AN EVALUATION OF RANDOMIZED ROUTING STRATEGIES FOR DECEPTION IN MOBILE NETWORKED CONTROL SYSTEMS

Networked unmanned autonomous systems will increasingly be employed to support ground force operations. Approaches to collaborative control can find near-optimal position recommendations that optimize over system parameters such as sensing and communication to increase mission effectiveness. However, over time these recommendations can create predictable paths that may provide leading indications of the force’s operational intent. We assume that the adversary’s goal is to identify a ground force’s operational intent. Using randomized routing strategies to generate deception plans for unmanned systems against the adversary, this red methodology has the potential to change many aspects of military operational planning, including operational and strategic level planning and wargaming. This topic builds on research from L. Wigington in 2021, which developed an adversarial assessment of unmanned mobile networked control systems. From that and based on prior research, this thesis applies and potentially extends prior methodologies to analyzing adversarial behaviors and manipulating their behaviors to NCS using randomized routing strategies.Lieutenant, United States NavyApproved for public release. Distribution is unlimited

The uptake of soluble and particulate antigens by epithelial cells in the mouse small intestine.

Intestinal epithelial cells (IECs) overlying the villi play a prominent role in absorption of digested nutrients and establish a barrier that separates the internal milieu from potentially harmful microbial antigens. Several mechanisms by which antigens of dietary and microbial origin enter the body have been identified; however whether IECs play a role in antigen uptake is not known. Using in vivo imaging of the mouse small intestine, we investigated whether epithelial cells (enterocytes) play an active role in the uptake (sampling) of lumen antigens. We found that small molecular weight antigens such as chicken ovalbumin, dextran, and bacterial LPS enter the lamina propria, the loose connective tissue which lies beneath the epithelium via goblet cell associated passageways. However, epithelial cells overlying the villi can internalize particulate antigens such as bacterial cell debris and inert nanoparticles (NPs), which are then found co-localizing with the CD11c+ dendritic cells in the lamina propria. The extent of NP uptake by IECs depends on their size: 20-40 nm NPs are taken up readily, while NPs larger than 100 nm are taken up mainly by the epithelial cells overlying Peyer's patches. Blocking NPs with small proteins or conjugating them with ovalbumin does not inhibit their uptake. However, the uptake of 40 nm NPs can be inhibited when they are administered with an endocytosis inhibitor (chlorpromazine). Delineating the mechanisms of antigen uptake in the gut is essential for understanding how tolerance and immunity to lumen antigens are generated, and for the development of mucosal vaccines and therapies

The presence of NPs in the IECs isolated from the mouse SI.

<p>40(red) were injected into the lumen of the SI and 30 minutes later the SI was excised, Peyer's patches were removed (discarded), and IECs were isolated from the SI sections. (A–D) Isolated IECs from mice that were administered NPs (A, B) or PBS (C, D) were fixed then placed on a glass slide and imaged with a fluorescent microscope at 630× magnification. (A, B) A patch of IECs isolated from NP-treated mouse imaged in the green channel (autofluorescence) (A) and the red channel (red: NPs) (B). Characteristic GAPs that are not highlighted by NPs appear as black holes in isolated IEC patches (white arrows), while IECs exhibit strong red fluorescence due to the presence of NPs (similar to images taken in vivo). (C, D) No red fluorescence was detected in IEC patches isolated from a control mouse. (E) Expression of E-cadherin (green) in isolated IECs imaged with a fluorescence microscope. (F) A confocal image of IECs isolated from NP-treated mouse showing strong red fluorescence in IEC cytoplasm. (G) Distribution of E-cadherin (green) in a section of SI. Actin staining with phalloidin-Alexa 350 (blue) highlights the tissue architecture. (H) Western blot analysis of E-cadherin (120 kDa) expression in isolated IECs (Lane 1) or spleen lymphocytes (Control, Lane 3). Lane 2: Spectra™ multicolor protein ladder. Each image is a representative of at least 3 experiments.</p

Distribution of NPs in the SI and MLNs of mice 30–40 minutes after administration in the SI.

<p>(A) A three color IFM image of a villus showing co-localization of NPs (red) with CD11c+ DCs (yellow) in the LP. Actin highlighted by phalloidin-Alexa 350 (blue). (B) A three color IFM image of a MLN 40 minutes after NP administration into the SI. NPs (red) seen in the capsule of the MLN. The lymphatics of MLN stained with Lyve-1 antibodies (green). Actin is highlighted by phalloidin-Alexa 350 (blue). (C) A higher resolution three color image of MLN capsule taken at 630×. Some NPs (red) appear to be cell-bound (arrows). (D) A three color IFM image of an intestinal section. NPs (red) colocalize with the lymphatics (Lyve-1 staining, green, circled) in the submucosa and highlight the serosa (white arrow). Actin is highlighted by phalloidin-Alexa 350 (blue). (E) A confocal image of the SI serosa taken in vivo 40 minutes after NP (red) and dextran-fluorescein (green) administration. (F) A confocal image of the SI serosa taken in vivo 40 minutes after administration of dextran-fluorescein (green, control). Each image is a representative of at least 3 experiments.</p

Inhibition of 40 nm NP uptake by CPZ leads to decreased concentration of NPs in the MLNs.

<p>NPs with or without CPZ were injected in the lumen of SI and 40 minutes later MLNs were snap-frozen. Tissue sections of MLNs from CPZ-treated and control mice were stained with phalloidin-Alexa 350 and imaged at 630×. The amount of NPs in MLN sections of CPZ-treated and control mice was quantified using Volocity software. Regions of MLNs with highest NP concentration from control and CPZ-treated mice (A–C) were analyzed separately from regions of MLN capsules from control and CPZ-treated mice (D–F). (A) The amount of NPs (red pixels per image) was significantly higher in control mice compared to CPZ-treated mice (p<0.05). (B, C) Stitched images of MLN regions with high NP concentration from tissues of control mice (B) and CPZ-treated mice (C). Eight images taken at 630× were stitched together to show large sections of MLNs. Insets: magnified representative images in which clumps of NPs and individual NPs can be visualized. (D) The amount of NPs (red pixels per image) was significantly higher in capsules of control mice compared to CPZ-treated mice (p<0.05). (E, F) Representative images of NP distribution in MLN capsules of control (E) and CPZ-treated mice (F). Data are representative of 3 experiments (6 mice). Group means were separated using Student's t-test and were considered significantly different at P<0.05. Data are expressed as mean ± SD of the mean.</p

Localization of 40 nm NPs in the circulation of the villi 30 minutes after administration in the SI lumen in vivo.

<p>To highlight the vasculature and lymph ducts of the villi, dextran (green) was injected in the tail vein while NPs (red) and DAPI (blue) were injected into the lumen of the SI. Thirty minutes later the SI was imaged with a confocal microscope and sequential Z-stacks were acquired. (A) A Z-stack image taken at the tip of the villi: nuclei of the IECs above the LP stained with DAPI (blue) and lumen (asterisks) stained with dextran (green) that has leaked from circulation. Inset: a magnified image showing the proximity of NPs to the IEC nuclei. (B) A Z-stack of the same villi taken at 70 µm depth showing NPs (red) that have accumulated in the LP and IEC nuclei stained with DAPI (blue) surrounding the LP. (C) Three color Z-stack shown in B in which NPs are seen co-localizing with vasculature of the villi (arrows). The images are representatives of at least 3 experiments.</p

Administration of CPZ inhibits the uptake of 40 nm NPs but does not affect the uptake of dextran via GAPs.

<p>(A) Green channel of a confocal image of SI villi taken in vivo showing the entry of dextran (green) into the LP via GAPs (arrows, inset) in CPZ-treated mouse SI. (B) The internalization of 40 nm NPs (red channel) is inhibited by CPZ, thus red fluorescence was detected only in the lumen of the SI (asterisk). (C–E) A representative IFM image of the villi from tissue sections of mice administered CPZ and lysine-fixable dextran (red). Goblet cells (GAPs) were stained with cytokeratin 18 (Cy-18) antibody (green). (C) Two color image showing actin staining (blue) and goblet cell staining (green). (D) A two color image showing the entry of dextran (red) via GAPs. (E) Overlap of image C and D showing co-localization of red dextran with Cy-18 positive GAPs (green). (F) Administration of CPZ does not alter the number of Cy-18+ cells in the villi. There were no differences in Cy-18+ cells present in the villi between CPZ-treated and control mice (p<0.05). (G) Administration of CPZ did not alter the entry of dextran into the LP via GAPs. There were no differences in the number of Cy-18+ GAPs co-localizing with dextran between CPZ-treated and control mice (p<0.05). Group means were separated using Student's t-test and were considered significantly different at P<0.05. Data (bars) are expressed as mean ± SD of the mean. In total over 200 villi and over 600 GAPs were counted per animal and per treatment group (+/− CPZ). For each treatment group 3 mice were used. Data are representative of 3 experiments.</p

Routes of uptake (entry) of soluble and particulate antigens in the small intestine (SI) of mice.

<p>Abbreviations: PO: Per-oral; IL: Intraluminal (injected in the lumen of the SI); N/E: Not evaluated. GAPs (Goblet Cell Associated Passageways); IECs (Intestinal Epithelial Cells).</p