Genotoxic Effects of Cylindrospermopsin,Microcystin-LR and Their Binary Mixture in Human Hepatocellular Carcinoma (HepG2) Cell Line

Simultaneous occurrence of cylindrospermopsin (CYN) and microcystin-LR (MCLR) has been reported in the aquatic environment and thus human exposure to such mixtures is possible. As data on the combined effects of CYN/MCLR are scarce, we aimed to investigate the adverse effects related to genotoxic activities induced by CYN (0.125, 0.25 and 0.5 µg/mL) and MCLR (1 µg/mL) as single compounds and their combinations in HepG2 cells after 24 and 72 h exposure. CYN and CYN/MCLR induced DNA double-strand breaks after 72 h exposure, while cell cycle analysis revealed that CYN and CYN/MCLR arrested HepG2 cells in G0/G1 phase. Moreover, CYN and the combination with MCLR upregulated CYP1A1 and target genes involved in DNA-damage response (CDKN1A, GADD45A). Altogether, the results showed that after 72 h exposure genotoxic activity of CYN/MCLR mixture was comparable to the one of pure CYN. On the contrary, MCLR (1 µg/mL) had no effect on the viability of cells and had no influence on cell division. It did not induce DNA damage and did not deregulate studied genes after prolonged exposure. The outcomes of the study confirm the importance of investigating the combined effects of several toxins as the effects can differ from those induced by single compounds.     

Role of molecular mimicry and polyclonal cell activation in the induction of pathogenic β2-glycoprotein I–directed immune response in Balb/c mice upon hyperimmunization with tetanus toxoid

It is known that tetanus toxoid (TTd)-hyperimmunization induces increased titer of sera β2-glycoprotein I (β2GPI)-specific antibodies (Abs) in Balb/c mice. The concentrations of such induced anti-β2GPI Abs as well as their pathogenic potential are strongly influenced by the context of TTd application. β2GPI-specific immune response is established as a part of TTd-specific immune response by molecular mimicry mechanism due to structural homology between TTd and β2GPI. This finding is supported by the following facts: (1) cross-reactive Abs that recognize both TTd and β2GPI epitopes are present in Balb/c mice sera; (2) anti-TTd Abs secretion in splenic cultures is induced after β2GPI stimulation and vice versa. However, analyses of (1) IL-10 production following in vitro stimulation of immunized Balb/c mice splenocytes by TTd, β2GPI or glutaraldehyde-treated β2GPI and (2) specific impact of ConA and agonists of TLR2, TLR4, and TLR9 on anti-TTd and autoreactive Abs secretion strongly imply that these two branches of the TTd-induced immune response do not use identical cell populations and are regulated in a different way. Results presented in this paper describe that structural homology between foreign and self-antigens could focus mounted autoreactive immune response toward specific self-structure, but the context of antigen application, including a history of previous immune stimulations and adjuvants applied together with the antigen, are the main factors which determine the outcome of the induced immune response.     

The site of allergen expression in hematopoietic cells determines the degree and quality of tolerance induced through molecular chimerism

The transplantation of allergens (e.g. Phl p 5 or Bet v 1) expressed on BM cells as membrane‐anchored full‐length proteins leads to permanent tolerance at the T‐cell, B‐cell, and effector‐cell levels. Since the exposure of complete allergens bears the risk of inducing anaphylaxis, we investigated here whether expression of Phl p 5 in the cytoplasm (rather than on the cell surface) is sufficient for tolerance induction. Transplantation of BALB/c BM retrovirally transduced to express Phl p 5 in the cytoplasm led to stable and durable molecular chimerism in syngeneic recipients (～20% chimerism at 6 months). Chimeras showed allergen‐specific T‐cell hyporesponsiveness. Further, Phl p 5‐specific T_H1‐dependent humoral responses were tolerized in several chimeras. Surprisingly, Phl p 5‐specific IgE and IgG₁ levels were significantly reduced but still detectable in sera of chimeric mice, indicating incomplete B‐cell tolerance. No Phl p 5‐specific sIgM developed in cytoplasmic chimeras, which is in marked contrast to mice transplanted with BM expressing membrane‐anchored Phl p 5. Thus, the expression site of the allergen substantially influences the degree and quality of tolerance achieved with molecular chimerism in IgE‐mediated allergy.     

The effects of sex steroids on thyroid C cells and trabecular bone structure in the rat model of male osteoporosis

Androgen deficiency is one of the major factors leading to the development of osteoporosis in men. Since calcitonin (CT) is a potent antiresorptive agent, in the present study we investigated the effects of androgen deficiency and subsequent testosterone and estradiol treatment on CT-producing thyroid C cells, skeletal and hormonal changes in middle-aged orchidectomized (Orx) rats. Fifteen-month-old male Wistar rats were either Orx or sham-operated (SO). One group of Orx rats received 5 mg kg⁻¹ b.w. testosterone propionate (TP) subcutaneously, while another group was injected with 0.06 mg kg⁻¹ b.w. estradiol dipropionate (EDP) once a day for 3 weeks. A peroxidase–antiperoxidase method was applied for localization of CT in the C cells. The studies included ultrastructural microscopic observation of these cells. The metaphyseal region of the proximal tibia was measured histomorphometrically using an imagej public domain image processing program. TP or EDP treatment significantly increased C cell volume (Vc), volume densities (Vv) and serum CT concentration compared with the Orx animals. Administration of both TP and EDP significantly enhanced cancellous bone area (B.Ar), trabecular thickness (Tb.Th) and trabecular number (Tb.N) and reduced trabecular separation (Tb.Sp). Serum osteocalcin (OC) and urinary Ca concentrations were significantly lower after these treatments in comparison with Orx rats. These data suggest that testosterone and estradiol treatment in Orx middle-aged rats affect calcitonin-producing thyroid C cells, which may contribute to the bone protective effects of sex hormones in the rat model of male osteoporosis.     

Rapid and Reliable Identification of Staphylococcus aureus Capsular Serotypes by Means of Artificial Neural Network-Assisted Fourier Transform Infrared Spectroscopy

Staphylococcus aureus capsular polysaccharides (CP) are important virulence factors and represent putative targets for vaccine development. Therefore, the purpose of this study was to develop a high-throughput method to identify and discriminate the clinically important S. aureus capsular serotypes 5, 8, and NT (nontypeable). A comprehensive set of clinical isolates derived from different origins and control strains, representative for each serotype, were used to establish a CP typing system based on Fourier transform infrared (FTIR) spectroscopy and chemometric techniques. By combining FTIR spectroscopy with artificial neuronal network (ANN) analysis, a system was successfully established, allowing a rapid identification and discrimination of all three serotypes. The overall accuracy of the ANN-assisted FTIR spectroscopy CP typing system was 96.7% for the internal validation and 98.2% for the external validation. One isolate in the internal validation and one isolate in the external validation failed in the classification procedure, but none of the isolates was incorrectly classified. The present study demonstrates that ANN-assisted FTIR spectroscopy allows a rapid and reliable discrimination of S. aureus capsular serotypes. It is suitable for diagnostic as well as large-scale epidemiologic surveillance of S. aureus capsule expression and provides useful information with respect to chronicity of infection.     

Cortical thickness as predictor of response to exercise in people with Parkinson's disease

We previously showed that dual‐task cost (DTC) on gait speed in people with Parkinson''s disease (PD) improved after 6 weeks of the Agility Boot Camp with Cognitive Challenge (ABC‐C) exercise program. Since deficits in dual‐task gait speed are associated with freezing of gait and gray matter atrophy, here we performed preplanned secondary analyses to answer two questions: (a) Do people with PD who are freezers present similar improvements compared to nonfreezers in DTC on gait speed with ABC‐C? (b) Can cortical thickness at baseline predict responsiveness to the ABC‐C? The DTC from 39 freezers and 43 nonfreezers who completed 6 weeks of ABC‐C were analyzed. A subset of 51 participants (21 freezers and 30 nonfreezers) with high quality imaging data were used to characterize relationships between baseline cortical thickness and delta (Δ) DTC on gait speed following ABC‐C. Freezers showed larger ΔDTC on gait speed than nonfreezers with ABC‐C program (p < .05). Cortical thickness in visual and fronto‐parietal areas predicted ΔDTC on gait speed in freezers, whereas sensorimotor‐lateral thickness predicted ΔDTC on gait speed in nonfreezers (p < .05). When matched for motor severity, visual cortical thickness was a common predictor of response to exercise in all individuals, presenting the largest effect size. In conclusion, freezers improved gait automaticity even more than nonfreezers from cognitively challenging exercise. DTC on gait speed improvement was associated with larger baseline cortical thickness from different brain areas, depending on freezing status, but visual cortex thickness showed the most robust relationship with exercise‐induced improvements in DTC.