Cytotoxic T-cell precursor frequencies to HER-2 (369 – 377) in patients with HER-2/neu-positive epithelial tumours

HER-2/neu oncoprotein contains several major histocompatibility complex class I-restricted epitopes, which are recognised by cytotoxic T lymphocyte (CTL) on autologous tumours and therefore can be used in immune-based cancer therapies. Of these, the most extensively studied is HER-2(9(369)). In the present report, we used dendritic cells pulsed with HER-2(9(369)) to stimulate, in the presence of IL-7 and IL-12, the production of IFN-gamma by patients' CTL detected by the enzyme-linked immunosorbent spot-assay. Frequencies of peptide-specific precursors were estimated in HLA-A2, HLA-A3 and HLA-A26 patients with HER-2/neu-positive (+) breast, ovarian, lung, colorectal and prostate cancers and healthy individuals. We found increased percentages of such precursors in HLA-A2 (25%) and HLA-A26 (30%) patients, which were significantly higher (60%) in HLA-A3 patients. Our results demonstrate for the first time that pre-existing immunity to HER-2(9(369)) occurs in patients with colorectal, lung and prostate cancer. They also suggest that HER-2(9(369)) can be recognised by CTL, besides HLA-A2, also in the context of HLA-A3 and HLA-A26, thus increasing the applicability of HER-2(9(369))-based vaccinations in a considerably broader patients' population.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Dysregulated Recruitment of the Histone Methyltransferase EZH2 to the Class II Transactivator (CIITA) Promoter IV in Breast Cancer Cells

One mechanism frequently utilized by tumor cells to escape immune system recognition and elimination is suppression of cell surface expression of Major Histocompatibility Class II (MHC II) molecules. Expression of MHC II is regulated primarily at the level of transcription by the Class II Transactivator, CIITA, and decreased CIITA expression is observed in multiple tumor types. We investigate here contributions of epigenetic modifications to transcriptional silencing of CIITA in variants of the human breast cancer cell line MDA MB 435. Significant increases in histone H3 lysine 27 trimethylation upon IFN-γ stimulation correlate with reductions in transcription factor recruitment to the interferon-γ inducible CIITA promoter, CIITApIV, and with significantly increased CIITApIV occupancy by the histone methyltransferase enhancer of zeste homolog 2 (EZH2). Most compelling is evidence that decreased expression of EZH2 in MDA MB 435 variants results in significant increases in CIITA and HLA-DRA mRNA expression, even in the absence of interferon-γ stimulation, as well as increased cell surface expression of MHC II. Together, these data add mechanistic insight to prior observations of increased EZH2 expression and decreased CIITA expression in multiple tumor types

Investigations and comparative detection of Cryptosporidium species by microscopy, nested PCR and LAMP in water supplies of Ordu, Middle Black Sea, Turkey

A total of 70 water samples, including tap, river, fountain and well water were collected in the Ordu province, Middle Black Sea, Turkey and investigated for the detection of Cryptosporidium oocysts. The samples were directly screened microscopically for Cryptosporidium oocysts' detection by immunofluorescence test and subsequently DNA was extracted for the molecular detection by loop-mediated isothermal amplification (LAMP) and nested polymerase chain reaction (PCR). Eighteen out of the 70 (25.7%) water samples were found positive for Cryptosporidium spp. by immunofluorescence test and 19 (27.1%) were found positive by LAMP. Nested PCR products were not generated in any of the investigated water samples. A total of 16 randomly selected pellets were spiked with 10 Cryptosporidium oocysts to test the efficiency of the applied method. All the samples were found positive by LAMP for the presence of Cryptosporidium DNA, while the nested PCR assay was positive in only seven (43.75%) out of the 16 examined spiked samples. This is the first report on the occurrence of Cryptosporidium species in environmental and drinking water supplies in the Black Sea area

Bench-scale experiments for the development of a unified loop-mediated isothermal amplification (LAMP) assay for the in vitro diagnosis of Leishmania species' promastigotes

We developed, in bench-scale experiments, a unified loop-mediated isothermal amplification (LAMP) assay for the detection of cutaneous, mucocutaneous and visceral leishmaniasis using DNA of cultivated promastigotes. Two primer sets for the LAMP assay were designed based on the 18S rRNA gene, and their sensitivity and specificity were tested and compared. Both of them were specific for Leishmania as the DNA of all ten Leishmania species tested was amplified, whereas the DNA of other parasites, including that of Trypanosoma, was not. The detection limit for primer set 1 ranged between 30 pg and 3.6 fg, depending on which Leishmania species tested. Primer set 2 showed high sensitivity, but was less sensitive than primer set 1. Our findings lead to the conclusion that the LAMP assay with primer set 1 is a promising and effective assay for the successful detection of a wide range of Leishmania infections using only a unified multiplex LAMP test