Patricia L. Chen

Novel transcriptome assembly and improved annotation of the whiteleg shrimp (Litopenaeus vannamei), a dominant crustacean in global seafood mariculture

We present a new transcriptome assembly of the Pacific whiteleg shrimp (Litopenaeus vannamei), the species most farmed for human consumption. Its functional annotation, a substantial improvement over previous ones, is provided freely. RNA-Seq with Illumina HiSeq technology was used to analyze samples extracted from shrimp abdominal muscle, hepatopancreas, gills and pleopods. We used the Trinity and Trinotate software suites for transcriptome assembly and annotation, respectively. The quality of this assembly and the affiliated targeted homology searches greatly enrich the curated transcripts currently available in public databases for this species. Comparison with the model arthropod Daphnia allows some insights into defining characteristics of decapod crustaceans. This large-scale gene discovery gives the broadest depth yet to the annotated transcriptome of this important species and should be of value to ongoing genomics and immunogenetic resistance studies in this shrimp of paramount global economic importance.

Quantum dots trigger immunomodulation of the NFκB pathway in human skin cells.

The immunological effects of quantum dots are dependent on a variety of factors including, but not limited to, exposure time and dosing concentrations. In this study, we investigated the influence of 15 nm CdSe/ZnS-COOH quantum dot nanocrystals (QDs) on cell density, viability, and morphology in human epidermal keratinocytes (HEK) and human dermal fibroblasts (HDF). Furthermore, inflammatory and non-inflammatory immune responses were measured using protein and real time PCR array analysis from HDF cells exposed to predetermined sub-lethal concentrations of QDs. CdSe/ZnS-COOH QDs caused concentration-dependent (1-120 nM exposure concentrations) and time-dependent (8 h or 48 h) cell death, as evidenced by metabolic activity and morphological changes. QD exposure induced upregulation of apoptotic, inflammatory and immunoregulatory proteins such as TNF-α, IL-1B and IL-10. HMOX1, an indicator of stress due to reactive oxygen intermediates (ROIs) and/or metals, was upregulated at the later time point as well. QDs also caused modulation of genes known to be associated with inflammatory (IL1-β, CCL2, IRAK-2), immune (IL-1, IL-6, PGLYRP1, SERPINA1, IL-10), stress due to ROIs and/or heavy metals (HMOX1), and apoptotic (CASP1, ADORA2A) responses. Cellular effects from QD exposure were found to primarily follow the NFκB pathway. In addition, QDs induced a differential cytotoxicity in keratinocytes and fibroblasts at different exposure concentrations and time points, even at physiologically relevant dosing concentrations, thus emphasizing the need to investigate potential mechanisms of action among different cell types within the same target organ.

Rapid elicitation of broadly neutralizing antibodies to HIV by immunization in cows

No immunogen to date has reliably elicited broadly neutralizing antibodies to HIV in humans or animal models. Advances in the design of immunogens that antigenically mimic the HIV envelope glycoprotein (Env), such as the soluble cleaved trimer BG505 SOSIP, have improved the elicitation of potent isolate-specific antibody responses in rabbits and macaques, but so far failed to induce broadly neutralizing antibodies. One possible reason for this failure is that the relevant antibody repertoires are poorly suited to target the conserved epitope regions on Env, which are somewhat occluded relative to the exposed variable epitopes. Here, to test this hypothesis, we immunized four cows with BG505 SOSIP. The antibody repertoire of cows contains long third heavy chain complementary determining regions (HCDR3) with an ultralong subset that can reach more than 70 amino acids in length. Remarkably, BG505 SOSIP immunization resulted in rapid elicitation of broad and potent serum antibody responses in all four cows. Longitudinal serum analysis for one cow showed the development of neutralization breadth (20%, n = 117 cross-clade isolates) in 42 days and 96% breadth (n = 117) at 381 days. A monoclonal antibody isolated from this cow harboured an ultralong HCDR3 of 60 amino acids and neutralized 72% of cross-clade isolates (n = 117) with a potent median IC50 of 0.028 μg ml−1. Breadth was elicited with a single trimer immunogen and did not require additional envelope diversity. Immunization of cows may provide an avenue to rapidly generate antibody prophylactics and therapeutics to address disease agents that have evolved to avoid human antibody responses.

Shark class II invariant chain reveals ancient conserved relationships with cathepsins and MHC class II.

The invariant chain (Ii) is the critical third chain required for the MHC class II heterodimer to be properly guided through the cell, loaded with peptide, and expressed on the surface of antigen presenting cells. Here, we report the isolation of the nurse shark Ii gene, and the comparative analysis of Ii splice variants, expression, genomic organization, predicted structure, and function throughout vertebrate evolution. Alternative splicing to yield Ii with and without the putative protease-protective, thyroglobulin-like domain is as ancient as the MHC-based adaptive immune system, as our analyses in shark and lizard further show conservation of this mechanism in all vertebrate classes except bony fish. Remarkable coordinate expression of Ii and class II was found in shark tissues. Conserved Ii residues and cathepsin L orthologs suggest their long co-evolution in the antigen presentation pathway, and genomic analyses suggest 450 million years of conserved Ii exon/intron structure. Other than an extended linker preceding the thyroglobulin-like domain in cartilaginous fish, the Ii gene and protein are predicted to have largely similar physiology from shark to man. Duplicated Ii genes found only in teleosts appear to have become sub-functionalized, as one form is predicted to play the same role as that mediated by Ii mRNA alternative splicing in all other vertebrate classes. No Ii homologs or potential ancestors of any of the functional Ii domains were found in the jawless fish or lower chordates.

Expressed IgH μ and τ transcripts share diversity segment in ranched Thunnus orientalis.

It is now appreciated that in addition to the immunoglobulin (Ig)M and D isotypes fish also make the mucosal IgT. In this study we sequenced the full length of Ig τ as well as μ in the commercially important Thunnus orientalis (Pacific bluefin tuna), the first molecular analysis of these two Ig isotypes in a member of the order Perciformes. Tuna IgM and IgT are each composed of four constant (CH) domains. We cloned and sequenced 48 different variable (VH) domain gene rearrangements of tuna immunoglobulins and grouped the VH gene sequences to four VH gene segment families based on 70% nucleotide identity. Three VH gene families were used by both IgM and IgT but one group was only found to be used by IgM. Most interestingly, both μ and τ clones appear to use the same diversity (DH) segment, unlike what has been described in other species, although they have dedicated IgT and IgM joining (JH) gene segments. We complemented this repertoire study with phylogenetic and tissue expression analysis. In addition to supporting the development of humoral vaccines in this important aquaculture species, these data suggest that the DH-JH recombination rather than the VH-DH recombination may be instructive for IgT versus IgM/D bearing lymphocyte lineages in some fish.

Distinct immunomodulatory effects of a panel of nanomaterials in human dermal fibroblasts

There are many efforts in understanding the effects of nanoparticles on cell viability and metabolism, however, not much is known regarding the distinct molecular mechanisms of inflammation and cellular stress using low dosing concentrations. To address this gap in the literature, we utilized a novel experimental design that specifically probes the effects of a panel of commonly studied engineered nanomaterials along immunomodulatory pathways, including NF-κB. The panel of particles selected for this study included quantum dot nanocrystals, titanium dioxide, hydroxylated fullerenes, and silver nanoparticles. Cell viability, antioxidant activity, select messenger RNA, and protein modulation were studied in primary human dermal fibroblasts (HDF) and NF-κB knockdown HDF cells. Inflammatory and non-inflammatory immune responses were measured using protein and real-time PCR array analysis from HDF cells exposed to sub-lethal concentrations of nanoparticles. Differences in cellular response to nanoparticles in protein and antioxidant experiments were evident in NF-κB knockdown cells. The methods used in the study, along with the resultant data sets, serve as a potential model for studying the complex pathway-specific biochemical responses in cell and tissue systems associated with nanoparticle exposures.

DNP-KLH Yields Changes in Leukocyte Populations and Immunoglobulin Isotype Use with Different Immunization Routes in Zebrafish

Distinct methods are required for inducing mucosal versus systemic immunity in mammals for vaccine protection at the tissues most commonly breached by pathogens. Understanding of mucosal immunization in teleost fish is needed to combat aquaculture disease, understand emerging ecological threats, and know how vertebrate adaptive immunity evolved. Here, we quantitatively measured expression levels of IgM as well as the teleost mucosal immunoglobulin, IgZ/IgT, in zebrafish given an antigen systemically via intraperitoneal (i.p.) injection or mucosally via bath immersion. Both immunoglobulin isotypes and the B cell activating factor gene transcription was induced in fish injected with antigen as compared to saline injected or antigen immersed fish, though these failed to reach statistical significance. Here we provide additional reference hematology for this model species. Differential blood counts revealed a greater lymphocyte percentage in both i.p. and immersed fish, with increase in large lymphocyte counts and decrease in neutrophils. These humoral adaptive gene transcription and cytological data should provide a foundation for more studies connecting immunology in this dominant developmental and genetic fish model to other species where mucosal immunization is of greater commercial importance.

Immunogenetic factors driving formation of ultralong VH CDR3 in Bos taurus antibodies

The antibody repertoire of Bos taurus is characterized by a subset of variable heavy (VH) chain regions with ultralong third complementarity determining regions (CDR3) which, compared to other species, can provide a potent response to challenging antigens like HIV env. These unusual CDR3 can range to over seventy highly diverse amino acids in length and form unique β-ribbon ‘stalk’ and disulfide bonded ‘knob’ structures, far from the typical antigen binding site. The genetic components and processes for forming these unusual cattle antibody VH CDR3 are not well understood. Here we analyze sequences of Bos taurus antibody VH domains and find that the subset with ultralong CDR3 exclusively uses a single variable gene, IGHV1-7 (VHBUL) rearranged to the longest diversity gene, IGHD8-2. An eight nucleotide duplication at the 3′ end of IGHV1-7 encodes a longer V-region producing an extended F β-strand that contributes to the stalk in a rearranged CDR3. A low amino acid variability was observed in CDR1 and CDR2, suggesting that antigen binding for this subset most likely only depends on the CDR3. Importantly a novel, potentially AID mediated, deletional diversification mechanism of the B. taurus VH ultralong CDR3 knob was discovered, in which interior codons of the IGHD8-2 region are removed while maintaining integral structural components of the knob and descending strand of the stalk in place. These deletions serve to further diversify cysteine positions, and thus disulfide bonded loops. Hence, both germline and somatic genetic factors and processes appear to be involved in diversification of this structurally unusual cattle VH ultralong CDR3 repertoire.

The Florida manatee (Trichechus manatus latirostris) immunoglobulin heavy chain suggests the importance of clan III variable segments in repertoire diversity

Abstract Manatees are a vulnerable, charismatic sentinel species from the evolutionarily divergent Afrotheria. Manatee health and resistance to infectious disease is of great concern to conservation groups, but little is known about their immune system. To develop manatee‐specific tools for monitoring health, we first must have a general knowledge of how the immunoglobulin heavy (IgH) chain locus is organized and transcriptionally expressed. Using the genomic scaffolds of the Florida manatee (Trichechus manatus latirostris), we characterized the potential IgH segmental diversity and constant region isotypic diversity and performed the first Afrotherian repertoire analysis. The Florida manatee has low V(D)J combinatorial diversity (3744 potential combinations) and few constant region isotypes. They also lack clan III V segments, which may have caused reduced VH segment numbers. However, we found productive somatic hypermutation concentrated in the complementarity determining regions. In conclusion, manatees have limited IGHV clan and combinatorial diversity. This suggests that clan III V segments are essential for maintaining IgH locus diversity. HighlightsT. m. latirostris has only four functional IgH constant region isotypes.There are few functional VH, no clan III VH, and low combinatorial diversity.The absence of clan III VH correlates with low VH numbers in three other species.This is the first IgH repertoire analysis within Afrotheria.Nonsynonymous somatic hypermutation is higher in the CDR regions.