STUDY OF THE PROPERTIES OF THE MIXED WATER/SALT HOUSE DUST MITE ALLERGEN AIMING TO DESIGN ITS SUBLINGUAL FORM

Сurrent allergen-specific immunotherapy (AIT) is the most pathogenetically justified method of the IgE-dependent treatment of allergic diseases. Subcutaneous (SCIT) and sublingual (SLIT) AIT are currently used in wide clinical practice. Sublingual immunotherapy is characterized by the convenience of use, decreased incidence of systemic and local side effects, and proven clinical efficacy. However, sublingual form of home dust mite allergen for AIT is not yet produced in Russia. Previously, the technology for sublingual form of D. farinae (Der f) mites allergen was developed in our laboratory, and this extract was shown to be non-toxic and free of any significant side effects. We obtained a sublingual form of mixed D. pteronyssinus (Der р)/D. farina mites allergen for sublingually allergen-specific immunotherapy. We studied physico-chemical and immunobiological properties of the Der р and Der f mites phenol-free water-salt mixed extract (WWSME). WWSME was studied after sterilizing filtration. WWSME was analyzed by electrophoresis in polyacrylamide gel in the presence of dodecyl-sodium sulfate and reverse-phase high-performance liquid chromatography. ELISA was performed with polyclonal rabbit serum, obtained by sensitization of animals with recombinant Der f1 and Der f2 allergens. ELISA shows positive reaction of WWSME with IgG-antibodies of rabbits. We studied acute and chronic toxicity of received mixed allergen in laboratory animals. Pathomorphological studies have shown that WWSME has no detectable toxicity and does not cause local irritation in the animals. The WWSME product contains native proteins, retains immunogenic properties, is non-toxic and does not cause significant side effects. This extract can be used as the basis for designing a sublingual form of dust mite mixed allergen, which is especially promising for pediatric applications

Long-Term Effects of Very Low Dose Particle Radiation on Gene Expression in the Heart: Degenerative Disease Risks

Compared to low doses of gamma irradiation (γ-IR), high-charge-and-energy (HZE) particle IR may have different biological response thresholds in cardiac tissue at lower doses, and these effects may be IR type and dose dependent. Three- to four-month-old female CB6F1/Hsd mice were exposed once to one of four different doses of the following types of radiation: γ-IR 137Cs (40-160 cGy, 0.662 MeV), 14Si-IR (4-32 cGy, 260 MeV/n), or 22Ti-IR (3-26 cGy, 1 GeV/n). At 16 months post-exposure, animals were sacrificed and hearts were harvested and archived as part of the NASA Space Radiation Tissue Sharing Forum. These heart tissue samples were used in our study for RNA isolation and microarray hybridization. Functional annotation of twofold up/down differentially expressed genes (DEGs) and bioinformatics analyses revealed the following: (i) there were no clear lower IR thresholds for HZE- or γ-IR; (ii) there were 12 common DEGs across all 3 IR types; (iii) these 12 overlapping genes predicted various degrees of cardiovascular, pulmonary, and metabolic diseases, cancer, and aging; and (iv) these 12 genes revealed an exclusive non-linear DEG pattern in 14Si- and 22Ti-IR-exposed hearts, whereas two-thirds of γ-IR-exposed hearts revealed a linear pattern of DEGs. Thus, our study may provide experimental evidence of excess relative risk (ERR) quantification of low/very low doses of full-body space-type IR-associated degenerative disease development

Immune response of macrophages from young and aged mice to the oral pathogenic bacterium Porphyromonas gingivalis

Periodontal disease is a chronic inflammatory gum disease that in severe cases leads to tooth loss. Porphyromonas gingivalis (Pg) is a bacterium closely associated with generalized forms of periodontal disease. Clinical onset of generalized periodontal disease commonly presents in individuals over the age of 40. Little is known regarding the effect of aging on inflammation associated with periodontal disease. In the present study we examined the immune response of bone marrow derived macrophages (BMM) from young (2-months) and aged (1-year and 2-years) mice to Pg strain 381. Pg induced robust expression of cytokines; tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10, chemokines; neutrophil chemoattractant protein (KC), macrophage colony stimulating factor (MCP)-1, macrophage inflammatory protein (MIP)-1α and regulated upon activation normal T cell expressed and secreted (RANTES), as well as nitric oxide (NO, measured as nitrite), and prostaglandin E2 (PGE2) from BMM of young mice. BMM from the 2-year age group produced significantly less TNF-α, IL-6 and NO in response to Pg as compared with BMM from 2-months and 1-year of age. We did not observe any difference in the levels of IL-1β, IL-10 and PGE2 produced by BMM in response to Pg. BMM from 2-months and 1-year of age produced similar levels of all chemokines measured with the exception of MCP-1, which was reduced in BMM from 1-year of age. BMM from the 2-year group produced significantly less MCP-1 and MIP-1α compared with 2-months and 1-year age groups. No difference in RANTES production was observed between age groups. Employing a Pg attenuated mutant, deficient in major fimbriae (Pg DPG3), we observed reduced ability of the mutant to stimulate inflammatory mediator expression from BMMs as compared to Pg 381, irrespective of age. Taken together these results support senescence as an important facet of the reduced immunological response observed by BMM of aged host to the periodontal pathogen Pg

Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells

Porphyromonas gingivalis is the primary etiologic agent of periodontal disease that is associated with other human chronic inflammatory diseases, including atherosclerosis. The ability of P. gingivalis to invade and persist within human aortic endothelial cells (HAEC) has been postulated to contribute to a low to moderate chronic state of inflammation, although how this is specifically achieved has not been well defined. In this study, we demonstrate that P. gingivalis infection of HAEC resulted in the rapid cleavage of receptor interacting protein 1 (RIPK1), a mediator of tumor necrosis factor (TNF) receptor-1 (TNF-R1)-induced cell activation or death, and RIPK2, a key mediator of both innate immune signaling and adaptive immunity. The cleavage of RIPK1 or RIPK2 was not observed in cells treated with apoptotic stimuli, or cells stimulated with agonists to TNF-R1, nucleotide oligomerization domain receptor 1(NOD1), NOD2, Toll-like receptor 2 (TLR2) or TLR4. P. gingivalis-induced cleavage of RIPK1 and RIPK2 was inhibited in the presence of a lysine-specific gingipain (Kgp) inhibitor. RIPK1 and RIPK2 cleavage was not observed in HAEC treated with an isogenic mutant deficient in the lysine-specific gingipain, confirming a role for Kgp in the cleavage of RIPK1 and RIPK2. Similar proteolysis of poly (ADP-ribose) polymerase (PARP) was observed. We also demonstrated direct proteolysis of RIPK2 by P. gingivalis in a cell-free system which was abrogated in the presence of a Kgp-specific protease inhibitor. Our studies thus reveal an important role for pathogen-mediated modification of cellular kinases as a potential strategy for bacterial persistence within target host cells, which is associated with low-grade chronic inflammation, a hallmark of pathogen-mediated chronic inflammatory disorders

Lifetime Evaluation of Left Ventricular Structure and Function in Male C57BL/6J Mice After Gamma and Space-Type Radiation Exposure

The lifetime effects of space irradiation (IR) on left ventricular (LV) function are unknown. The cardiac effects induced by space-type IR, specifically 5-ion simplified galactic cosmic ray simulation (simGCRsim), are yet to be discovered. Three-month-old, age-matched, male C57BL/6J mice were irradiated with Cs gamma (γ; 100, 200 cGy) and simGCRsim (50 and 100 cGy). LV function was assessed via transthoracic echocardiography at 14 and 28 days (early), and at 365, 440, and 660 (late) days post IR. We measured the endothelial function marker brain natriuretic peptide in plasma at three late timepoints. We assessed the mRNA expression of the genes involved in cardiac remodeling, fibrosis, inflammation, and calcium handling in LVs harvested at 660 days post IR. All IR groups had impaired global LV systolic function at 14, 28, and 365 days. At 660 days, 50 cGy simGCRsim-IR mice exhibited preserved LV systolic function with altered LV size and mass. At this timepoint, the simGCRsim-IR mice had elevated levels of cardiac fibrosis, inflammation, and hypertrophy markers , , , and , suggesting that space-type IR may induce the cardiac remodeling processes that are commonly associated with diastolic dysfunction. IR groups showing statistical significance were modeled to calculate the Relative Biological Effectiveness (RBE) and Radiation Effects Ratio (RER). The observed dose-response shape did not indicate a lower threshold at these IR doses. A single full-body IR at doses of 100-200 cGy for γ-IR, and 50-100 cGy for simGCRsim-IR decreases the global LV systolic function in WT mice as early as 14 and 28 days after exposure, and at 660 days post IR. Interestingly, there is an intermediate time point (365 days) where the impairment in LV function is observed. These findings do not exclude the possibility of increased acute or degenerative cardiovascular disease risks at lower doses of space-type IR, and/or when combined with other space travel-associated stressors such as microgravity

Human tankyrases are aberrantly expressed in colon tumors and contain multiple epitopes that induce humoral and cellular immune responses in cancer patients

PURPOSE: Tankyrases 1 and 2 are telomere-associated poly(ADP-ribose) polymerases (PARP) that can positively regulate telomere elongation and interact with multiple cellular proteins. Recent reports implicated tankyrases as tumor antigens and potential targets of anticancer treatment. We examined expression of tankyrases in colon tumors and immune response to these enzymes in patients with different types of cancer. METHODS: mRNA and protein expression was evaluated by quantitative real-time RT-PCR and Western blotting, respectively. Humoral immune response to recombinant tankyrases was investigated by modified enzyme-linked immunoassays. Cellular immune response was analysed by ELISPOT and (51)Cr release assays. RESULTS: We found that both mRNA and protein levels of tankyrase 2 (TNKL) are upregulated in colon tumors. In contrast, protein level of tankyrase 1 (TNKS) is downregulated, while mRNA level shows variable changes. More than a quarter of colon cancer patients develop humoral immune response to at least one of the two tankyrases. In this study we mapped common and unique B-cell epitopes located in different domains of the two proteins. Additionally, we present evidence for T-cell responses both to epitopes that are unique for TNKL and to those shared between TNKL and TNKS. CONCLUSION: Our study favors a biomarker usage of antibody response to tankyrases. Spontaneous CD8(+) T-cell responses to these enzymes are rare and further investigation is needed to evaluate tankyrases as potential targets for cancer immunotherapy