Durability of Vacuum Infusion Tooling Produced from Fused Granular Fabrication Additive Manufacturing.

Fused Granular Fabrication Additive Manufacturing (FGF AM) has the capability to create tooling that is lower cost than conventionally manufactured tooling and still has sufficient properties for many applications. A vacuum infusion (VI) mold was printed from fiberglass-acrylonitrile butadiene styrene (ABS) and evaluated for wear and suitability for small VI runs. The mold was designed to accentuate high wear as a "worst case" scenario. The mold was able to produce 10 parts successfully before any noticeable change occurred to the surface finish. By 14 parts, the surface finish had roughened sufficiently that demolding was difficult and resulted in damage to the part. Profilometry measurements showed a 7 × increase in roughness over the run. No significant tool wear or change in geometry was detected. Even longer life would be expected for typical tooling designs since the test mold was deliberately designed to accentuate wear and demolding issues. Based on these results, similar FGF molds are a feasible option for short run VI production for prototyping or low-volume composites manufacturing, possibly at lower cost and quicker turnaround time than machined aluminum molds.

"Intermission!" A short-term social media fast reduces self-objectification among pre-teen and teen dancers.

Social media use is pervasive among youth and is associated with body image disturbance and self-objectification. The present study investigated whether a 3-day social media fast in a sample for whom social media is especially salient, female adolescent dancers, can mitigate such negative effects. Through an online survey, 65 pre-teen and teen girls, aged 10-19, completed measures of self-objectification (body surveillance and body shame), self-esteem and self-compassion both prior to and following three days of abstaining from all social media. During the fast, girls reflected on their experiences in group messages on the messaging app, WhatsApp. Overall, the fast had positive effects on participants, for whom body surveillance and body shame was significantly reduced after the fast. Self-compassion significantly mediated the change in both body surveillance and body shame, and self-esteem was a significant mediator of improvements in body shame. The content of girls' group messages revealed a number of themes, such as more positive mental states during the fast. Future research should continue to examine the potential of brief social media fasts as a means to alleviate appearance pressures adolescent girls face on these platforms in daily life.

Interface Joint Strength between SS316L Wrought Substrate and Powder Bed Fusion Built Parts.

Metal powder bed fusion (PBF) additive manufacturing (AM) builds metal parts layer by layer upon a substrate material. The strength of this interface between the substrate and the printed material is important to characterize, especially in applications where the substrate is retained and included in the finished part. Ensuring that this interface between the original and the printed material has adequate material properties enables the use of this PBF AM process to repair existing structures and create new parts using both AM and conventional manufacturing. This paper studies the tensile and torsional shear strengths of wrought and PBF-built SS316L specimens and compares them to specimens that are composed of half wrought material and half PBF material. These specimens were created by building new material via PBF onto existing wrought SS316L blocks, then cutting the specimens to include both materials. The specimens are also examined using optical microscopy and electron backscatter diffraction (EBSD). The PBF specimens consistently exhibited higher strength and lower ductility than the wrought specimens. The hybrid PBF/wrought specimens performed similarly to the wrought material. In none of the specimens did any failure appear to occur at or near the interface between the wrought substrate and the PBF material. In addition, most of the deformation in the PBF/wrought specimens appeared to be limited to the wrought portion of the specimens. These results are consistent with optical microscopy and EBSD showing smaller grain size in the PBF material, which correlates to increased strength in SS316L due to the Hall-Petch relationship. With the strength at the interface meeting or exceeding the strength of the original wrought material, this process shows great promise as a method for adding additional features or repairing existing structures using metal PBF AM.

Cytokine diversity in the Th1-dominated human anti-influenza response caused by variable cytokine expression by Th1 cells, and a minor population of uncommitted IL-2+IFNγ- Thpp cells.

Within overall Th1-like human memory T cell responses, individual T cells may express only some of the characteristic Th1 cytokines when reactivated. In the Th1-oriented memory response to influenza, we have tested the contributions of two potential mechanisms for this diversity: variable expression of cytokines by a uniform population during activation, or different stable subsets that consistently expressed subsets of the Th1 cytokine pattern. To test for short-term variability, in vitro-stimulated influenza-specific human memory CD4+ T cells were sorted according to IL-2 and IFNγ expression, cultured briefly in vitro, and cytokine patterns measured after restimulation. Cells that were initially IFNγ+ and either IL-2+ or IL-2- converged rapidly, containing similar proportions of IL-2-IFNγ+ and IL-2+IFNγ+ cells after culture and restimulation. Both phenotypes expressed Tbet, and similar patterns of mRNA. Thus variability of IL-2 expression in IFNγ+ cells appeared to be regulated more by short-term variability than by stable differentiated subsets. In contrast, heterogeneous expression of IFNγ in IL-2+ influenza-specific T cells appeared to be due partly to stable T cell subsets. After sorting, culture and restimulation, influenza-specific IL-2+IFNγ- and IL-2+IFNγ+ cells maintained significantly biased ratios of IFNγ+ and IFNγ- cells. IL-2+IFNγ- cells included both Tbetlo and Tbethi cells, and showed more mRNA expression differences with either of the IFNγ+ populations. To test whether IL-2+IFNγ-Tbetlo cells were Thpp cells (primed but uncommitted memory cells, predominant in responses to protein vaccines), influenza-specific IL-2+IFNγ- and IL-2+IFNγ+ T cells were sorted and cultured in Th1- or Th2-generating conditions. Both cell types yielded IFNγ-secreting cells in Th1 conditions, but only IL-2+IFNγ- cells were able to differentiate into IL-4-producing cells. Thus expression of IL-2 in the anti-influenza response may be regulated mainly by short term variability, whereas different T cell subsets, Th1 and Thpp, may contribute to variability in IFNγ expression.

SWIFT-scalable clustering for automated identification of rare cell populations in large, high-dimensional flow cytometry datasets, part 2: biological evaluation.

A multistage clustering and data processing method, SWIFT (detailed in a companion manuscript), has been developed to detect rare subpopulations in large, high-dimensional flow cytometry datasets. An iterative sampling procedure initially fits the data to multidimensional Gaussian distributions, then splitting and merging stages use a criterion of unimodality to optimize the detection of rare subpopulations, to converge on a consistent cluster number, and to describe non-Gaussian distributions. Probabilistic assignment of cells to clusters, visualization, and manipulation of clusters by their cluster medians, facilitate application of expert knowledge using standard flow cytometry programs. The dual problems of rigorously comparing similar complex samples, and enumerating absent or very rare cell subpopulations in negative controls, were solved by assigning cells in multiple samples to a cluster template derived from a single or combined sample. Comparison of antigen-stimulated and control human peripheral blood cell samples demonstrated that SWIFT could identify biologically significant subpopulations, such as rare cytokine-producing influenza-specific T cells. A sensitivity of better than one part per million was attained in very large samples. Results were highly consistent on biological replicates, yet the analysis was sensitive enough to show that multiple samples from the same subject were more similar than samples from different subjects. A companion manuscript (Part 1) details the algorithmic development of SWIFT.

Increase in IFNγ(-)IL-2(+) cells in recent human CD4 T cell responses to 2009 pandemic H1N1 influenza.

Human CD4 T cell recall responses to influenza virus are strongly biased towards Type 1 cytokines, producing IFNγ, IL-2 and TNFα. We have now examined the effector phenotypes of CD4 T cells in more detail, particularly focusing on differences between recent versus long-term, multiply-boosted responses. Peptides spanning the proteome of temporally distinct influenza viruses were distributed into pools enriched for cross-reactivity to different influenza strains, and used to stimulate antigen-specific CD4 T cells representing recent or long-term memory. In the general population, peptides unique to the long-circulating influenza A/New Caledonia/20/99 (H1N1) induced Th1-like responses biased toward the expression of IFNγ(+)TNFα(+) CD4 T cells. In contrast, peptide pools enriched for non-cross-reactive peptides of the pandemic influenza A/California/04/09 (H1N1) induced more IFNγ(-)IL-2(+)TNFα(+) T cells, similar to the IFNγ(-)IL-2(+) non-polarized, primed precursor T cells (Thpp) that are a predominant response to protein vaccination. These results were confirmed in a second study that compared samples taken before the 2009 pandemic to samples taken one month after PCR-confirmed A/California/04/09 infection. There were striking increases in influenza-specific TNFα(+), IFNγ(+), and IL-2(+) cells in the post-infection samples. Importantly, peptides enriched for non-cross-reactive A/California/04/09 specificities induced a higher proportion of Thpp-like IFNγ(-)IL-2(+)TNFα(+) CD4 T cells than peptide pools cross-reactive with previous influenza strains, which induced more Th1 (IFNγ(+)TNFα(+)) responses. These IFNγ(-)IL-2(+)TNFα(+) CD4 T cells may be an important target population for vaccination regimens, as these cells are induced upon infection, may have high proliferative potential, and may play a role in providing future effector cells during subsequent infections.

Abortive activation of CD4 T cell responses during competitive priming in vivo.

Immunodominance refers to the highly selective peptide reactivity of T cells during an immune response. In this study, we tested the hypothesis that persistence of peptide:class II complexes is one key parameter that selects the final specificity of CD4 T cells. We found that low-stability peptide:class II complexes support the initial priming and expansion of CD4 T cells, but the expansion becomes strikingly aborted in the presence of competitive T cell responses to unrelated peptides. Our experiments revealed that for inhibition to occur, the competitive responses must be initiated by the same antigen presenting cell, and it is not because of competition for MHC binding. These studies not only provide an insight into the events that regulate competitive CD4 T cell priming in vivo, but also provide a previously undescribed conceptual framework to understand the parameters that select the final specificity of the T cell repertoire during pathogen or vaccine-induced immune responses.

Understanding the focused CD4 T cell response to antigen and pathogenic organisms.

Immunodominance is a term that reflects the final, very limited peptide specificity of T cells that are elicited during an immune response. Recent experiments in our laboratory compel us to propose a new paradigm for the control of immunodominance in CD4 T cell responses, stating that immunodominance is peptide-intrinsic and is dictated by the off-rate of peptides from MHC class II molecules. Our studies have revealed that persistence of peptide:class II complexes both predicts and controls CD4 T cell immunodominance and that this parameter can be rationally manipulated to either promote or eliminate immune responses. Mechanistically, we have determined that DM editing in APC is a key event that is influenced by the kinetic stability of class II:peptide complexes and that differential persistence of complexes also impacts the expansion phase of the immune response. These studies have important implications for rational vaccine design and for understanding the immunological mechanisms that limit the specificity of CD4 T cell responses.

Immunodominance of CD4 T cells to foreign antigens is peptide intrinsic and independent of molecular context: implications for vaccine design.

Immunodominance refers to the restricted peptide specificity of T cells that are detectable after an adaptive immune response. For CD4 T cells, many of the mechanisms used to explain this selectivity suggest that events related to Ag processing play a major role in determining a peptide's ability to recruit CD4 T cells. Implicit in these models is the prediction that the molecular context in which an antigenic peptide is contained will impact significantly on its immunodominance. In this study, we present evidence that the selectivity of CD4 T cell responses to peptides contained within protein Ags is not detectably influenced by the location of the peptide in a given protein or the primary sequence of the protein that bears the test peptide. We have used molecular approaches to change the location of peptides within complex protein Ags and to change the flanking sequences that border the peptide epitope to now include a protease site, and find that immunodominance or crypticity of a peptide observed in its native protein context is preserved. Collectively, these results suggest immunodominance of peptides contained in complex Ags is due to an intrinsic factor of the peptide, based upon the affinity of that peptide for MHC class II molecules. These findings are discussed with regard to implications for vaccine design.

Immunodominance in CD4 T-cell responses: implications for immune responses to influenza virus and for vaccine design.

CD4 T cells play a primary role in regulating immune responses to pathogenic organisms and to vaccines. Antigen-specific CD4 T cells provide cognate help to B cells, a requisite event for immunoglobulin switch and affinity maturation of B cells that produce neutralizing antibodies and also provide help to cytotoxic CD8 T cells, critical for their expansion and persistence as memory cells. Finally, CD4 T cells may participate directly in pathogen clearance via cell-mediated cytotoxicity or through production of cytokines. Understanding the role of CD4 T-cell immunity to viruses and other pathogens, as well as evaluation of the efficacy of vaccines, requires insight into the specificity of CD4 T cells. This review focuses on the events within antigen-presenting cells that focus CD4 T cells toward a limited number of peptide antigens within the pathogen or vaccine. The molecular events are discussed in light of the special challenges that the influenza virus poses, owing to the high degree of genetic variability, unpredictable pathogenicity and the repeated encounters that human populations face with this highly infectious pathogenic organism.

Effects of receptor activator of NFkappaB (RANK) signaling blockade on fracture healing.

As dominant regulators of osteoclastogenesis and bone resorption, receptor activator of NFkappaB (RANK), receptor activator of NFkappaB ligand, and OPG have been identified as ideal drug targets for the treatment of metabolic bone disease. One concern regarding the therapeutic use of RANK signaling inhibitors is their effect on fracture healing. Therefore we tested if uncoupling and osteoclast depletion via RANK blockade affects callus formation, maturation and matrix remodeling, as well as union rates in a mouse tibia fracture model. Low dose (1 mg/kg i.p.) RANK:Fc therapy had no effect on callus formation, matrix maturation and remodeling, and resulted in 100% bony union by day 28. High dose RANK:Fc treatment (10 mg/kg i.p.) effectively eliminated osteoclasts at the fracture site on day 14, with no significant effects on fracture healing. When therapy was discontinued, normal numbers of osteoclasts were observed at the fracture site by day 28. However, continuous therapy resulted in a large osteopetrotic callus consisting of both mineralized and unmineralized matrix that was void of osteoclasts, but bony union was unaffected at day 28. We also evaluated this process in the complete absence of RANK signaling using RANK -/- mice. These animals exhibited significant radiographic and histologic evidence of callus formation, indicating that RANK signaling is not required for fracture callus formation. However, only 33% of RANK -/- animals formed bony unions compared to 100% of the osteopetrotic control mice. This defect was most likely a result of decreased blood flow, as evidenced by fewer blood vessels in the RANK -/- animals. Together, these data imply that osteoclast depletion via inhibition of RANK signaling is a viable option for the treatment of pathological bone loss since no adverse effects on fracture healing are observed when therapy is discontinued.