Survey of autoantibody responses against tumor-associated antigens in thyroid cancer.

BACKGROUND: Autoantibodies against tumor-associated antigens (TAAs) have been shown to serve as highly specific serological biomarkers for the diagnosis of various solid cancers. Although the autoimmunity against thyroid tissue specific antigens has been studied extensively, so far, the autoantibody responses against common TAAs such as cancer-testis antigens (CTAs), mutated or differentiation antigens have not been comprehensively analyzed in patients with thyroid cancer. OBJECTIVE: The current study aims to characterize the frequency of autoantibody responses against common TAAs in patients with thyroid cancer and benign thyroid nodules. METHODS: A phage-displayed antigen microarray comprising 65 TAAs was produced and tested with sera from 53 patients with thyroid cancer, 90 patients with benign thyroid nodules and 96 cancer-free individuals, 100 melanoma, 54 breast cancer and 14 lung cancer patients as controls. RESULTS: A panel of 6 TAAs was identified that preferentially reacted with sera from patients with thyroid cancer. The top ranked antigen in this panel was GAGE1 eliciting autoantibody response in 6% of patients with thyroid cancer but not with benign nodules, whereas no reactivity to other CTAs was detected in the sera from patients with thyroid cancer. CONCLUSIONS: Although six TAAs, including one CTA, showed thyroid cancer-associated reactivity, overall, spontaneous humoral immune responses against TAAs are rare in thyroid cancer and their utility for the development of non-invasive assay for the differential diagnosis of thyroid nodules is limited.

ELISPOT assays provide reproducible results among different laboratories for T-cell immune monitoring--even in hands of ELISPOT-inexperienced investigators.

Measurements of antibodies in bodily fluids (e.g., by ELISA) have provided robust and reproducible results for decades and such assays have been validated for monitoring of B-cell immunity. In contrast, measuring T-cell immunity has proven to be a challenge due to the need to test live cells in functional assays ex vivo. Several previous efforts looking into the reproducibility of ex vivo T-cell assays between different laboratories, or even within the same laboratory, have provided rather discouraging results. The hypothesis we tested in this study is that those poor results are due to the lack of assay and data analysis standardization, rather than the inherent complexity of T-cell assays. In this study, 11 laboratories across Europe and the United States were provided identical reagents and were asked to follow the same protocol while testing aliquots of the same three cryopreserved peripheral blood mononuclear cells (PBMC) in an interferon-gamma (IFNgamma) ELISPOT assay measuring the antigen-specific T-cell response to a CMV peptide. All individuals performing the assays were ELISPOT novices. At their first attempt, while three of these individuals failed with the basic logistics of the trial, eight detected the peptide-specific CD8+ T-cells in frequencies approximating the values established by the Reference Laboratory. The data show that ELISPOT assays provide reproducible results among different laboratories when the assay procedure and data analysis is standardized. Since ELISPOT assays have been qualified and validated for regulated studies, they are ideal candidates for robust and reproducible monitoring of T-cell activity in vivo.

Molecular characterisation and expression analysis of SEREX-defined antigen NUCB2 in gastric epithelium, gastritis and gastric cancer.

NUCB2 is an EF-hand Ca2+ binding protein that has been implicated in various physiological processes like calcium homeostasis, hypothalamic regulation of feeding and TNF receptor shedding. In our previous study we identified NUCB2 as a potential tumour antigen eliciting autoantibody responses in 5.4% of gastric cancer patients but not in the healthy individuals.The current study aimed to elucidate the molecular mechanism underlying NUCB2 immunogenicity and to gain an insight into the physiological functions of NUCB2 in the stomach. mRNA expression analysis demonstrated that NUCB2 is ubiquitously expressed in normal tissues, including lymphoid tissues, and downregulated in gastric tumours when compared with the adjacent relatively normal stomach tissues.The search for molecular alterations resulted in the identification of novel mRNA variants transcribed from an alternative promoter and expressed predominantly in gastric cancers. Western blot analysis demonstrated that the protein levels correspond to mRNA levels and revealed that NUCB2 is phosphorylated in gastric mucosa. Furthermore, a 55 kDa isoform,generated presumably by yet an unidentified post-translational modification was detected in gastric tumours and AGS gastric cancer cells but was absent in the relatively normal gastric mucosa and thereby might have served as a trigger for the immune response against NUCB2. Staining of stomach tissue microarray with anti-NUCB2 antibody revealed that it is expressed in the secretory granules of chief cells and in the cytoplasm of parietal cells in the functioning gastric glands which are lost in atrophic glands and tumour cells. Hence we propose that NUCB2 may be implicated in gastric secretion by establishing an agonist-releasable Ca2+ store in ER or Golgi apparatus, signalling via heterotrimeric Galpha proteins and/or mediating the exocytosis of the secretory granules.

Molecular characterisation and expression analysis of SEREX-defined antigen NUCB2 in gastric epithelium, gastritis and gastric cancer.

NUCB2 is an EF-hand Ca2+ binding protein that has been implicated in various physiological processes like calcium homeostasis, hypothalamic regulation of feeding and TNF receptor shedding. In our previous study we identified NUCB2 as a potential tumour antigen eliciting autoantibody responses in 5.4% of gastric cancer patients but not in the healthy individuals. The current study aimed to elucidate the molecular mechanism underlying NUCB2 immunogenicity and to gain an insight into the physiological functions of NUCB2 in the stomach. mRNA expression analysis demonstrated that NUCB2 is ubiquitously expressed in normal tissues, including lymphoid tissues, and downregulated in gastric tumours when compared with the adjacent relatively normal stomach tissues. The search for molecular alterations resulted in the identification of novel mRNA variants transcribed from an alternative promoter and expressed predominantly in gastric cancers. Western blot analysis demonstrated that the protein levels correspond to mRNA levels and revealed that NUCB2 is phosphorylated in gastric mucosa. Furthermore, a 55 kDa isoform, generated presumably by yet an unidentified post-translational modification was detected in gastric tumours and AGS gastric cancer cells but was absent in the relatively normal gastric mucosa and thereby might have served as a trigger for the immune response against NUCB2. Staining of stomach tissue microarray with anti-NUCB2 antibody revealed that it is expressed in the secretory granules of chief cells and in the cytoplasm of parietal cells in the functioning gastric glands which are lost in atrophic glands and tumour cells. Hence we propose that NUCB2 may be implicated in gastric secretion by establishing an agonist-releasable Ca2+ store in ER or Golgi apparatus, signalling via heterotrimeric Gα proteins and/or mediating the exocytosis of the secretory granules.

[Electron microscope study of haemolymph cells of Tettigonia cantans (Orthoptera, Tettigonidae)]. / Electronno-mikroskopicheskoe issledovanie kletok gemolimfy kuznechika pevchego Tettigonia cantans (Orthoptera, Tettigonidae).

The haemolymph of Tettigonia cantans (Fuess) has been studied at the preimago and imago stages with both electron and light microscopic methods. PAS-negative granules were detected in histochemical reactions. On electronograms, one type of haemocytes was referred to as granulocytes recognized in haemolymph. In the cytoplasm of granulocytes, two types of granules were found: those of mitochondrial origin, and those being derivatives of the Golgi apparatus. Secret discharge is realized by the merocrine way. Four stages of granulocyte development are distinguished: the formation of granules and organelle development; the formation and accumulation of granules, active secretion, and cell destruction.

[Electron microscope study of haemolymph cells of Decticus verrucivorus (Orthoptera, Tettigoniidae) in larva and imago stages]. / Elektonno-mikroskopicheskoe issledovanie kletok gemolimfy skakuna serogo Decticus verrucivorus (Orthoptera, Tettigoniidae) na stadiiakh lichinok i imago.

The haemolymph of larvae and imago stages of Decticus verrucirus was studied with electron and light microscope. PAS-positive and PAS-negative granules were detected in haemocytes. On the electronograms, granulocytes were recognized as the only type of haemocytes. In the cytoplasm of granulocytes, granules of two types were found: those of mitochondrial origin, and originating from the Golgi apparatus, respectively. The discharge of a secret is realized by the merocrine way. Four stages of granulocyte development have been distinguished: 1) granule formation and organelle development, 2) granule formation and accumulation, 3) active secretion, and 4) cell destruction.

[Electron microscope study of haemolymph cells of Metrioptera roeselii (Orthoptera, Tettigoniidae)]. / Elektronno-mikroskopicheskoe issledovanie kletok gemolimfy skachka zelenogo Metrioptera roeselii (Orthoptera, Tettigoniidae).

On the basis of patterns of haemocyte ultrastructure and functions at preimago and imago stages of Metrioptera roeselii, secretory cells of granulocyte type were recognized in the haemolymph. The development of granulocytes was traced starting from their formation up to cell death and destruction. The haemocytes develop as "dark" and "light" cells, differing in their functional activity, although their ultrastructure is similar. In the cytoplasm of granulocytes, granules of both mitochondrial and nuclear origin were detected. Simultaneously two processes occur in the cells--the accumulation and discharge of granules.