Copper Oxide Nanoparticles Stimulate the Immune Response and Decrease Antioxidant Defense in Mice After Six-Week Inhalation.

Copper oxide nanoparticles (CuO NPs) are increasingly used in various industry sectors. Moreover, medical application of CuO NPs as antimicrobials also contributes to human exposure. Their toxicity, including toxicity to the immune system and blood, raises concerns, while information on their immunotoxicity is still very limited. The aim of our work was to evaluate the effects of CuO NPs (number concentration 1.40×106 particles/cm3, geometric mean diameter 20.4 nm) on immune/inflammatory response and antioxidant defense in mice exposed to 32.5 µg CuO/m3 continuously for 6 weeks. After six weeks of CuO NP inhalation, the content of copper in lungs and liver was significantly increased, while in kidneys, spleen, brain, and blood it was similar in exposed and control mice. Inhalation of CuO NPs caused a significant increase in proliferative response of T-lymphocytes after mitogenic stimulation and basal proliferative activity of splenocytes. CuO NPs significantly induced the production of IL-12p70, Th1-cytokine IFN-γ and Th2-cytokines IL-4, IL-5. Levels of TNF-α and IL-6 remained unchanged. Immune assays showed significantly suppressed phagocytic activity of granulocytes and slightly decreased respiratory burst. No significant differences in phagocytosis of monocytes were recorded. The percentage of CD3+, CD3+CD4+, CD3+CD8+, and CD3-CD19+ cell subsets in spleen, thymus, and lymph nodes did not differ between exposed and control animals. No changes in hematological parameters were found between the CuO NP exposed and control groups. The overall antioxidant protection status of the organism was expressed by evaluation of GSH and GSSG concentrations in blood samples. The experimental group exposed to CuO NPs showed a significant decrease in GSH concentration in comparison to the control group. In summary, our results indicate that sub-chronic inhalation of CuO NPs can cause undesired modulation of the immune response. Stimulation of adaptive immunity was indicated by activation of proliferation and secretion functions of lymphocytes. CuO NPs elicited pro-activation state of Th1 and Th2 lymphocytes in exposed mice. Innate immunity was affected by impaired phagocytic activity of granulocytes. Reduced glutathione was significantly decreased in mice exposed to CuO NPs.

Seasonal changes of circulating 25-hydroxyvitamin D correlate with the lower gut microbiome composition in inflammatory bowel disease patients.

Higher probability of the development of Crohn's disease (CD) and ulcerative colitis (UC) as a possible consequence of the north-south gradient has been recently suggested. Living far north or south of the equator is manifested in fluctuation of vitamin D (vitD) levels depending on the season in both healthy and affected individuals. In the present study we investigate the possible link between the seasonal serum vitD level to the microbial composition of the lower gut of Inflammatory Bowel disease (IBD) patients using 16S rRNA sequencing. Decrease of serum vitD level in winter/spring season in a cohort of 35 UC patients and 39 CD patients was confirmed. Low gut microbiota composition of patients with IBD correlated with the serum level of 25(OH)D that directly coupled to seasonal variability of the sunshine in the central European countries. It is supposed to be related to increased abundance of Actinobacteria and Proteobacteria in UC and Actinobacteria, Fusobacteria, Firmicutes and Bacteroidetes in CD. In summer/autumn period, we observed a reduction in abundance of bacterial genera typical for inflammation like Eggerthella lenta, Fusobacterium spp., Bacteroides spp., Collinsella aerofaciens, Helicobacter spp., Rhodococcus spp., Faecalibacterium prausnitzii; and increased abundance of Pediococcus spp. and Clostridium spp. and of Escherichia/Shigella spp.

Polymorphisms in the genes encoding TGF-beta1, TNF-alpha, and IL-6 show association with type 1 diabetes mellitus in the Slovak population.

Numerous cytokines have been shown to participate in the pathogenesis of type 1 diabetes (T1D). As gene polymorphisms can influence cytokine production or function, they may potentially contribute to genetic predisposition to the disease. The aim of this study was therefore to investigate the role of 22 single nucleotide polymorphisms (SNPs) in 13 cytokine and cytokine receptor genes in genetic susceptibility to T1D. Polymerase chain reaction with sequence-specific primers was used to genotype cytokine SNPs and HLA-DRB1 alleles in 151 diabetics and 140 healthy individuals of Slovak origin. Univariate analysis showed that transforming growth factor (TGF)-beta1 codon 10 TT homozygotes were significantly more susceptible to developing T1D than C allele carriers (P (c) = 0.0066, OR = 2.46). Furthermore, tumor necrosis factor (TNF)-alpha -308 A allele carriers were also significantly overrepresented among the diabetics (P (c) = 0.0031, OR = 2.62); however, the association of the -308 A allele with T1D might be due to its strong linkage disequilibrium with the susceptibility allele HLA-DRB1*0301. An association was also found with interleukin (IL)-6 -174 G/C and nt565 G/A SNPs; however, its significance was lost when statistical correction was applied. These data suggest that the TGF-beta1 codon 10 SNP is among numerous genetic variations with small individual effects on T1D development. Moreover, a possible role of TNF-alpha and IL-6 SNPs cannot be ruled out, although their association with T1D was due to strong LD with the HLA class II susceptibility allele or did not withstand statistical correction, respectively.

The use of transformed Escherichia coli for experimental angiogenesis induced by regulated in situ production of vascular endothelial growth factor--an alternative gene therapy.

BACKGROUND: Defects in angiogenesis (blood vessel formation) are responsible for two most important causes of death in developed countries (ischemic heart disease and cancer). Vascular endothelial growth factor (VEGF) plays a pivotal role in physiological and pathological regulation of angiogenesis. In the last years several studies have indicated the possibilities of VEGF in the therapy of ischemic heart disease. However, especially VEGF gene therapy (naked DNA, plasmids and adenovirus mediated) is associated with adverse side effects regarding the expression regulation. AIM: To prepare bacterial strains producing VEGF using plasmids containing the VEGF cDNA for the use in experimental angiogenesis. METHODS AND RESULTS: Escherichia coli strain BL21(DE3) was transformed with Bluescript vector containing the inserts with cDNA sequences coding VEGF-A isoforms (VEGF121, VEGF164, VEGF189). Selection of recombinants was achieved by cultivating E. coli cells on ampicillin-added medium. The expression of target genes in the T7 expression system was induced by isopropyl-beta-D-thiogalactoside (IPTG). Polyacrylamide gel electrophoresis of the cell lysates showed the presence of polypeptides of molecular weight corresponding with known values of VEGF isoforms. Blood vessel formation induced by bacterial VEGF production was proved in vivo in mice seven days after intraperitoneal injection of transformed bacteria by light microscopy. CONCLUSION AND HYPOTHESIS: In summary, E. coli strain expressing VEGF was prepared and its biological effect confirmed. Bacteria, which produce angiogenic factors, provide a new modality for experimental angiogenesis and may be also suitable for clinical use. The in situ production of therapeutic proteins using optimalized prokaryotic expression systems can represent a useful tool for treatment based on molecular biomedicine. The main advantage of the described approach lies in the enhanced regulation control--bacterial expression can be regulated positively (induction by exogenous low molecular weight agents) and negatively (application of antibiotics). The hypothesis of alternative gene therapy should be proved in further studies.