Alley Cropping: An Agroforestry Practice

Alley cropping is an agroforestry practice intended to place trees within agricultural cropland systems. The purpose is to enhance or add income diversity (both long and short range), reduce wind and water erosion, improve crop production, improve utilization of nutrients, improve wildlife habitat or aesthetics, and/or convert cropland to forest. The practice is especially attractive to landowners wishing to add economic stability to their farming system while protecting soil from erosion, water from contamination, and improving wildlife habitat

Alley Cropping: An Agroforestry Practice

Alley cropping is an agroforestry practice intended to place trees within agricultural cropland systems. The purpose is to enhance or add income diversity (both long and short range), reduce wind and water erosion, improve crop production, improve utilization of nutrients, improve wildlife habitat or aesthetics, and/or convert cropland to forest. The practice is especially attractive to landowners wishing to add economic stability to their farming system while protecting soil from erosion, water from contamination, and improving wildlife habitat

Outdoor Living Barn: A Specialized Windbreak

In April of 1987, a spring blizzard swept through northern Kansas and southern Nebraska killing nearly 60,000 newborn calves and other winter stressed animals. This tremendous loss could have been lessened had protection, such as outdoor living barns (OLB), been provided to reduce the windchill. An outdoor living barn is a specialized windbreak, typically composed of trees and shrubs, and strategically located in open grasslands, center pivot irrigation corners, and pasture areas to protect livestock during severe weather situations. The purpose of an OLB is to: 1) defuse and deflect cold winds away from livestock, moderating the windchill; and 2) trap and hold blowing snow, preventing it from covering feed, water, and livestock. Outdoor living barns pay for themselves by cutting livestock losses, lowering feed costs, and sustaining animal health during stressful weather conditions

Extracellular DNA release induced by <i>M</i>. <i>abscessus</i>.

<p>Micrococcal nuclease-releasable extracellular DNA was quantified in <b>(A)</b> non-stimulated neutrophils (NS) and in neutrophils stimulated with <b>(B)</b> smooth (Sm) and <b>(C)</b> rough (R) <i>M</i>. <i>abscessus</i> for the indicted times. Neutrophils were pre-incubated with DPI (<i>red</i>), cytochalasin D (<i>blue)</i>, <i>and</i> DNase (<i>green</i>), as indicated. Data was analyzed by two-way ANOVA and Bonferroni post test versus the control (untreated) condition; n = 5; *p<0.05, and ***p<0.001 for all time points; †p<0.001 at 4h only. <b>(D-F</b>) NET formation by <i>M</i>. <i>abscessus</i> morphotypes. Neutrophils were left non-stimulated (<b>D</b>) or exposed to smooth (<b>E</b>) and rough (<b>F</b>) <i>M</i>. <i>abscessus</i> in suspension for 1h followed by plating on glass slides for an additional 2h. Sytox Red was added for 10 min, and cells were fixed and permeabilized. Slides were immunostained for elastase (<i>green</i>) and visualized under a 40X objective. Colocalized staining of extracellular elastase and extracellular DNA is observed in yellow.</p

Reactive oxygen species (ROS) generated by neutrophils exposed to <i>M</i>. <i>abscessus</i>.

<p><b>(A)</b> Intracellular ROS production is enhanced in the presence of smooth (<i>circles</i>) and rough (<i>squares</i>) <i>M</i>. <i>abscessus</i>; <i>small diamonds</i>, non-stimulated neutrophils; n = 8. <b>(B)</b> Areas-under-the-curve (AUC) were calculated for each condition in <b>A</b>; data analyzed by t-test; *p<0.05. Areas-under-the-curve were calculated for intracellular ROS curves of <i>M</i>. <i>abscessus</i>-infected neutrophils treated with <b>C)</b> cytochalasin D (<i>hatched</i>), and <b>D)</b> DPI (<i>hatched</i>); data analyzed by t-test; n = 3–9; *p<0.05, †p<0.001.</p

Intra- and extracellular neutrophil killing of <i>M</i>. <i>abscessus</i>.

<p>Neutrophils (n = 7) were pre-incubated with DNase (100 units/ml) or DPI (10 μM), or left untreated (C). Neutrophils were exposed to <b>(A)</b> smooth (n = 6) or <b>(B)</b> rough <i>M</i>. <i>abscessus</i> (n = 7) for 1h, and surviving mycobacteria were compared to the initial infection. <b>(C)</b> Neutrophils (n = 7) were treated with cytochalasin D (cytoD; 5 μg/ml) or left untreated (C) and killing of smooth (<i>closed bars</i>) and rough (<i>open bars</i>) <i>M</i>. <i>abscessus</i> was assessed after 1h. Panels A-C were analyzed by t-test; *P<0.05. <b>(D)</b> Targeting of <i>M</i>. <i>abscessus</i>, without regard for morphotype, by cell-free conditioned media (CM) from neutrophils treated with smooth <i>M</i>. <i>abscessus</i> (Sm) or rough <i>M</i>. <i>abscessus</i> (R), and represents composite data from <b>E</b> and <b>F</b>. Data analysis by paired t-test; n = 14. Morphotype-specific targeting of <b>(E)</b> smooth or <b>(F)</b> rough <i>M</i>. <i>abscessus</i> by conditioned media (CM) from neutrophils treated with smooth <i>M</i>. <i>abscessus</i> (Sm), rough <i>M</i>. <i>abscessus</i> (R), or peptidoglycan (PGN); conditioned media were incubated at 95°C, as indicated, before killing assays. Killing of each morphotype was normalized to that of <i>M</i>. <i>abscessus</i> exposed to supernatants from untreated neutrophils. Data were analyzed by two-way ANOVA; n = 7; an interaction (P = 0.01) was found for heat-treatment of CM for targeting rough <i>M</i>. <i>abscessus</i> (F), but not smooth (E).</p

Neutrophil killing of <i>M</i>. <i>abscessus</i>.

<p><b>(A)</b> Neutrophils were incubated with smooth (<i>closed circles</i>) and rough (<i>open circles</i>) <i>M</i>. <i>abscessus</i> for 1h, and surviving mycobacteria were compared to the inoculum. Connecting lines represent results from the same donor neutrophils; n = 26. Mean values are depicted by the gray bars. <b>(B)</b> Time course of killing of <i>M</i>. <i>abscessus</i> morphotypes by neutrophils; n = 6. <b>(C)</b> <i>M</i>. <i>abscessus</i> clinical isolates were assayed for killing by neutrophils for 1 h; smooth morphotypes (<i>closed bars</i>); rough morphotypes (<i>open bars</i>); n = 4–10. None of the differences were significantly different for the Fig 1 data by paired t-test.</p

Phagocytosis of <i>M</i>. <i>abscessus</i> by neutrophils.

<p><b>(A-C)</b> FITC-<i>M</i>. <i>abscessus</i> (<i>green</i>) was incubated with neutrophils for 1 h, and cells were cytocentrifuged on to glass slides, stained for elastase (<i>red</i>), and visualized by confocal microcopy. <b>(A)</b> Non-stimulated neutrophils, or after incubation with <b>(B)</b> smooth, and <b>(C)</b> rough FITC-<i>M</i>. <i>abscessus</i>. <b>(D)</b> Representative flow cytometry plots of isolated non-stimulated neutrophils showing forward scatter (FSC) and side-scatter (SSC) of non-stimulated neutrophils, and AlexaFluor-450-conjugated CD16. <b>(E)</b> Flow cytometric analysis of FITC-Mab association with neutrophils at 5 and 60 min; trypan blue was included to quench extracellular fluorescence. Examples of smooth (Sm-Mab; upper panels) and rough (R-Mab; lower panels) <i>M</i>. <i>abscessus</i> uptake are shown. <b>(F)</b> Time-dependent uptake of FITC-Mab. Neutrophils were incubated for the indicated time with smooth FITC-Mab (<i>left</i>) and rough FITC-Mab (<i>right</i>). Data represents the percentage of neutrophils positive for FITC for 3 independent experiments.</p

Extracellular DNA release induced by <i>M</i>. <i>abscessus</i>.

<p>Micrococcal nuclease-releasable extracellular DNA was quantified in <b>(A)</b> non-stimulated neutrophils (NS) and in neutrophils stimulated with <b>(B)</b> smooth (Sm) and <b>(C)</b> rough (R) <i>M</i>. <i>abscessus</i> for the indicted times. Neutrophils were pre-incubated with DPI (<i>red</i>), cytochalasin D (<i>blue)</i>, <i>and</i> DNase (<i>green</i>), as indicated. Data was analyzed by two-way ANOVA and Bonferroni post test versus the control (untreated) condition; n = 5; *p<0.05, and ***p<0.001 for all time points; †p<0.001 at 4h only. <b>(D-F</b>) NET formation by <i>M</i>. <i>abscessus</i> morphotypes. Neutrophils were left non-stimulated (<b>D</b>) or exposed to smooth (<b>E</b>) and rough (<b>F</b>) <i>M</i>. <i>abscessus</i> in suspension for 1h followed by plating on glass slides for an additional 2h. Sytox Red was added for 10 min, and cells were fixed and permeabilized. Slides were immunostained for elastase (<i>green</i>) and visualized under a 40X objective. Colocalized staining of extracellular elastase and extracellular DNA is observed in yellow.</p

Mycobacterium abscessus induces a limited pattern of neutrophil activation that promotes pathogen survival.

Mycobacterium abscessus is a rapidly growing mycobacterium increasingly detected in the neutrophil-rich environment of inflamed tissues, including the cystic fibrosis airway. Studies of the immune reaction to M. abscessus have focused primarily on macrophages and epithelial cells, but little is known regarding the neutrophil response despite the predominantly neutrophillic inflammation typical of these infections. In the current study, human neutrophils released less superoxide anion in response to M. abscessus than to Staphylococcus aureus, a pathogen that shares common sites of infection. Exposure to M. abscessus induced neutrophil-specific chemokine and proinflammatory cytokine genes. Although secretion of these protein products was confirmed, the quantity of cytokines released, and both the number and level of gene induction, was reduced compared to S. aureus. Neutrophils mediated killing of M. abscessus, but phagocytosis was reduced when compared to S. aureus, and extracellular DNA was detected in response to both bacteria, consistent with extracellular trap formation. In addition, M. abscessus did not alter cell death compared to unstimulated cells, while S. aureus enhanced necrosis and inhibited apoptosis. However, neutrophils augment M. abscessus biofilm formation. The response of neutrophils to M. abscessus suggests that the mycobacterium exploits neutrophil-rich settings to promote its survival and that the overall neutrophil response was reduced compared to S. aureus. These studies add to our understanding of M. abscessus virulence and suggest potential targets of therapy