The NF-{kappa}B negative regulator TNFAIP3 (A20) is inactivated by somatic mutations and genomic deletions in marginal zone lymphomas

Unique and shared cytogenetic abnormalities have been documented for marginal zone lymphomas (MZLs) arising at different sites. Recently, homozygous deletions of the chromosomal band 6q23, involving the tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20) gene, a negative regulator of NF-kappaB, were described in ocular adnexal MZL, suggesting a role for A20 as a tumor suppressor in this disease. Here, we investigated inactivation of A20 by DNA mutations or deletions in a panel of extranodal MZL (EMZL), nodal MZL (NMZL), and splenic MZL (SMZL). Inactivating mutations encoding truncated A20 proteins were identified in 6 (19%) of 32 MZLs, including 2 (18%) of 11 EMZLs, 3 (33%) of 9 NMZLs, and 1 (8%) of 12 SMZLs. Two additional unmutated nonsplenic MZLs also showed monoallelic or biallelic A20 deletions by fluorescent in situ hybridization (FISH) and/or SNP-arrays. Thus, A20 inactivation by either somatic mutation and/or deletion represents a common genetic aberration across all MZL subtypes, which may contribute to lymphomagenesis by inducing constitutive NF-kappaB activation.

Patterns of morphological changes and hybridization between sympatric whitefish morphs (Coregonus spp.) in a Swiss lake: a role for eutrophication?

Whitefish, genus Coregonus, show exceptional levels of phenotypic diversity with sympatric morphs occurring in numerous postglacial lakes in the northern hemisphere. Here, we studied the effects of human-induced eutrophication on sympatric whitefish morphs in the Swiss lake, Lake Thun. In particular, we addressed the questions whether eutrophication (i) induced hybridization between two ecologically divergent summer-spawning morphs through a loss of environmental heterogeneity, and (ii) induced rapid adaptive morphological changes through changes in the food web structure. Genetic analysis based on 11 microsatellite loci of 282 spawners revealed that the pelagic and the benthic morph represent highly distinct gene pools occurring at different relative proportions on all seven known spawning sites. Gill raker counts, a highly heritable trait, showed nearly discrete distributions for the two morphs. Multilocus genotypes characteristic of the pelagic morph had more gill rakers than genotypes characteristic of benthic morph. Using Bayesian methods, we found indications of recent but limited introgressive hybridization. Comparisons with historical gill raker data yielded median evolutionary rates of 0.24 haldanes and median selection intensities of 0.27 for this trait in both morphs for 1948-2004 suggesting rapid evolution through directional selection at this trait. However, phenotypic plasticity as an alternative explanation for this phenotypic change cannot be discarded. We hypothesize that both the temporal shifts in mean gill raker counts and the recent hybridization reflect responses to changes in the trophic state of the lake induced by pollution in the 1960s, which created novel selection pressures with respect to feeding niches and spawning site preferences.

Hybridization between distant lineages increases adaptive variation during a biological invasion: stickleback in Switzerland

The three-spined stickleback is a widespread Holarctic species complex that radiated from the sea into freshwaters after the retreat of the Pleistocene ice sheets. In Switzerland, sticklebacks were absent with the exception of the far northwest, but different introduced populations have expanded to occupy a wide range of habitats since the late 19th century. A well-studied adaptive phenotypic trait in sticklebacks is the number of lateral plates. With few exceptions, freshwater and marine populations in Europe are fixed for either the low plated phenotype or the fully plated phenotype, respectively. Switzerland, in contrast, harbours in close proximity the full range of phenotypic variation known from across the continent. We addressed the phylogeographic origins of Swiss sticklebacks using mitochondrial partial cytochrome b and control region sequences. We found only five different haplotypes but these originated from three distinct European regions, fixed for different plate phenotypes. These lineages occur largely in isolation at opposite ends of Switzerland, but co-occur in a large central part. Across the country, we found a strong correlation between a microsatellite linked to the high plate ectodysplasin allele and the mitochondrial haplotype from a region where the fully plated phenotype is fixed. Phylogenomic and population genomic analysis of 481 polymorphic amplified fragment length polymorphism loci indicate genetic admixture in the central part of the country. The same part of the country also carries elevated within-population phenotypic variation. We conclude that during the recent invasive range expansion of sticklebacks in Switzerland, adaptive and neutral between-population genetic variation was converted into within-population variation, raising the possibility that hybridization between colonizing lineages contributed to the ecological success of sticklebacks in Switzerland.

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.

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.

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.

Differential expression of IGF-I mRNA and peptide in the male and female gonad during early development of a bony fish, the tilapia Oreochromis niloticus

Insulin-like growth factor I (IGF-I) plays a key role in the complex system that regulates bony fish growth, differentiation, and reproduction. The major source of circulating IGF-I is liver, but IGF-I-producing cells also occur in other organs, including the gonads. Because no data are available on the potential production sites of IGF-I in gonad development, developmental stages of monosex breedings of male and female tilapia from 0 day postfertilization (DPF) to 90 DPF were investigated for the production sites of IGF-I at the peptide (immunohistochemistry) and mRNA (in situ hybridization) level. IGF-I mRNA first appeared in somatic cells of the male and female gonad anlage at 7 DPF followed by IGF-I peptide around 9-10 DPF. Gonad anlagen were detected from 7 DPF. Starting at 7 DPF, IGF-I peptide but no IGF-I mRNA was observed in male and female primordial germ cells (PGCs) provided that IGF-I mRNA was not under the detection level, this observation may suggest that IGF-I originates from the somatic cells and is transferred to the PGCs or is of maternal origin. While in female germ cells IGF-I mRNA and peptide appeared at 29 DPF, in male germ cells both were detected as late as at 51-53 DPF. It is assumed that the production of IGF-I in the germ cells is linked to the onset of meiosis that in tilapia ovary starts at around 28 DPF and in testes at around 52-53 DPF. In adult testis, IGF-I mRNA and peptide occurred in the majority of spermatogonia and spermatocytes as well as in Leydig cells, the latter indicating a role of IGF-I in the synthesis of male sex steroids. In adult ovary, IGF-I mRNA and IGF-I peptide were always present in small and previtellogenic oocytes but only IGF-I peptide infrequently occurred in oocytes at the later stages. IGF-I expression appeared in numerous granulosa and some theca cells of follicles at the lipid stage and persisted in follicles with mature oocytes. The results suggest a crucial role of local IGF-I in the formation, differentiation and function of tilapia gonads.

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.