Comparison of ELISA and capillary electrophoresis with laser-induced fluorescence detection in the analysis of Ochratoxin A in low volumes of human blood serum

In this paper the determination of Ochratoxin A (OTA) in low volumes of human blood serum by enzyme-linked immunosorbent assay (ELISA) is compared with an appropriate capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) method. In order to use ELISA for high-throughput analysis in epidemiological studies no sample cleanup was performed. Both methods showed a limit of detection (LOD) of 0.5 ng/mL. Comparing the precisions of both methods, the data show that the quantified concentrations in ELISA are higher than the corresponding concentrations in the CE-LIF method. Using a matrix calibration curve instead of a standard calibration curve the reproducibilities of both methods are comparable. No additional matrix effect could be observed by adding phenylalanine as probable matrix compound to the serum.     

Effect of early thermal experience on pituitary-gonadal axis in female rats

The reproductive system fully develops during postnatal stages of life, and as such it may be susceptible to environmental cues such as high temperature. Thus, the purpose of the study was to compare how exposure to 34 degrees Celsius ambient temperature affects pituitary-gonadal axis of immature and adult female rats. Three groups of females at proestrous or metaestrous (n=38) were used in the study. The females were housed in ambient temperature of 34 degrees Celsius (WR group, n=10) or room temperature (CR group, n=16) from birth to adulthood. The females from the third group were acclimated to 34 degrees Celsius as adults (WA group, n=12). In the WR group the onset of puberty was delayed in comparison to the CR group. The plasma PRL level was lower during proestrous and higher during metaestrous in the WR group compared to the CR group. During metaestrous, lower FSH and higher progesterone (P(4)) plasma concentrations were found in the WR females. No changes in LH and oestradiol (E(2)) plasma concentrations were demonstrated. Higher in vitro E(2) and lower P(4) secretions under FSH stimulation were observed in the WR follicles compared to those of the CR group. The WR group also demonstrated higher basal and LH-stimulated luteal in vitro secretion of P(4) than controls. Plasma LH and FSH concentrations during metaestrous were higher in WA females than in the WR group, but PRL level was lower. Follicles of the WA group were unresponsive to FSH with respect to steroid secretion. In addition, LH stimulated luteal E(2) secretion in this group. P(4) release by luteal cells was lower in the WA than in the WR group. We concluded that WR females differ from WA in reproductive system adjustments to rearing temperature and that early thermal experience is more effective in antagonizing the effect of high temperature than acclimation of adult females.     

Early Innate Recognition of Herpes Simplex Virus in Human Primary Macrophages Is Mediated via the MDA5/MAVS-Dependent and MDA5/MAVS/RNA Polymerase III-Independent Pathways

Innate recognition of viruses is mediated by pattern recognition receptors (PRRs) triggering expression of antiviral interferons (IFNs) and proinflammatory cytokines. In mice, Toll-like receptor 2 (TLR2) and TLR9 as well as intracellular nucleotide-sensing pathways have been shown to recognize herpes simplex virus (HSV). Here, we describe how human primary macrophages recognize early HSV infection via intracellular pathways. A number of inflammatory cytokines, IFNs, and IFN-stimulated genes were upregulated after HSV infection. We show that early recognition of HSV and induction of IFNs and inflammatory cytokines are independent of TLR2 and TLR9, since inhibition of TLR2 using TLR2 neutralizing antibodies did not affect virus-induced responses and the macrophages were unresponsive to TLR9 stimulation. Instead, HSV recognition involves intracellular recognition systems, since induction of tumor necrosis factor alpha (TNF-α) and IFNs was dependent on virus entry and replication. Importantly, expression of IFNs was strongly inhibited by small interfering RNA (siRNA) knockdown of MAVS, but this MAVS-dependent IFN induction occurred independently of the recently discovered polymerase III (Pol III)/RIG-I DNA sensing system. In contrast, induction of TNF-α was largely independent of MAVS, suggesting that induction of inflammatory cytokines during HSV infection proceeds via a novel pathway. Transfection with ODN2006, a broad inhibitor of intracellular nucleotide recognition, revealed that nucleotide-sensing systems are employed to induce both IFNs and TNF-α. Finally, using siRNA knockdown, we found that MDA5, but not RIG-I, was the primary mediator of HSV recognition. Thus, innate recognition of HSV by human primary macrophages occurs via two distinct intracellular nucleotide-sensing pathways responsible for induction of IFNs and inflammatory cytokine expression, respectively.Virus recognition is essential for activation of innate antiviral immune defense and the subsequent induction of acquired immunity. Conserved pathogen motifs, termed pathogen-associated molecular patterns (PAMPs), are recognized by pattern recognition receptors (PRRs). Virus-recognizing PRRs include Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and a number of intracellular DNA receptors. Several TLRs have been attributed roles in the recognition of virus. TLR2 and TLR4 recognize viral surface structures (3, 6, 18, 31), TLR3 recognizes double-stranded RNA (dsRNA) (2), and TLR7/8 and TLR9 function as signaling receptors for viral single-stranded RNA (ssRNA) (8, 11, 21) and CpG DNA (12, 20), respectively.Within the cell, cytoplasmic RLRs RIG-I and MDA5 both recognize accumulation of virus-derived dsRNA; in addition, RIG-I recognizes 5′-triphosphated RNA (14, 27, 39, 40). In addition to the RLRs, a number of receptors recognize foreign DNA. Presently, three DNA receptors have been identified: Z-DNA binding protein 1 (ZBP-1, or DAI) (36) and RNA polymerase III (Pol III) (1, 4) both mediate interferon (IFN) and cytokine production, whereas the AIM2 inflammasome is involved in caspase 1 activation in response to cytoplasmic dsDNA (13).Herpes simplex virus type 1 (HSV-1) and HSV-2 are two closely related human DNA viruses associated with a number of serious diseases, including orofacial infections, encephalitis, and genital infections (34). Macrophages play an important role in the first line of defense against viral infection via production of IFNs, cytokines, and chemokines that regulate the progress of the virus infection and activate and support appropriate defense mechanisms (9, 10, 24).TLR2, TLR3, and TLR9 have been identified as mediators of proinflammatory cytokine production during HSV infections. TLR2 mediates an overzealous inflammatory cytokine response following HSV-1 infection in mice, promoting mononuclear cell infiltration of the brain and development of encephalitis (18). TLR3 mediates type I and III IFN production in human fibroblasts (41). TLR9 recognizes genomic DNA from HSV-1 and HSV-2 in murine plasmacytoid dendritic cells (DCs) (17, 20) and mediates tumor necrosis factor alpha (TNF-α) and CCL5 production in murine macrophages (22). Both TLR2 and TLR9 mediate recognition of HSV and cytokine production in murine conventional DCs (35). HSV is recognized by an RLR/MAVS-dependent mechanism in murine macrophages and mouse embryonic fibroblasts (MEFs) (5, 29, 30). Recent data suggest that RNA Pol III mediates IFN production following HSV-1 infection and transfection with HSV-1 DNA in macrophage-like RAW 264.7 cells (4). Finally, murine L929 fibroblast-like cells are moderately inhibited in their ability to produce IFN after HSV-1 infection when ZBP-1 is knocked down (19, 36). Thus, several PRRs have been reported to recognize HSV-1 in murine cells and different cell lines, but the pathways responsible for sensing this virus in human primary macrophages and their impact on cytokine expression have not previously been described.In this work, we investigate the recognition pathways underlying HSV-induced cytokine and chemokine expression in human primary macrophages. We demonstrate that HSV-1-induced IFN and cytokine expression is independent of TLR2 and TLR9 but highly dependent on virus replication and intracellular nucleotide recognition systems. Specifically, induction of IFNs is dependent on MAVS and MDA5, whereas TNF-α is induced by a novel intracellular nucleotide-sensing system.     

Acute Oxidative Stress Can Reverse Insulin Resistance by Inactivation of Cytoplasmic JNK

Chronic oxidative stress results in decreased responsiveness to insulin, eventually leading to diabetes and cardiovascular disease. Activation of the JNK signaling pathway can mediate many of the effects of stress on insulin resistance through inhibitory phosphorylation of insulin receptor substrate 1. By contrast, exercise, which acutely increases oxidative stress in the muscle, improves insulin sensitivity and glucose tolerance in patients with Type 2 diabetes. To elucidate the mechanism underlying the contrasting effects of acute versus chronic oxidative stress on insulin sensitivity, we used a cellular model of insulin-resistant muscle to induce either chronic or acute oxidative stress and investigate their effects on insulin and JNK signaling. Chronic oxidative stress resulted in increased levels of phosphorylated (activated) JNK in the cytoplasm, whereas acute oxidative stress led to redistribution of JNK-specific phosphatase MKP7 from the nucleus into the cytoplasm, reduction in cytoplasmic phospho-JNK, and a concurrent accumulation of phospho-JNK in the nucleus. Acute oxidative stress restored normal insulin sensitivity and glucose uptake in insulin-resistant muscle cells, and this effect was dependent on MKP7. We propose that the contrasting effects of acute and chronic stress on insulin sensitivity are driven by changes in subcellular distribution of MKP7 and activated JNK.     

Protein expression pattern in experimental pneumococcal meningitis

In this study, we investigated cytokine expression during experimental pneumococcal meningitis. Mice were intracisternally infected with Streptococcus pneumoniae and treated with ceftriaxone starting at 24 h after infection. At different time points before and after antibiotic therapy, the cytokine expression pattern was determined in mouse brains using protein arrays. Underlining the power of this method, the meningitis-relevant cytokines interleukin-1beta (IL-1beta), IL-6, KC, macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractant protein-1 (MCP-1/CCL2) were markedly elevated in infected animals. Newly identified proteins during the acute stage of the disease (until 30 h after infection) included lymphotactin (XCL-1), MIP-1gamma (CCL9) and MCP-5 (CCL12), cytokine responsive gene- 2 (CRG-2/CXCL10) and CXCL16, and insulin-like growth factor binding protein 3 (IGFBP3). During later stages, an induction of T-cell activation-3 (TCA-3/CCL1), platelet factor-4 (PF-4/CXCL4) and stromal derived factor-1alpha (SDF-1alpha/CXCL13), and IL-4 was observed. The validity of this method was supported by an additional ELISA analysis of the expression profile of CXCL16 and IGFBP3, which was identical to that observed by protein array. In conclusion, the use of protein array technology led to an extension of the current picture of protein expression in pneumococcal meningitis. Most important, new factors that might play a role in pneumococcal meningitis were identified.     

Redox-regulated ion channel activity of a cysteine-containing gramicidin A analogue

According to recent data, gramicidin A analogues having positively charged amino acid sequences at the C-termini exhibit two types of channel activity in lipid membranes: classical cation-selective channels and large unselective pores. The induction of unselective pores was shown here to strongly depend on the redox state of the membrane-bathing solution, if the gramicidin analogue contained a cysteine residue in the sequence GSGPKKKRKVC attached to the C-terminus. In particular, the addition of H2O2 led to an increase in the transmembrane current and the loss of cationic selectivity on planar bilayer lipid membranes and an increase in the carboxyfluorescein leakage of liposomes. The effect was observed at high concentration of the peptide while was absent at the single-channel level. It was concluded that oxidation led to possible formation of dimers of the peptide, which promoted the formation of large unselective pores.     

An assessment of potential exposure to bioaerosols among swine farm workers with particular reference to airborne microorganisms in the respirable fraction under various breeding conditions

The project was aimed at evaluating the potential occupational exposure of swine farm workers to dust and microorganisms present in piggery bioaerosols (especially in its respirable fraction) under various breeding conditions. Sampling was carried out in 14 buildings located at 13 pig breeding and production farms in Poland. Concentrations of inhalable and respirable dusts in the air of the piggeries were low (means, respectively, 1.76 and 0.23 mg/m³). The concentration of microorganisms was generally high (mean = 3.53 × 10⁵ cfu/m³). More than 96% of determined microorganisms were bacteria (mean = 3.42 × 10⁵ cfu/m³). The fungal concentration was distinctly lower (mean = 2.71 × 10³ cfu/m³). The concentration of bacteria in the respirable fraction of bioaerosol (mean = 1.51 × 10⁵ cfu/m³) made up for 48.2% of their total concentration, while the level of fungi in that fraction (mean = 1.50 × 10³ cfu/m³) formed 68.8% of the total fungal concentration. The concentration of inhalable dust was significantly modified by the type of breeding system. The factors that significantly affected the total concentrations of microbes and bacteria, as well as their levels in the bioaerosols’ respirable fraction were as follows: herd size, breeding system, feeding method and the type of ventilation system. In the case of fungi, these were the livestock breeding system and the feeding method. Moreover, there was a high positive correlation of inhalable dust concentrations with the fungal concentration, CO₂ and relative humidity. A negative correlation was found between concentrations of each microbe group and the airflow velocity. Swine farm workers are exposed to relatively low dust concentrations and high concentrations of microorganisms, bacteria in particular. Fungi, to a much larger extent than bacteria, are correlated with the respirable particles of a piggery bioaerosol, which may harm the respiratory system of exposed workers.     

Histone H3 methylation patterns in Brassica nigra, Brassica juncea, and Brassica carinata species

Core histones are subjected to various post-translational modifications, and one of them, most intensively studied in plants, is the methylation of histone H3. In the majority of analyzed plant species, dimethylation of H3 at lysine 9 (H3K9me2) is detected in heterochromatin domains, whereas methylation of H3 at lysine 4 (H3K4me2) is detected in euchromatin domains. The distribution of H3K9me2 in the interphase nucleus seems to be correlated with genome size, chromatin organization, but also with tissue specificity. In this paper, we present the analysis of the pattern and level of histone H3 methylation for two allotetraploid and one diploid Brassica species. We have found that the pattern of H3K9me2 in interphase nuclei from root meristematic tissue is comparable within the analyzed species and includes both heterochromatin and euchromatin, but the level of modification differs not only among species but even among nuclei in the same phase of the cell cycle within one species. Moreover, the differences in the level of H3K9me2 are not directly coupled with DNA content in the nuclei and are probably tissue specific.