Epigenetic regulation of somatic angiotensin-converting enzyme by DNA methylation and histone acetylation

Somatic angiotensin-converting enzyme (sACE) is crucial in cardiovascular homeostasis and displays a tissue-specific profile. Epigenetic patterns modulate genes expression and their alterations were implied in pathologies including hypertension. However, the influence of DNA methylation and chromatin condensation state on the expression of sACE is unknown. We examined whether such epigenetic mechanisms could participate in the control of sACE expression in vitro and in vivo. We identified two CpG islands in the human ace-1 gene 3 kb proximal promoter region. Their methylation abolished the luciferase activity of ace-1 promoter/reporter constructs transfected into human liver (HepG2), colon (HT29), microvascular endothelial (HMEC-1) and lung (SUT) cell lines (p < 0.001). Bisulphite sequencing revealed a cell-type specific basal methylation pattern of the ace-1 gene -1,466/+25 region. As assessed by RT-qPCR, inhibition of DNA methylation by 5-aza-2'-deoxycytidine and/or of histone deacetylation by trichostatin A highly stimulated sACE mRNA expression cell-type specifically (p < 0.001 vs. vehicle treated cells). In the rat, in vivo 5-aza-cytidine injections demethylated the ace-1 promoter and increased sACE mRNA expression in the lungs and liver (p = 0.05), but not in the kidney. In conclusion, the expression level of somatic ACE is modulated by CpG-methylation and histone deacetylases inhibition. The basal methylation pattern of the promoter of the ace-1 gene is cell-type specific and correlates to sACE transcription. DNMT inhibition is associated with altered methylation of the ace-1 promoter and a cell-type and tissue-specific increase of sACE mRNA levels. This study indicates a strong influence of epigenetic mechanisms on sACE expression.

Monomeric and dimeric IgG fractions show differential reactivity against pathogen-derived antigens

Polyvalent Ig preparations, derived from the pooled plasma of thousands of healthy donors, contain a complex mix of both 'acquired' and natural antibodies directed against pathogens as well as foreign and self/auto antigens (Ag). Depending on their formulation, donor pool size, etc., liquid Ig preparations contain monomeric and dimeric IgG. The dimeric IgG fraction is thought to represent mainly idiotype-antiidiotype Ab pairs. Treatment of all IgG fractions at pH 4 effectively monomerizes the IgG dimers resulting in separated idiotype-antiidiotype Ab pairs and thus in a comparable F(ab')(2) binding site availability of the different IgG fractions. Previously, we identified an increased anti-self-reactivity within the monomerized dimer fraction. This study addressed if, among the different IgG fractions, an analogous preferential reactivity was evident in the response against different pathogen-derived protein and carbohydrate antigens. Therefore, we assessed the activity of total unseparated IgG, the monomeric and dimeric IgG fractions against antigenic structures of bacterial and viral antigens/virulence factors. All fractions showed similar reactivity to protein antigens except for exotoxin A of Pseudomonas aeruginosa, where the dimeric fraction, especially when monomerized, showed a marked increase in reactivity. This suggests that the production of antiidiotypic IgG antibodies contributes to controlling the immune response to certain categories of pathogens. In contrast, the monomeric IgG fractions showed increased reactivity towards pathogen-associated polysaccharides, classically regarded as T-independent antigens. Taken together, the differential reactivity of the IgG fractions seems to indicate a preferential segregation of antibody reactivities according to the nature of the antigen.

In vitro induction of lymph node cell proliferation by mouse bone marrow dendritic cells following stimulation with different Echinococcus multilocularis antigens

The immune response of mice experimentally infected with Echinococcus multilocularis metacestodes becomes impaired so as to allow parasite survival and proliferation. Our study tackled the question on how different classes of E. multilocularis antigens (crude vesicular fluid (VF); purified proteinic rec-14-3-3; purified carbohydrate Em2(G11)) are involved in the maturation process of bone-marrow-derived dendritic cells (BMDCs) and subsequent exposure to lymph node (LN) cells. In our experiments, we used BMDCs cultivated from either naïve (control) or alveolar echinococcosis (AE)-infected C57BL/6 mice. We then tested surface markers (CD80, CD86, MHC class II) and cytokine expression levels (interleukin (IL)-10, IL-12p40 and tumour necrosis factor (TNF)-α) of non-stimulated BMDCs versus BMDCs stimulated with different Em-antigens or lipopolysaccharide (LPS). While LPS and rec-14-3-3-antigen were able to induce CD80, CD86 and (to a lower extent) MHC class II surface expression, Em2(G11) and, strikingly, also VF-antigen failed to do so. Similarly, LPS and rec-14-3-3 yielded elevated IL-12, TNF-α and IL-10 expression levels, while Em2(G11) and VF-antigen didn't. When naïve BMDCs were loaded with VF-antigen, they induced a strong non-specific proliferation of uncommitted LN cells. For both, BMDCs or LN cells, isolated from AE-infected mice, proliferation was abrogated. The most striking difference, revealed by comparing naïve with AE-BMDCs, was the complete inability of LPS-stimulated AE-BMDCs to activate lymphocytes from any LN cell group. Overall, the presenting activity of BMDCs from AE-infected mice seemed to trigger unresponsiveness in T cells, especially in the case of VF-antigen stimulation, thus contributing to the suppression of clonal expansion during the chronic phase of AE infection.

Comparative expression profiling of E. coli and S. aureus inoculated primary mammary gland cells sampled from cows with different genetic predispositions for somatic cell score

BACKGROUND: During the past ten years many quantitative trait loci (QTL) affecting mastitis incidence and mastitis related traits like somatic cell score (SCS) were identified in cattle. However, little is known about the molecular architecture of QTL affecting mastitis susceptibility and the underlying physiological mechanisms and genes causing mastitis susceptibility. Here, a genome-wide expression analysis was conducted to analyze molecular mechanisms of mastitis susceptibility that are affected by a specific QTL for SCS on Bos taurus autosome 18 (BTA18). Thereby, some first insights were sought into the genetically determined mechanisms of mammary gland epithelial cells influencing the course of infection. METHODS: Primary bovine mammary gland epithelial cells (pbMEC) were sampled from the udder parenchyma of cows selected for high and low mastitis susceptibility by applying a marker-assisted selection strategy considering QTL and molecular marker information of a confirmed QTL for SCS in the telomeric region of BTA18. The cells were cultured and subsequently inoculated with heat-inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus, respectively. After 1, 6 and 24 h, the cells were harvested and analyzed using the microarray expression chip technology to identify differences in mRNA expression profiles attributed to genetic predisposition, inoculation and cell culture. RESULTS: Comparative analysis of co-expression profiles clearly showed a faster and stronger response after pathogen challenge in pbMEC from less susceptible animals that inherited the favorable QTL allele 'Q' than in pbMEC from more susceptible animals that inherited the unfavorable QTL allele 'q'. Furthermore, the results highlighted RELB as a functional and positional candidate gene and related non-canonical Nf-kappaB signaling as a functional mechanism affected by the QTL. However, in both groups, inoculation resulted in up-regulation of genes associated with the Ingenuity pathways 'dendritic cell maturation' and 'acute phase response signaling', whereas cell culture affected biological processes involved in 'cellular development'. CONCLUSIONS: The results indicate that the complex expression profiling of pathogen challenged pbMEC sampled from cows inheriting alternative QTL alleles is suitable to study genetically determined molecular mechanisms of mastitis susceptibility in mammary epithelial cells in vitro and to highlight the most likely functional pathways and candidate genes underlying the QTL effect.

Intraperitoneal and intra-nasal vaccination of mice with three distinct recombinant Neospora caninum antigens results in differential effects with regard to protection against experimental challenge with Neospora caninum tachyzoites

Recombinant NcPDI(recNcPDI), NcROP2(recNcROP2), and NcMAG1(recNcMAG1) were expressed in Escherichia coli and purified, and evaluated as potential vaccine candidates by employing the C57Bl/6 mouse cerebral infection model. Intraperitoneal application of these proteins suspended in saponin adjuvants lead to protection against disease in 50% and 70% of mice vaccinated with recNcMAG1 and recNcROP2, respectively, while only 20% of mice vaccinated with recNcPDI remained without clinical signs. In contrast, a 90% protection rate was achieved following intra-nasal vaccination with recNcPDI emulsified in cholera toxin. Only 1 mouse vaccinated intra-nasally with recNcMAG1 survived the challenge infection, and protection achieved with intra-nasally applied recNcROP2 was at 60%. Determination of cerebral parasite burdens by real-time PCR showed that these were significantly reduced only in recNcROP2-vaccinated animals (following intraperitoneal and intra-nasal application) and in recNcPDI-vaccinated mice (intra-nasal application only). Quantification of viable tachyzoites in brain tissue of intra-nasally vaccinated mice showed that immunization with recNcPDI resulted in significantly decreased numbers of live parasites. These data show that, besides the nature of the antigen, the protective effect of vaccination also depends largely on the route of antigen delivery. In the case of recNcPDI, the intra-nasal route provides a platform to generate a highly protective immune response.

Serodiagnosis of Echinococcus spp. infection: explorative selection of diagnostic antigens by peptide microarray

BACKGROUND: Production of native antigens for serodiagnosis of helminthic infections is laborious and hampered by batch-to-batch variation. For serodiagnosis of echinococcosis, especially cystic disease, most screening tests rely on crude or purified Echinococcus granulosus hydatid cyst fluid. To resolve limitations associated with native antigens in serological tests, the use of standardized and highly pure antigens produced by chemical synthesis offers considerable advantages, provided appropriate diagnostic sensitivity and specificity is achieved. METHODOLOGY/PRINCIPAL FINDINGS: Making use of the growing collection of genomic and proteomic data, we applied a set of bioinformatic selection criteria to a collection of protein sequences including conceptually translated nucleotide sequence data of two related tapeworms, Echinococcus multilocularis and Echinococcus granulosus. Our approach targeted alpha-helical coiled-coils and intrinsically unstructured regions of parasite proteins potentially exposed to the host immune system. From 6 proteins of E. multilocularis and 5 proteins of E. granulosus, 45 peptides between 24 and 30 amino acids in length were designed. These peptides were chemically synthesized, spotted on microarrays and screened for reactivity with sera from infected humans. Peptides reacting above the cut-off were validated in enzyme-linked immunosorbent assays (ELISA). Peptides identified failed to differentiate between E. multilocularis and E. granulosus infection. The peptide performing best reached 57% sensitivity and 94% specificity. This candidate derived from Echinococcus multilocularis antigen B8/1 and showed strong reactivity to sera from patients infected either with E. multilocularis or E. granulosus. CONCLUSIONS/SIGNIFICANCE: This study provides proof of principle for the discovery of diagnostically relevant peptides by bioinformatic selection complemented with screening on a high-throughput microarray platform. Our data showed that a single peptide cannot provide sufficient diagnostic sensitivity whereas pooling several peptide antigens improved sensitivity; thus combinations of several peptides may lead the way to new diagnostic tests that replace, or at least complement conventional immunodiagnosis of echinococcosis. Our strategy could prove useful for diagnostic developments in other pathogens.

Immune response of bovine milk somatic cells to endotoxin in healthy quarters with normal and very low cell counts

Low somatic cell count (SCC) is a reliable indicator of high-quality milk free of pathogenic microorganisms. Thus, an important goal in dairy practice is to produce milk with low SCC. Selection for cows with low SCC can sometimes lead to extremely low SCC in single quarters. The cells in milk are, however, predominantly immune cells with important immune functions. To investigate the mammary immune competence of quarters with very low SCC, healthy udder quarters of cows with normal SCC of (40-100) x 10(3) cells/ml and very low SCC of < 20 x 10(3) cells/ml were challenged with lipopolysaccharide (LPS) from Escherichia coli. In the first experiment, SCC and cell viability after a challenge with 50 ng of LPS/quarter was investigated. In the second experiment, tumour necrosis factor alpha (TNF-alpha) concentration and lactate dehydrogenase (LDH) activity in milk, and mRNA expression of various innate immune factors in milk cells were measured after a challenge with 100 mug LPS/quarter. LPS challenge induced an increase of SCC. SCC levels reached were higher in quarters with normal SCC and maximum SCC was reached 1 h earlier than in very low SCC quarters. The increase of TNF-alpha concentrations in milk in response to LPS challenge was lower in quarters with very low SCC than in quarters with normal SCC. The viability of cells and the LDH activity in milk increased in response to LPS challenge, however, without a difference between the groups. The mRNA expression of IL-1beta and IL-8 was increased in milk cells at 12 h after LPS challenge, whereas that of TNF-alpha and lactoferrin was not increased at the measured time points (12, 24 and 36 h after LPS challenge). No differences of mRNA expression of measured immune factors between normal and very low SCC samples were detected. The study showed that udder quarters with very low SCC responded with a less marked increase of SCC compared with quarters with normal SCC. This difference corresponded with simultaneously lower TNF-alpha concentrations in milk. However, the immune competence of the cells themselves based on mRNA expression of TNF-alpha, IL-8, IL-1beta, and lactoferrin, did not differ. The results may indicate that very low SCC can impair the immune competence of udder quarters, because the immune response in udder quarters with lower SCC is less efficient as fewer cells contribute to the production of immunoregulators.

Human leukocyte antigens (HLA) associated drug hypersensitivity: consequences of drug binding to HLA

Recent publications have shown that certain human leukocyte antigen (HLA) alleles are strongly associated with hypersensitivity to particular drugs. As HLA molecules are a critical element in T-cell stimulation, it is no surprise that particular HLA alleles have a direct functional role in the pathogenesis of drug hypersensitivity. In this context, a direct interaction of the relevant drug with HLA molecules as described by the p-i concept appears to be more relevant than presentation of hapten-modified peptides. In some HLA-associated drug hypersensitivity reactions, the presence of a risk allele is a necessary but incomplete factor for disease development. In carbamazepine and HLA-B*15:02, certain T-cell receptor (TCR) repertoires are required for immune activation. This additional requirement may be one of the 'missing links' in explaining why most individuals carrying this allele can tolerate the drug. In contrast, abacavir generates polyclonal T-cell response by interacting specifically with HLA-B*57:01 molecules. T cell stimulation may be due to presentation of abacavir or of altered peptides. While the presence of HLA-B*58:01 allele substantially increases the risk of allopurinol hypersensitivity, it is not an absolute requirement, suggesting that other factors also play an important role. In summary, drug hypersensitivity is the end result of a drug interaction with certain HLA molecules and TCRs, the sum of which determines whether the ensuing immune response is going to be harmful or not.

Assessing the expression of enterotoxigenic Escherichia coli-specific surface antigens in recombinant strains by transmission electron microscopy and immunolabeling

Infections with enterotoxigenic Escherichia coli (ETEC) are a major cause of travelers' diarrhea worldwide. Colonization of the small intestine mucosa is dependent on specific colonization factor antigens (CFA) and coli surface (CS) antigens. CFA/1, CS3, and CS6 are the most prevalent fimbrial antigens found in clinical isolates. The goal of our study was to visualize the morphology of CS3 and CS6 fimbriae in wild-type and recombinant E. coli strains by means of transmission electron microscopy in conjunction with negative staining and immunolabeling. Corresponding ETEC genes were cloned into E. coli K12 strain DH10B. Expression of fimbriae was dependent on culture conditions and sample handling. Specific immunolabeling of fimbriae unequivocally demonstrated the presence of all types of surface antigens investigated. Negative staining was effective in revealing CS3 but not CS6. In addition, this technique clearly demonstrated differences in the morphology of genetically and immunologically identical CS3 surface antigens in wild-type and recombinant strains. This paper provides a basis for the assessment of recombinant vaccines.

Leukocyte populations and mRNA expression of inflammatory factors in quarter milk fractions at different somatic cell score levels in dairy cows

The effect of somatic cell count (SCC) and milk fraction on milk composition, distribution of cell populations, and mRNA expression of various inflammatory parameters was studied. Therefore, quarter milk samples were defined as cisternal (C), first 400 g of alveolar (A1), and remaining alveolar milk (A2) during the course of milking. Quarters were assigned to 4 groups according to their total SCC: 1) <12 x 10(3)/mL, 2) 12 to 100 x 10(3)/mL, 3) 100 to 350 x 10(3)/mL, and 4) >350 x 10(3)/mL. Milk constituents of interest were SCC, fat, protein, lactose sodium, and chloride ions as well as electrical conductivity. Cell populations were classified into lymphocytes, macrophages, and neutrophils (PMN). The mRNA expression of the inflammatory factors tumor necrosis factor-alpha, interleukin-1beta, cyclooxygenase-2, lactoferrin, and lysozyme was measured via real-time, quantitative reverse transcription PCR. Somatic cell count decreased from highest levels in C to lowest levels in A1 and increased thereafter to A2 in all groups. Fat content increased from C to A2 and with increasing SCC level. Lactose decreased with increasing SCC level but remained unchanged during milking. Concentrations of sodium and chloride, and electrical conductivity increased with increasing SCC but were higher in C than in A1 and A2. Protein was not affected by milk fraction or SCC level. The distribution of leukocytes was dramatically influenced by milk fraction and SCC. Lymphocytes were the dominating cell population in group 1, but the proportion of lymphocytes was low in groups 2, 3, and 4. Macrophage proportion was highest in group 2 and decreased in groups 3 and 4, whereas that of PMN increased from group 2 to 4. The content of macrophages decreased during milking in all SCC groups whereas that of PMN increased. The proportion of lymphocytes was not affected by milk fraction. The mRNA expression of all inflammatory factors showed an increase with increasing SCC but minor changes occurred during milking. In conclusion, milk fraction and SCC level have a crucial influence on the distribution of leukocyte populations and several milk constituents. The surprisingly high content of lymphocytes and concomitantly low mRNA expression of inflammatory factors in quarters with SCC <12 x 10(3)/mL indicates a different and possibly reduced readiness of the immune system to respond to invading pathogens.

Importance of the sampled milk fraction for the prediction of total quarter somatic cell count

This study investigated the changes in somatic cell counts (SCC) in different fractions of milk, with special emphasis on the foremilk and cisternal milk fractions. Therefore, in Experiment 1, quarter milk samples were defined as strict foremilk (F), cisternal milk (C), first 400 g of alveolar milk (A1), and the remaining alveolar milk (A2). Experiment 2 included 6 foremilk fractions (F1 to F6), consisting of one hand-stripped milk jet each, and the remaining cisternal milk plus the entire alveolar milk (RM). In Experiment 1, changes during milking indicated the importance of the sampled milk fraction for measuring SCC because the decrease in the first 3 fractions (F, C, and A1) was enormous in milk with high total quarter SCC. The decline in SCC from F to C was 50% and was 80% from C to A1. Total quarter SCC presented a value of approximately 20% of SCC in F or 35% of SCC in C. Changes in milk with low or very low SCC were marginal during milking. Fractions F and C showed significant differences in SCC among different total SCC concentrations. These differences disappeared with the alveolar fractions A1 and A2. In Experiment 2, a more detailed investigation of foremilk fractions supported the findings of Experiment 1. A significant decline in the foremilk fractions even of F1 to F6 was observed in high-SCC milk at concentrations >350 x 10(3) cells/mL. Although one of these foremilk fractions presented only 0.1 to 0.2% of the total milk, the SCC was 2- to 3-fold greater than the total quarter milk SCC. Because the trait of interest (SCC) was measured directly by using the DeLaval cell counter (DCC), the quality of measurement was tested. Statistically interesting factors (repeatability, recovery rate, and potential matrix effects of milk) proved that the DCC is a useful tool for identifying the SCC of milk samples, and thus of grading udder health status. Generally, the DCC provides reliable results, but one must consider that SCC even in strict foremilk can differ dramatically from SCC in the total cisternal fraction, and thus also from SCC in the alveolar fraction.