considered as useful tools in biological control of salmonellosis.

Glucosylceramide and galactosylceramide, small glycosphingolipids with significant impact on health and disease.

Numerous clinical observations and exploitation of cellular and animal models indicate that glucosylceramide (GlcCer) and galactosylceramide (GalCer) are involved in many physiological and pathological phenomena. In many cases, the biological importance of these monohexosylcermides has been shown indirectly as the result of studies on enzymes involved in their synthesis and degradation. Under physiological conditions, GalCer plays a key role in the maintenance of proper structure and stability of myelin and differentiation of oligodendrocytes. On the other hand, GlcCer is necessary for the proper functions of epidermis. Such an important lysosomal storage disease as Gaucher disease (GD) and a neurodegenerative disorder as Parkinson's disease are characterized by mutations in the GBA1 gene, decreased activity of lysosomal GBA1 glucosylceramidase and accumulation of GlcCer. In contrast, another lysosomal disease, Krabbe disease, is associated with mutations in the GALC gene, resulting in deficiency or decreased activity of lysosomal galactosylceramidase and accumulation of GalCer and galactosylsphingosine. Little is known about the role of both monohexosylceramides in tumor progression; however, numerous studies indicate that GlcCer and GalCer play important roles in the development of multidrug-resistance by cancer cells. It was shown that GlcCer is able to provoke immune reaction and acts as a self-antigen in GD. On the other hand, GalCer was recognized as an important cellular receptor for HIV-1. Altogether, these two molecules are excellent examples of how slight differences in chemical composition and molecular conformation contribute to profound differences in their physicochemical properties and biological functions.

Catabolic/Anabolic Imbalance Is Accompanied by Changes of Left Ventricular Steroid Nuclear Receptor Expression in Tachycardia-Induced Systolic Heart Failure in Male Pigs.

BACKGROUND: Steroid hormones play an important role in heart failure (HF) pathogenesis, and clinical data have revealed disordered steroidogenesis in male patients with HF. However, there is still a lack of studies on steroid hormones and their receptors during HF progression. Therefore, a porcine model of tachycardia-induced cardiomyopathy corresponding to HF was used to assess steroid hormone concentrations in serum and their nuclear receptor levels in heart tissue during the consecutive stages of HF. METHODS AND RESULTS: Male pigs underwent right ventricular pacing and developed a clinical picture of mild, moderate, or severe HF. Serum concentrations of dehydroepiandrosterone, testosterone, dihydrotestosterone, estradiol, aldosterone, and cortisol were assessed by enzyme-linked immunosorbent assay. Androgen receptor, estrogen receptor alpha, mineralocorticoid receptor, and glucocorticoid receptor messenger RNA levels in the left ventricle were determined by qPCR.The androgen level decreased in moderate and severe HF animals, while the corticosteroid level increased. The estradiol concentration remained stable. The quantitative real-time polymerase chain reaction revealed the downregulation of androgen receptor in consecutive stages of HF and increased expression of mineralocorticoid receptor messenger RNA under these conditions. CONCLUSIONS: In the HF pig model, deteriorated catabolic/anabolic balance, manifested by upregulation of aldosterone and cortisol and downregulation of androgen signaling on the ligand level, was augmented by changes in steroid hormone receptor expression in the heart tissue.

Antitumor and antioxidant activities of purple potato ethanolic extract and its interaction with liposomes, albumin and plasmid DNA.

The aim of the study was to broadly determine the biological activities of purple potato ethanolic extract of the Blue Congo variety (BCE). The antioxidant activity of BCE was determined in relation to liposome membranes, and peroxidation was induced by UVB and AAPH. To clarify the antioxidant activity of BCE, we investigated its interactions with hydrophilic and hydrophobic regions of a membrane using fluorimetric and FTIR methods. Next, we investigated the cytotoxicity and pro-apoptotic activities of BCE in two human colon cancer cell lines (HT-29 and Caco-2) and in normal cells (IPEC-J2). In addition, the ability to inhibit enzymes that are involved in pro-inflammatory reactions was examined. Furthermore, BCE interactions with serum albumin and plasmid DNA were investigated using steady state fluorescence spectroscopy and a single molecule fluorescence technique (TCSPC-FCS). We proved that BCE effectively protects lipid membranes against the process of peroxidation and successfully inhibits the cyclooxygenase and lipoxygenase enzymes. Furthermore, it interacts with the hydrophilic and hydrophobic parts of lipid membranes as well as with albumin and plasmid DNA. It was observed that BCE is more cytotoxic against colon cancer cell lines than normal IPEC-J2 cells; it also induces apoptosis in cancer cell lines, but does not induce cell death in normal cells.

Retinol-Binding Protein 4 Accelerates Metastatic Spread and Increases Impairment of Blood Flow in Mouse Mammary Gland Tumors.

Retinol-binding protein 4 (RBP4) is proposed as an adipokine that links obesity and cancer. We analyzed the role of RBP4 in metastasis of breast cancer in patients and in mice bearing metastatic 4T1 and nonmetastatic 67NR mammary gland cancer. We compared the metastatic and angiogenic potential of these cells transduced with Rbp4 (4T1/RBP4 and 67NR/RBP4 cell lines). Higher plasma levels of RBP4 were observed in breast cancer patients with metastatic tumors than in healthy donors and patients with nonmetastatic cancer. Increased levels of RBP4 were observed in plasma, tumor tissue, liver, and abdominal fat. Moreover, the blood vessel network was highly impaired in mice bearing 4T1 as compared to 67NR tumors. RBP4 transductants showed further impairment of blood flow and increased metastatic potential. Exogenous RBP4 increased lung settlement by 67NR and 4T1 cells. In vitro studies showed increased invasive and clonogenic potential of cancer cells treated with or overexpressing RBP4. This effect is not dependent on STAT3 phosphorylation. RBP4 enhances the metastatic potential of breast cancer tumors through a direct effect on cancer cells and through increased endothelial dysfunction and impairment of blood vessels within the tumor.

Production of Recombinant EAV with Tagged Structural Protein Gp3 to Study Artervirus Minor Protein Localization in Infected Cells.

Equine arteritis virus (EAV) is a prototype member of the Arterivirus family, comprising important pathogens of domestic animals. Minor glycoproteins of Arteriviruses are responsible for virus entry and cellular tropism. The experimental methods for studying minor Arterivirus proteins are limited because of the lack of antibodies and nested open reading frames (ORFs). In this study, we generated recombinant EAV with separated ORFs 3 and 4, and Gp3 carrying HA-tag (Gp3-HA). The recombinant viruses were stable on passaging and replicated in titers similar to the wild-type EAV. Gp3-HA was incorporated into the virion particles as monomers and as a Gp2/Gp3-HA/Gp4 trimer. Gp3-HA localized in ER and, to a lesser extent, in the Golgi, it also co-localized with the E protein but not with the N protein. The co-localization of Gp3-HA and the E protein with ERGIC was reduced. Moreover, EAV with Gp3-HA could become a valuable research tool for identifying host cell factors during infection and the role of Gp3 in virus attachment and entry.

Everything You Always Wanted to Know About Salmonella Type 1 Fimbriae, but Were Afraid to Ask.

Initial attachment to host intestinal mucosa after oral infection is one of the most important stages during bacterial pathogenesis. Adhesive structures, widely present on the bacterial surface, are mainly responsible for the first contact with host cells and of host-pathogen interactions. Among dozens of different bacterial adhesins, type 1 fimbriae (T1F) are one of the most common adhesive organelles in the members of the Enterobacteriaceae family, including Salmonella spp., and are important virulence factors. Those long, thin structures, composed mainly of FimA proteins, are responsible for recognizing and binding high-mannose oligosaccharides, which are carried by various glycoproteins and expressed at the host cell surface, via FimH adhesin, which is presented at the top of T1F. In this review, we discuss investigations into the functions of T1F, from the earliest work published in 1958 to operon organization, organelle structure, T1F biogenesis, and the various functions of T1F in Salmonella-host interactions. We give special attention to regulation of T1F expression and their role in binding of Salmonella to cells, cell lines, organ explants, and other surfaces with emphasis on biofilm formation and discuss T1F role as virulence factors based on work using animal models. We also discuss the importance of allelic variation in fimH to Salmonella pathogenesis, as well as role of FimH in Salmonella host specificity.

Syntheses and cytotoxicity of phosphatidylcholines containing ibuprofen or naproxen moieties.

In this study, novel phosphatidylcholines containing ibuprofen or naproxen moieties were synthesized in good yields and high purities. Under the given synthesis conditions, the attached drug moieties racemized, which resulted in the formation of phospholipid diastereomers. The comperative studies of the cytotoxicity of ibuprofen, naproxen and their phosphatidylcholine derivatives against human promyelocytic leukemia HL-60, human colon carcinoma Caco-2, and porcine epithelial intestinal IPEC-J2 cells were carried out. The results of these studies indicated that phospholipids with NSAIDs at both sn-1 and sn-2 positions (15 and 16) were more toxic than ibuprofen or naproxen themselves, whereas 2-lysophosphatidylcholines (7 and 8) were less toxic against all tested cell lines. Phospholipids with NSAIDs at sn-1 and palmitic acid at sn-2 (9 and 10) were also less toxic against Caco-2 and normal cells (IPEC-J2).

Prolactin-induced protein (PIP)-characterization and role in breast cancer progression.

Prolactin-induced protein (PIP) is a small secreted glycoprotein carrying several N-linked carbohydrate chains. The expression of PIP is generally restricted to cells with apocrine properties. It was found in apocrine glands of the axilla, vulva, eyelid, ear canal, and seminal vesicle. Being a secretory protein, PIP is present in seminal plasma, saliva, lacrimal fluid, tears, sweat gland secretion. Little is known about the biological role of PIP. It binds to numerous proteins, however, in most cases the biological role of such interactions is poorly understood. A notable exception is its binding to CD4 receptors present on the surface of T lymphocytes, macrophages, and spermatozoa. The available data suggest that PIP can have immunomodulatory functions and plays an important role in cell-mediate adoptive immunity. PIP binds to bacteria from several genera, which suggests that this glycoprotein may participate also in innate immunity and protection of hosts against microbial infections. Increased levels of PIP were found in several types of human cancer (prostate, sweat and salivary gland cancers). It is especially common in breast cancer, however, data on the expression of PIP in normal and cancerous breast cancer tissues are to some degree conflicting. In early studies, it was shown that PIP is absent or its expression is very low in normal breast epithelium, whereas in breast cancers PIP is frequently expressed and present in large amounts. On the other hand, later study showed that expression of PIP is lower in advanced apocrine carcinomas and invasive carcinomas than in, respectively, in situ carcinomas and adjacent normal tissue. The most recent study revealed that PIP gene expression decreased gradually along with higher stage and grade of breast cancer. In agreement with these data, it was shown that that low levels or the lack of PIP expression are associated with a worse response of breast cancer cells to chemotherapy. It was proposed that PIP plays important role in the development and progression of breast cancer. However, its role in these processes is both unclear and controversial. In this review, the role of PIP in both physiological processes and carcinogenesis is discussed.

Sulfatide decreases the resistance to stress-induced apoptosis and increases P-selectin-mediated adhesion: a two-edged sword in breast cancer progression.

BACKGROUND: We have previously shown that galactosylceramide (GalCer) affects the tumourigenic and metastatic properties of breast cancer cells by acting as an anti-apoptotic molecule. Since GalCer is a precursor molecule in the synthesis of sulfatides, the present study was aimed to define the role of sulfatides in apoptosis and breast cancer progression. METHODS: Expression of GAL3ST1 in breast cancer cell lines and breast cancer tissue specimens was analysed using real-time PCR, western blotting and immunohistochemistry analysis. The amount of sulfatide, GalCer and ceramide was analysed by thin-layer chromatography binding assay and by the modified hydrophilic interaction liquid chromatography coupled with electrospray mass spectrometry methodology. The tumourigenicity of cancer cells was analysed by an in-vivo tumour growth assay. Apoptotic cells were detected based on caspase-3 activation and the TUNEL assay. The interaction of breast cancer cells with P-selectin or E-selectin was analysed using the flow adhesion assay. The ability of sulfatide-expressing cells to activate and aggregate platelets was studied using the flow-cytometry-based aggregation assay. RESULTS: Using two models of breast cancer, T47D cells with blocked synthesis of sulfatide and MDA-MB-231 cells with neosynthesis of this glycosphingolipid, we showed that high sulfatide levels resulted in increased sensitivity of cancer cells to apoptosis induced by hypoxia and doxorubicin in vitro, and decreased their tumourigenicity after transplantation into athymic nu/nu mice. Accordingly, a clinical study on GAL3ST1 expression in invasive ductal carcinoma revealed that its elevated level is associated with better prognosis. Using MDA-MB-231 cells with neosynthesis of sulfatide we also showed that sulfatide is responsible for adhesion of breast cancer cells to P-selectin-expressing cells, including platelets. Sulfatide also acted as an activating molecule, increasing the expression of P-selectin. CONCLUSIONS: This study demonstrates that increased synthesis of sulfatide sensitises cancer cells to microenvironmental stress factors such as hypoxia and anticancer drugs such as doxorubicin. However, sulfatide is probably not directly involved in apoptotic cascades, because its increased synthesis by GAL3ST1 decreased the amounts of its precursor, GalCer, a known anti-apoptotic molecule. On the other hand, our data support the view that sulfatides are malignancy-related adhesive molecules involved in activating and binding P-selectin-expressing platelets to breast cancer cells.

Development of novel monoclonal antibodies to dog leukocyte antigen DR displaying direct and immune-mediated cytotoxicity toward canine lymphoma cell lines.

Spontaneous canine lymphoma (CL) has become a promising, nonrodent model for advancing the therapeutic strategies of human hematological malignancies. As new resources for veterinary and comparative studies on CL-associated antigens, we developed 2 novel mouse monoclonal antibodies, denoted B5 and E11, that recognized the canine major histocompatibility Class II DR antigens (dog leukocyte antigen DR). Using flow cytometry and solid phase immunoenzymatic assays, we showed that the antigens recognized by B5 and E11 were strongly expressed in several CL cell lines and the ex vivo canine neoplastic cells of B and mixed B/T immunophenotypes. Additionally, we evaluated a minimal cross-reactivity of B5 and E11 with the human B-cell line, Raji. By the ectopic expression of the hybrid murine/canine I-E/DR dimers in the HEK293 cells, we demonstrated that the epitope of B5 was localized to the invariant DRα chain, whereas the epitope of E11 was collectively formed by the DRα and DRß chains. Both epitopes were conformational and conserved in all the tested unrelated individuals of different dog breeds. In vitro treatment of 2 CL B-cell lines (CLBL1 and CLB70) with B5 and E11 rapidly induced a direct apoptotic cell death. Similarily, both mouse monoclonal antibodies efficiently killed the above cell lines through the mechanisms of complement-dependent and antibody-mediated cellular phagocytosis. Collectively, our data support the further development of B5 and E11 as novel tools for dog leukocyte antigen DR-targeted, preclinical trials involving CL.

Podoplanin increases the migration of human fibroblasts and affects the endothelial cell network formation: A possible role for cancer-associated fibroblasts in breast cancer progression.

In our previous studies we showed that in breast cancer podoplanin-positive cancer-associated fibroblasts correlated positively with tumor size, grade of malignancy, lymph node metastasis, lymphovascular invasion and poor patients' outcome. Therefore, the present study was undertaken to assess if podoplanin expressed by fibroblasts can affect malignancy-associated properties of breast cancer cells. Human fibroblastic cell lines (MSU1.1 and Hs 578Bst) overexpressing podoplanin and control fibroblasts were co-cultured with breast cancer MDA-MB-231 and MCF7 cells and the impact of podoplanin expressed by fibroblasts on migration and invasiveness of breast cancer cells were studied in vitro. Migratory and invasive properties of breast cancer cells were not affected by the presence of podoplanin on the surface of fibroblasts. However, ectopic expression of podoplanin highly increases the migration of MSU1.1 and Hs 578Bst fibroblasts. The present study also revealed for the first time, that podoplanin expression affects the formation of pseudo tubes by endothelial cells. When human HSkMEC cells were co-cultured with podoplanin-rich fibroblasts the endothelial cell capillary-like network was characterized by significantly lower numbers of nodes and meshes than in co-cultures of endothelial cells with podoplanin-negative fibroblasts. The question remains as to how our experimental data can be correlated with previous clinical data showing an association between the presence of podoplanin-positive cancer-associated fibroblasts and progression of breast cancer. Therefore, we propose that expression of podoplanin by fibroblasts facilitates their movement into the tumor stroma, which creates a favorable microenvironment for tumor progression by increasing the number of cancer-associated fibroblasts, which produce numerous factors affecting proliferation, survival and invasion of cancer cells. In accordance with this, the present study revealed for the first time, that such podoplanin-mediated effects can affect tube formation by endothelial cells and participate in their pathological properties in the tumor context. Our experimental data were supported by clinical studies. First, when IDC and DCIS were analyzed by immunohistochemistry according to the presence of podoplanin-expressing cells, the numbers of cancer-associated fibroblasts with high expression of this glycoprotein were significantly higher in IDC than in DCIS cases. Second, using immunofluorescence, the co-localization of PDPN-positive CAFs with blood vessels stained with antibody directed against CD34 was observed in tumor stroma of IDC samples.

Expression of periostin in breast cancer cells.

Periostin (POSTN) is a protein involved in multiple processes important for cancer development, both at the stage of cancer initiation and progression, as well as metastasis. The aim of this study was to determine the expression of POSTN in the cells of non-invasive ductal breast carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) and to correlate it with clinicopathological data. Immunohistochemical studies (IHC) were conducted on 21 cases of fibrocystic breast change (FC), 44 cases of DCIS and 92 cases of IDC. POSTN expression at mRNA (real-time PCR) and protein level (western blot analysis) was also confirmed in selected breast cancer cell lines (MCF-7, SK-BR-3, MDA-MB-231 and BO2). Statistically significant higher level of POSTN expression in IDC and DCIS cancer cells compared to FC was noted. Also, the level of POSTN expression in the cytoplasm of IDC cells was shown to increase with the increasing degree of tumour malignancy (G) and significantly higher expression of POSTN was observed in each degree of tumour malignancy (G) relative to FC. Statistically significant higher POSTN expression was observed in tumours with estrogen receptor-negative (ER-) and progesterone receptor-negative (PR-) phenotypes in comparison to estrogen receptor-positive (ER+) and progesterone receptor-positive (PR+) cases, as well as significant negative correlation between POSTN expression in cancer cells and expression of ER and PR (p<0.05). Additionally, statistically significant differences in POSTN expression were shown between particular breast cancer cell lines, both at mRNA and protein level. Observed POSTN expression was the lowest in the case of MCF-7, and the highest in MDA-MB-231 and BO2 of the most aggressive potential clinically corresponding to G3 tumours. POSTN expression in the cytoplasm of IDC cancer cells may play an important role in cancer transformation mechanism.

The Novel Type 1 Fimbriae FimH Receptor Calreticulin Plays a Role in Salmonella Host Specificity.

It was suggested that minor differences in the structure of FimH are most likely associated with differences in its adhesion specificities and may determine the tropism of various Salmonella serovars to different species and tissues. We have recently shown that FimH adhesins from host-adapted serovars, e.g., Salmonella Choleraesuis (SCh), bind to other glycoprotein receptors compared to FimH from host-unrestricted Salmonella Enteritidis (SE). Here we identify porcine calreticulin expressed by swine intestinal cells as a host-specific receptor for SCh FimH adhesin, suggesting that such an interaction may contribute to SCh host specificity. Calreticulin was identified by 2D electrophoresis and mass spectrometry as a glycoprotein that was bound specifically by recombinant SCh FimH protein, but not by FimH from SE. The functionality of calreticulin as a specific receptor of SCh FimH adhesin was further confirmed by adhesion and invasion of mutated strains of SCh carrying different variants of FimH proteins to IPEC-J2 cells with overexpression and silenced expression of calreticulin. It was found that SCh carrying the active variant of FimH adhered and invaded IPEC-J2 cells with calreticulin overexpression at significantly higher numbers than those of SCh expressing the non-active variant or SE variant of FimH. Moreover, binding of SCh carrying the active variant of FimH to IPEC-J2 with silenced calreticulin expression was significantly weaker. Furthermore, we observed that SCh infection induces translocation of calreticulin to cell membrane. All of the aforementioned results lead to the general conclusion that Salmonella host specificity requires not only special mechanisms and proteins expressed by the pathogen but also specifically recognized receptors expressed by a specific host.