Serum anti-flagellin and anti-lipopolysaccharide immunoglobulins as predictors of linear growth faltering in Pakistani infants at risk for environmental enteric dysfunction

Background: Environmental Enteric Dysfunction (EED) in children from low-income countries has been linked to linear growth declines. There is a critical need to identify sensitive and early EED biomarkers.Objective: Determine whether levels of antibodies against bacterial components flagellin (flic) and lipopolysaccharide (LPS) predict poor growth.Design/Methods: In a prospective birth cohort of 380 children in rural Pakistan blood and stool samples were obtained at ages 6 and 9 months. Linear mixed effects models were used to examine longitudinal associations between quartiles of anti-flic and anti-LPS antibodies and changes in LAZ, WAZ and WLZ scores. Spearman\u27s correlations were measured between anti-flic and anti-LPS immunoglobulins with measures of systemic/enteric inflammation and intestinal regeneration.Results: Anti-LPS IgA correlated significantly with CRP, AGP and Reg1 serum at 6mo and with MPO at 9mo. In multivariate analysis at 6mo of age, higher anti-LPS IgA levels predicted greater declines in LAZ scores over subsequent 18mo (comparing highest to lowest quartile, β (SE) change in LAZ score/year = -0.313 (0.125), p-value = 0.013). Anti-flic Ig A in the two highest quartiles measured at 9mo of age had declines in LAZ of -0.269 (0.126), p = 0.033; and -0.306 (0.129), p = 0.018 respectively, during the subsequent 18mo of life, compared to those in the lowest quartile of anti-flic IgA.Conclusions and Relevance: Elevated anti-flic IgA and anti-LPS IgA antibodies at 6 and 9mo, predict declines in linear growth. Systemic and enteric inflammation correlated with anti-LPS IgA provides mechanistic considerations for potential future interventions

Morphological adaptations in breast cancer cells as a function of prolonged passaging on compliant substrates

<div><p>Standard tissue culture practices involve propagating cells on tissue culture polystyrene (TCP) dishes, which are flat, 2-dimensional (2D) and orders of magnitude stiffer than most tissues in the body. Such simplified conditions lead to phenotypical cell changes and altered cell behaviors. Hence, much research has been focused on developing novel biomaterials and culture conditions that more closely emulate <i>in vivo</i> cell microenvironments. In particular, biomaterial stiffness has emerged as a key property that greatly affects cell behaviors such as adhesion, morphology, proliferation and motility among others. Here we ask whether cells that have been conditioned to TCP, would still show significant dependence on substrate stiffness if they are first pre-adapted to a more physiologically relevant environment. We used two commonly utilized breast cancer cell lines, namely MDA-MB-231 and MCF-7, and examined the effect of prolonged cell culturing on polyacrylamide substrates of varying compliance. We followed changes in cell adhesion, proliferation, shape factor, spreading area and spreading rate. After pre-adaptation, we noted diminished differences in cell behaviors when comparing between soft (1 kPa) and stiff (103 kPa) gels as well as rigid TCP control. Prolonged culturing of cells on complaint substrates further influenced responses of pre-adapted cells when transferred back to TCP. Our results have implications for the study of stiffness-dependent cell behaviors and indicate that cell pre-adaptation to the substrate needs consideration.</p></div

Histograms of MDA-MB-231 cell spreading area and circularity expressed as percent occurrence.

<p>Spreading area and circularity were measured at 72 h of each passage (P1, P2 and P3). (A) Percent occurrence of cell spreading area and circularity on TCP. (B) Percent occurrence of cell spreading area and (C) circularity on 103 kPa gels. (D) Percent occurrence of cell spreading area and (E) circularity on 1 kPa gels.</p

Cell spreading area of MDA-MB-231 cells.

<p>Cell spreading area was measured after cells were transferred onto TCP from 3 difference’ conditions: TCP control (TCP→TCP), P3 on 103 kPa gel (103→TCP), and P3 on 1 kPa gel (1→TCP). *represent significant difference from 24 h, ^#represents significant difference from TCP→TCP (p<0.05, n = 3).</p

Live imaging of MCF-7 cells on TCP and 1 kPa PA gels immediately post-seeding on the substrate.

<p>(A) Cells were harvested from TCP and seeded onto TCP. (B) Cells were harvested from TCP and seeded onto a 1 kPa gel. (C) Pre-adapted cells at P3 were harvested from a 1 kPa gel and seeded onto a 1 kPa gel. Scale bar is 100 μm (<b>A-C</b>). (D) Spreading area for cells seeded onto 1 kPa gel, where cells were either harvested from TCP (TCP→1) or pre-adapted on a 1 kPa gel for 3 passages (1→1). Cells harvested from TCP and seeded onto TCP were used as control (TCP→TCP). (E) Cell spreading rate calculated as per <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187853#pone.0187853.e005" target="_blank">Eq 5</a>. *represent significant difference between all conditions (p<0.05, n = 3).</p

Cell spreading area and circularity of MCF-7A cells as a function of adaptation to substrate stiffness.

<p>Cells were seeded onto the PA substrates for 9 consecutive days without re-passaging. (A) Representative phase contrast images of cells on TCP and 1 kPa PA gels. Scale bar is 100 μm. (B) Cell spreading area and (C) circularity of cells on TCP and 1 kPa PA gels as a function of culture time.</p

MDA-MB-231 cells on PA gels of varying stiffness.

<p>(A) Representative phase contrast cell images taken at 24 h post-seeding. Scale bar is 50 μm. (B) Cell spreading area and (C) circularity at 24 h post-seeding. The dashed line represents values on TCP. *^,#,$ represent significant difference from each other, where same symbol represents no difference (p<0.05, n = 3).</p

Live imaging of MDA-MB-231 cells on TCP, 1 kPa and 103 kPa PA gels immediately post-seeding on the substrate.

<p>(A) Cells were harvested from TCP and seeded onto TCP. (B) Cells were harvested from TCP and seeded onto a 103 kPa gel. (C) Pre-adapted cells at P3 were harvested from a 103 kPa gel and seeded onto a 103 kPa gel. (D) Cells were harvested from TCP and seeded onto a 1 kPa gel. (E) Pre-adapted cells at P3 were harvested from a 1 kPa gel and seeded onto a 1 kPa gel. Scale bar is 100 μm.</p

Cell proliferation of MDA-MB-231 cells.

<p>Cell proliferation was measured after cells were transferred onto TCP from 3 difference conditions: TCP control (TCP→TCP), P3 on 103 kPa gel (103→TCP), and P3 on 1 kPa gel (1→TCP). *represent significant difference from 103→TCP (p<0.05, n = 3).</p

Composition and Young’s moduli of polyacrylamide gels.

<p>Composition and Young’s moduli of polyacrylamide gels.</p