Tissue Phthalate Levels Correlate With Changes in Immune Gene Expression in a Population of Juvenile Wild Salmon.

Phthalates have detrimental effects on health and have been shown to dysregulate the immune system of mammals, birds, and fish. We recently reported that di(2-ethylhexyl) phthalate exposure reduces the abundance and inhibits the proliferation of rainbow trout (Oncorhynchus mykiss) IgM(+) B lymphocytes and expression of secreted immunoglobulin heavy-chain mu transcripts in an in vitro culture system. We proposed that phthalates act as immunomodulators by modifying the normal B cell-activation pathways by accelerating B cell differentiation while suppressing plasmablast expansion, thus resulting in fewer IgM-secreting plasma cells. This hypothesis was tested here in an in vivo field study of juvenile Dolly Varden (Salvelinus malma) from a plastic-polluted lake in the Gulf of Alaska. Fish tissues were analyzed for both phthalate levels using liquid chromatography-coupled tandem mass spectrometry and for changes in immune gene expression using reverse transcriptase-real time polymerase chain reaction. Results showed that fish with higher tissue levels of di(2-ethylhexyl) phthalate, di(n-butyl) phthalate, and/or dimethyl phthalate expressed significantly fewer secreted and membrane-bound immunoglobulin heavy-chain mu and Blimp1 transcripts in their hematopoietic tissue. This suggests that in vivo uptake of phthalates in fish changes the expression of B cell-specific genes. Chronic exposure to phthalates likely dysregulates normal B-lymphoid development and antibody responses in salmonids and may increase susceptibility to infection. Given the conserved nature of B-lineage cells in vertebrate animals, other marine species may be similarly affected by chronic phthalate exposure.

Innate immune cell signatures in a BCWD-Resistant line of rainbow trout before and after in vivo challenge with Flavobacterium psychrophilum.

Phenotypes of myeloid-lineage cells remain poorly understood in the rainbow trout, and were the focus of this study, including effects of in vivo challenge to Flavobacterium psychrophilum (Fp), the cause of Bacterial Cold Water Disease (BCWD). A genetic line was used that is highly resistant to BCWD (R-line) as well as a susceptible control line (S-line). Using flow cytometry, we describe two Pax5-negative, myeloid-lineage populations: Population 1 consisted of small cells with high SSC and strong staining for Q4E, MPO, Pu1, EBF, and IL- 1ß, which we named "neutrophil-like" cell. Population 2 had high Q4E, but weaker MPO, Pu1, EBF, and IL-1ß staining. Five days after Fp-challenge, both genetic lines had a reduced abundance of neutrophil-like cells in anterior kidney, PBL, and spleen. Pop. 2 abundance was reduced in anterior kidney, and increased in spleen. S-line fish responded more strongly to Fp-challenge compared to R-line fish. Challenged fish with a higher abundance of neutrophil-like cells had significantly lower Fp-loads after challenge, suggesting that these cells aid in the resistance to BCWD.

Consensus characterization of 16 FMR1 reference materials: a consortium study.

Fragile X syndrome, which is caused by expansion of a (CGG)(n) repeat in the FMR1 gene, occurs in approximately 1:3500 males and causes mental retardation/behavioral problems. Smaller (CGG)(n) repeat expansions in FMR1, premutations, are associated with premature ovarian failure and fragile X-associated tremor/ataxia syndrome. An FMR1-sizing assay is technically challenging because of high GC content of the (CGG)(n) repeat, the size limitations of conventional PCR, and a lack of reference materials available for test development/validation and routine quality control. The Centers for Disease Control and Prevention and the Association for Molecular Pathology, together with the genetic testing community, have addressed the need for characterized fragile X mutation reference materials by developing characterized DNA samples from 16 cell lines with repeat lengths representing important phenotypic classes and diagnostic cutoffs. The alleles in these materials were characterized by consensus analysis in nine clinical laboratories. The information generated from this study is available on the Centers for Disease Control and Prevention and Coriell Cell Repositories websites. DNA purified from these cell lines is available to the genetics community through the Coriell Cell Repositories. The public availability of these reference materials should help support accurate clinical fragile X syndrome testing.

The genetic insulator RiboJ increases expression of insulated genes.

A primary objective of synthetic biology is the construction of genetic circuits with behaviors that can be predicted based on the properties of the constituent genetic parts from which they are built. However a significant issue in the construction of synthetic genetic circuits is a phenomenon known as context dependence in which the behavior of a given part changes depending on the choice of adjacent or nearby parts. Interactions between parts compromise the modularity of the circuit, impeding the implementation of predictable genetic constructs. To address this issue, investigators have devised genetic insulators that prevent these unintended context-dependent interactions between neighboring parts. One of the most commonly used insulators in bacterial systems is the self-cleaving ribozyme RiboJ. Despite its utility as an insulator, there has been no systematic quantitative assessment of the effect of RiboJ on the expression level of downstream genetic parts. Here, we characterized the impact of insulation with RiboJ on expression of a reporter gene driven by a promoter from a library of 24 frequently employed constitutive promoters in an Escherichia coli model system. We show that, depending on the strength of the promoter, insulation with RiboJ increased protein abundance between twofold and tenfold and increased transcript abundance by an average of twofold. This result demonstrates that genetic insulators in E. coli can impact the expression of downstream genes, information that is essential for the design of predictable genetic circuits and constructs.

Sockeye salmon immunoglobulin VH usage and pathogen loads differ between spawning sites.

The Immunological Imprinting Hypothesis proposes that juvenile anadromous fish respond to the pathogen fingerprint specific to their natal site by producing protective long lived plasma cells (LLPCs) that constitutively produce antibodies against those pathogens. Hence, fish returning to their natal streams have immunological protection from pathogens at that specific location. Here, we tested the hypothesis through analysis of antibody composition and usage in sockeye salmon populations in Alaska. Spleen and anterior kidney were sampled from salmon from six sites, and relative usage levels of six different Immunoglobulin VH gene families determined using RT-qPCR. Additionally, prevalence and pathogen loads were measured in each fish for Renibacterium salmoninarum, Flavobacterium psychrophilum, and Infectious Hematopoietic Necrosis Virus. Results revealed differences in VH usage, pathogen loads, and infection rates between spawning sites, while probability of infection was dependent on location for each pathogen analyzed. Further, several negative correlations between specific VH usage patterns and pathogen loads were uncovered. Greater understanding of site-dependent VH usage in spawning fish potentially suggests a method of natural immunization against common fish pathogens and thus protection of both farmed and wild populations.

A BCWD-resistant line of rainbow trout exhibits higher abundance of IgT+ B cells and heavy chain tau transcripts compared to a susceptible line following challenge with Flavobacterium psychrophilum.

Bacterial Cold Water Disease (BCWD) is a common, chronic disease in rainbow trout, and is caused by the gram-negative bacterium Flavobacterium psychrophilum (Fp). Through selective breeding, the National Center for Cool and Cold Water Aquaculture has generated a genetic line that is highly resistant to Fp challenge, designated ARS-Fp-R (or R-line), as well as a susceptible "control" line, ARS-Fp-S (S-line). In previous studies, resistance to Fp had been shown to correlate with naive animal spleen size, and further, naïve R-line trout had been shown to have a lower abundance of IgM+ and IgM++ cells compared to S-line fish. Here we wished to first determine whether the abundance of IgT+ and/or IgT++ cells differed between the two lines in naïve fish, and if so, how these patterns differed after in vivo challenge with Fp. Fp challenge was by intramuscular injection of live Fp and tissue collections were on days 5, 6, and/or 28 post-challenge, in two independent challenge experiments. Flow cytometric and gene expression analyses revealed that naïve R-line fish had a higher abundance of IgT+ B cells in their anterior kidney, spleen, and blood, compared to S line fish. Further, that after Fp challenge, this difference was maintained between the two lines. Lastly, abundance of IgT+ B cells and expression of secHCtau correlated with lower Fp pathogen loads in challenged fish. In the anterior kidney, IgM+ B cell abundance correlated with increased Fp loads. Together, these results suggest that IgT+ B lineage cells may have a protective function in the immune response to Fp.

Quantitation of HERV-K env gene expression and splicing in human breast cancer.

Human endogenous retroviruses (HERVs) comprise up to 8% of the human genome. In previous studies, we demonstrated that type 1 HERV-K envelope (env) transcripts are expressed in most human breast cancers, but not in normal breast tissues. In the current study, we report that type 2 HERV-K env transcripts are also present in human breast cancers. By real-time RT-PCR, the expression of HERV-K env transcripts was 5-10-fold higher in breast cancer cell lines treated with estradiol and progesterone than in cells without treatment, and expression was significantly higher in most breast cancer tissues than in normal breast tissues. Furthermore, both types of HERV-K env transcripts were capable of being spliced into subgenomic env transcripts and various splice donor and acceptor sites were detected in breast cancers. The selective expression and distribution of multiple HERV-K endogenous retroviral element splice variants in breast cancer, but not in normal controls, suggests that they are novel breast tumor markers.

B cell signatures of BCWD-resistant and susceptible lines of rainbow trout: a shift towards more EBF-expressing progenitors and fewer mature B cells in resistant animals.

Bacterial cold water disease (BCWD) is a chronic disease of rainbow trout, and is caused by the Gram-negative bacterium Flavobacterium psychrophilum (Fp), a common aquaculture pathogen. The National Center for Cool and Cold Water Aquaculture has bred two genetic lines of rainbow trout: a line of Fp-resistant trout (ARS-Fp-R or R-line trout) and a line of susceptible trout (ARS-Fp-S, or S-line). Little is known about how phenotypic selection alters immune response parameters or how such changes relate to genetic disease resistance. Herein, we quantify interindividual variation in the distribution and abundance of B cell populations (B cell signatures) and examine differences between genetic lines of naive animals. There are limited trout-specific cell surface markers currently available to resolve B cell subpopulations and thus we developed an alternative approach based on detection of differentially expressed transcription factors and intracellular cytokines. B cell signatures were compared between R-line and S-line trout by flow cytometry using antibodies against transcription factors early B cell factor-1 (EBF1) and paired domain box protein Pax5, the pro-inflammatory cytokine IL-1ß, and the immunoglobulin heavy chain mu. R-line trout had higher percentages of EBF(+) B myeloid/ progenitor and pre-B cells in PBL, anterior and posterior kidney tissues compared to S-line trout. The opposite pattern was detected in more mature B cell populations: R-line trout had lower percentages of both IgM(+) mature B cells and IgM-secreting cells in anterior kidney and PBL compared to S-line trout. In vitro LPS-activation studies of PBL and spleen cell cultures revealed no significant induction differences between R-line and S-line trout. Together, our findings suggest that selective resistance to BCWD may be associated with shifts in naive animal developmental lineage commitment that result in decreased B lymphopoiesis and increased myelopoiesis in BCWD resistant trout relative to susceptible trout.

Sockeye salmon retain immunoglobulin-secreting plasma cells throughout their spawning journey and post-spawning.

Antibody-producing plasma cells are a major source of protective immunity in vertebrates, including salmon. During the spawning phase, salmon undergo drastic, hormonally driven changes in their physiology, including elevated levels of cortisol, which are known to suppress the immune system. As adult fish need to survive their long journey to the spawning grounds, we hypothesized that humoral immunity, in the form of IgM-secreting plasma cells, remains functional until post-spawning. This was investigated by measuring changes in membrane and secreted immunoglobulin heavy chain mu and Pax5 transcripts in spleen and kidney from migrating sockeye salmon, using real-time qPCR. As an additional measurement, the abundance of developing B, mature B, and plasma cells was determined in spawning fish, using flow cytometry. Immune tissue samples were collected from fish from the Kenai River drainage and Main Bay, Prince William Sound. Our results reveal that spawning fish express high levels of secreted heavy chain mu transcripts in their spleen and anterior kidney throughout the spawning journey. Furthermore, we show that IgM-secreting PCs (HCmu++/Pax5-) remain abundant in anterior kidney and spleen of post-spawning sockeye salmon, with a concomitant loss in developing B cells (HCmu-/Pax5+). This suggests that successful spawners retain their PCs throughout the spawning journey and post-spawning.

Alternative splicing of the trout Pax5 gene and identification of novel B cell populations using Pax5 signatures.

Pax5 is an alternatively spliced transcription factor that regulates B cell development and activation. The function of specific Pax5 isoforms is unknown. Here we report the existence of seven alternatively spliced isoforms of Pax5 in the rainbow trout. We hypothesized that B cells differentially express specific Pax5 isoforms as a means of modulating Pax5 activity during cell maturation. Flow cytometric analyses using Pax5-specific antibodies recognizing the paired domain, a central (exon 6-encoding) domain, or the C-terminus, revealed the existence of distinct Pax5-expressing cell populations in trout immune tissues. Additionally, using the transcription factor EBF, we show that Pax5 isoforms lacking a paired domain are already expressed at the earliest stages of trout (B) lymphopoiesis, and unexpectedly, that minor populations of such cells reside in blood and spleen. These data support use of differentially expressed Pax5 isoforms to identify novel B cell subsets in the form of Pax5 tissue signatures, and as such, provides new biomarkers for malignancy, infectious disease, and disease resistance in trout and humans.