Browsing by Subject "Human"

*Designated as an exemplary master's project for 2015-16*

B lymphocytes are multifunctional and play important roles in both innate and adaptive immunity. The diverse roles of B cells can be attributed to the various and distinct types of B cells as determined by their origin, developmental stage, antigen specificity, and function.

Evidence suggests that human innate-like B cells (i.e., marginal zone and/or B1-like B cells) develop during fetal life. However, the characteristics of human fetal B-lineage cells are less understood. Recent studies of fetal and human umbilical cord B cells indicated that CD27, a well-established marker of human memory B cells, may also be expressed on human B1-like B cells. Indeed, CD27+ B cells are present in patients with hyper-IgM 1 (HIGM1) syndrome who are unable to generate GCs or memory B cells. In order to define the origin of naïve CD27+IgD+ human B cells, I studied B-cell development in both fetal and adult tissues.

In human fetal liver, most CD19+ cells co-express CD10, a marker of human developing B cells. Some CD19+CD10+ B cells express CD27, and these fetal CD27+ cells are present in the pro-B, pre-B, and immature/transitional B-cell compartments. Lower frequencies of phenotypically identical cells are also identified in adult bone marrow. CD27+ pro-B, pre-B, and immature/transitional B cells express recombination activating gene-1, terminal deoxynucleotidyl transferase, and Vpre-B mRNA comparable to their CD27− counterparts. CD27+ and CD27− developing B cells show similar immunoglobulin heavy chain gene usage with low levels of mutations, suggesting that CD27+ developing B cells are distinct from mutated memory B cells. Despite these similarities, CD27+ developing B cells differ from CD27− developing B cells by their increased expression of LIN28B, a transcription factor associated with the fetal lymphoid lineages of mice. Furthermore, CD27+ pro-B cells efficiently generate IgM+IgD+ immature/transitional B cells in vitro. Our observations suggest that CD27 expression during B-cell development identifies a physiologic state or lineage for human B-cell development distinct from the memory B-cell compartment.

Regarding B-cell repertoire, due to the random recombination of immunoglobulin V, D, and J gene segments during B-cell development, B cells are highly diversified in their antigen specificity. Through their specific B-cell antigen receptors (BCRs), B cells recognize foreign (and self-) antigens, and present these antigens to cognate T cells to elicit/establish humoral responses, such as germinal centers, immunological memory, and long-lasting circulating antibodies. Some bacteria and viruses escape the host’s immune system by mimicking host antigens, as B cells that recognize shared epitopes on self- and foreign antigens may provide protection against such pathogens; however, these B cells are normally eliminated by tolerance mechanisms during development. The extent of tolerization manifest among human B cells that recognize both self- and foreign antigens is unknown. Here, I and my colleagues use an efficient single B-cell culture method and multiplexed antigen-binding assays to determine the specificity of about 2,300 clonal IgG antibodies produced by the progeny of single transitional and mature B cells. We show that in healthy individuals, half of the self-reactive B cells crossreact with foreign antigen, and that the frequencies of crossreactive B cells decrease by half between the transitional and mature B-cell stages, indicating that a substantial fraction of foreign specificities is lost by the second tolerance mechanisms. In SLE patients, who show defective peripheral tolerance, frequencies of crossreactive B cells are unchanged between the B-cell stages. The crossreactive, mature B cells in SLE patients show distinct reactivity to foreign antigens. We propose that activating forbidden B cells may be a good strategy for protection against host-mimicking pathogens if we can control tolerance.

Activated B cells can present antigen to T cells, as well as differentiate into memory B cells and plasma cells. Indeed, activated B cells express high levels of MHCII and are considered to be professional antigen presenting cells (APC), along with dendritic cells and macrophages. APC can be used to discover the epitopes targeted in T-cell responses; T cells are co-cultured with autologous APC in the presence of antigens and T-cell responses are evaluated. With numerous epitope candidates, mapping T-cell epitopes requires large numbers of APC; the availability of APC in blood is a limiting component and leukapheresis is often required. Since B cells can be expanded in vitro more easily than other APC, they represent a solution for the challenge of isolating adequate numbers of APC from blood in order to determine T-cell antigen specificity. I modified our single B-cell culture to support efficient activation and proliferation of both naïve and memory human B cells for the purpose of generating large numbers of autologous APC. Briefly, naïve or memory B cells recovered from blood are cultured with recombinant human IL-2, IL-4, IL-21, and BAFF on CD154+ feeder cells; this culture supports extensive B-cell proliferation, with approximately 103 fold increases following 8 days in culture, and 106 fold increases when cultures are split and cultured for 8 more days. The capacity for continued proliferation is stable for at least another week. In culture, a significant fraction of naïve B cells undergo isotype switching and terminally differentiate into plasmacytes. Culture-derived (CD) B cells are readily cryopreserved, and when recovered, retain their ability to proliferate and differentiate. Significantly, proliferating CD B cells express high levels of MHCII, CD80, and CD86. I have examined the APC function of CD B cells and found that they present both allo- and microbial antigens to autologous T cells with comparable efficiency to PBMC. Moreover, I am able to activate and expand antigen-specific memory B cells; these cultured cells are highly effective in presenting antigen to T cells. This culture method provides a platform for studying the BCR and TCR repertoires within a single individual.

Deciphering the molecular mechanisms driving the phenotypic differences between humans and primates remains a daunting challenge. Mutations found in protein coding DNA alone has not been able to explain these phenotypic differences. The hypothesis that mutations in non-coding regulatory DNA are responsible for altered gene expression leading to these phenotypic changes has now been widely supported by differential gene expression experiments. Yet, comprehensive identification of all regulatory DNA elements across different species has not been performed. To identify the genetic source of regulatory change, genome-wide DNaseI hypersensitivity assays, marking all types of active gene regulatory element sites, were performed in human, chimpanzee, macaque, orangutan, and mouse. Many DNaseI hypersensitive (DHS) sites were conserved among all 5 species, but we also identified hundreds of novel human- and chimpanzee-specific DHS gains and losses that showed signatures of positive selection. Species-specific DHS gains were enriched in distal non-coding regions, associated with active histone modifications, and positively correlated with increased expression - indicating that these are likely to be functioning as enhancers. Comparison to mouse DHS data indicate that human or chimpanzee DHS gains are likely to have been a result of single events that occurred primarily on the human- or chimpanzee-specific branch, respectively. In contrast, DHS losses are associated with events that occurred on multiple branches. At least one mechanism contributing to DHS gains and losses are species-specific variants that lead to sequence changes at transcription factor binding motifs, affecting the binding of TFs such as AP1. These variants were functionally verified by DNase footprinting and ChIP-qPCR analyses.

Scientists expect temperatures on Earth to get substantially warmer over the course of the 21st century, causing storm systems to intensify and sea-level rise to accelerate--these changes will likely have dramatic impacts on how the coastlines of tomorrow will evolve. Humans are also playing an increasingly important role in shaping Earth's coastal systems. Coastal scientists have only a general understanding of how these three factors--humans, storms, and sea-level rise--will alter the evolution of coastlines over the coming century, however. I conduct numerical modeling experiments to shed light on the relative importance of these factors on the evolution of coastline geomorphology.

In a series of experiments using a numerical model of large-scale (1 to 100's km) and long-term (years to centuries) coastline evolution that results from gradients in alongshore sediment transport, I explore how the patterns and rates of shoreline erosion and accretion are affected by shifts in 'wave climate' (the mix of influences on alongshore sediment transport of waves approaching from different directions) induced by intensified storm systems and the direct manipulation of the shoreline system by humans through beach nourishment (periodically placing sand on an eroding beach). I use a cuspate-cape coastline, similar to the Outer Banks, North and South Carolina, USA, as an important case study in my experiments. I observe that moderate shifts in the wave climate can alter the patterns of shoreline erosion and accretion, potentially increasing migration rates by several times that which we see today, and nearly an order-of-magnitude larger than sea-level rise-related erosion alone. I also find that under possible wave climate futures, beach nourishment may also induce shoreline change on the same order of magnitude as does sea-level rise.

The decision humans make whether or not to nourish their beach often depends upon a favorable economic outcome in the endeavor. In further experiments, I couple a cost-benefit economic model of human decision making to the numerical model of coastline evolution and test a hypothetical scenario where two communities (one 'rich' and one 'poor') nourish their beaches in tandem, under different sets of economic and wave climate parameters. I observe that two adjacent communities can benefit substantially from each other's nourishment activity, and these effects persist even if the two communities are separated by several tens of kilometers.

In a separate effort, I employ techniques from dynamic capital theory coupled to a physically-realistic model of coastline evolution to find the optimum time a community should wait between beach nourishment episodes ('rotation length') to maximize the utility to beach-front property owners. In a series of experiments, I explore the sensitivity of the rotation length to economic parameters, including the discount rate, the fixed and variable costs of beach nourishment, and the benefits from beach nourishment, and physical parameters including the background erosion rate and the exponential rate at which both the cross-shore profile and the plan-view coastline shape re-adjusts following a beach nourishment episode ('decay rate' of nourishment sand). Some results I obtained were expected: if property values, the hedonic value of beach width, the baseline retreat rate, the fixed cost of beach nourishment, and the discount rate increase, then the rotation length of nourishment decreases. Some results I obtained, however, were unexpected: the rotation length of nourishment can either increase or decrease when the decay rate of nourishment sand varies versus the discount rate and when the variable costs of beach nourishment increase.

The development of a functional tissue-engineered human skeletal muscle model in vitro would provide an excellent platform on which to study the process of myogenesis, various musculoskeletal disease states, and drugs and therapies for muscle toxicity. We developed a protocol to culture human skeletal muscle bundles in a fibrin hydrogel under static conditions capable of exerting active contractions. Additionally, we demonstrated the use of joint miR-133a and miR-696 inhibition for acceleration of muscle differentiation, elevation of active contractile force amplitudes, and increasing Type II myofiber formation in vitro.

The global hypothesis that motivated this research was that joint inhibition of miR-133a and miR-696 in isolated primary human skeletal myoblasts would lead to accelerated differentiation of tissue-engineered muscle constructs with higher proportion of Type I myofibers and that are capable of significantly increased active contractile forces when subjected to electrical stimulus. The proposed research tested the following specific hypotheses: (1) that HSkM would require different culture conditions than those optimal for C2C12 culture (8% equine serum in differentiation medium on uncoated substrates), as measured by miR expression, (2) that joint inhibition of miR-133a and miR-696 would result in 2D human skeletal muscle cultures with accelerated differentiation and increased Type I muscle fibers compared to control and individual inhibition of each miR, as measured by protein and gene expression, (3) that joint inhibition of miR-133a and miR-696 in this functional 3D human skeletal muscle model would result in active contraction significantly higher than control and individual inhibition by each miR, as measured by isometric force testing, and finally (4) that specific co-culture conditions could support a lamellar co-culture model in 3D of human cord blood-derived endothelial cells (hCB-ECs) and HSkM capable of active contraction, as measured by isometric force testing and immunofluorescence.

Major results of the dissertation are as follows. Culture conditions of 100 μg/mL growth factor reduced-Matrigel-coated substrates and 2% equine serum in differentiation medium were identified to improve human skeletal myoblast culture, compared to conditions optimal for C2C12 cell culture (uncoated substrates and 8% equine serum media). Liposomal transfection of human skeletal myoblasts with anti-miR-133a and anti-miR-696 led to increased protein presence of sarcomeric alpha-actinin and PGC-1alpha when cells were cultured in 2D for 2 weeks. Presence of mitochondria and distribution of fiber type did not change with miR transfection in a 2D culture. Joint inhibition also resulted in increased PPARGC1A gene expression after 2 weeks of 2D culture. For muscle bundles in 3D, results suggest there exists a myoblast seeding density threshold for the production of functional muscle. 5 x 106 myoblasts/mL did not produce active contraction, while 10 x 106 myoblasts/mL and above were successful. Of the seeding densities studied, 15 x 106 myoblasts/mL resulted in constructs that exerted the highest twitch and tetanus forces. Engineering of human skeletal muscle from transfected cells led to significant increases in force amplitude in joint inhibition compared to negative control (transfection with scrambled miR sequence). Joint inhibition in myoblasts seeded into 3D constructs led to decreased presence of slow myosin heavy chain and increased fast myosin heavy chain. Finally, co-culture of functional human skeletal muscle with human cord blood-derived endothelial cells is possible in 3.3% FBS in DMEM culture conditions, with significant increases in force amplitudes at 48 and 96 hours of co-culture.