Plantar fascia segmentation and thickness estimation in ultrasound images

Ultrasound (US) imaging offers significant potential in diagnosis of plantar fascia (PF) injury and monitoring treatment. In particular US imaging has been shown to be reliable in foot and ankle assessment and offers a real-time effective imaging technique that is able to reliably confirm structural changes, such as thickening, and identify changes in the internal echo structure associated with diseased or damaged tissue. Despite the advantages of US imaging, images are difficult to interpret during medical assessment. This is partly due to the size and position of the PF in relation to the adjacent tissues. It is therefore a requirement to devise a system that allows better and easier interpretation of PF ultrasound images during diagnosis. This study proposes an automatic segmentation approach which for the first time extracts ultrasound data to estimate size across three sections of the PF (rearfoot, midfoot and forefoot). This segmentation method uses artificial neural network module (ANN) in order to classify small overlapping patches as belonging or not-belonging to the region of interest (ROI) of the PF tissue. Features ranking and selection techniques were performed as a post-processing step for features extraction to reduce the dimension and number of the extracted features. The trained ANN classifies the image overlapping patches into PF and non-PF tissue, and then it is used to segment the desired PF region. The PF thickness was calculated using two different methods: distance transformation and area-length calculation algorithms. This new approach is capable of accurately segmenting the PF region, differentiating it from surrounding tissues and estimating its thickness

Identification of an Arginine-Rich Motif in Human Papillomavirus Type 1 E1^E4 Protein Necessary for E4-Mediated Inhibition of Cellular DNA Synthesis In Vitro and in Cells ▿

Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G2-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1^E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification

ICOS expression by activated human Th cells is enhanced by IL-12 and IL-23: Increased ICOS expression enhances the effector function of both Th1 and Th2 cells

Previous mouse studies have shown that IL-4 increases the expression of ICOS on activated Th cells, resulting in enhanced ICOS expression on Th2 cells. In this study, we show that ICOS expression on human Th cells is not increased by IL-4, but by IL-12 and by IL-23 instead. Consequently, ICOS expression during IL-12-driven Th1 cell polarization was transiently increased compared with the levels on Th0 cells and IL-4-driven Th2 cells. Addition of IL-12 and/or IL-23 during restimulation increased ICOS expression to the same extent on pre-established Th1, Th2, and Th0 cells, indicating that ICOS levels are not stably imposed by prior polarization. In contrast to the findings in the mouse, IL-4 significantly suppressed the ICOS-enhancing effects of IL-12 and IL-23. The functional, consequence of variable ICOS levels was shown in coculture experiments with cells expressing the ICOS-ligand B7-related protein 1 (either transfected Chinese hamster ovary cells or autologous dendritic cells). Ligation of ICOS on 2-day-preactivated effector cells increased their cytokine production to an extent proportional to their ICOS expression levels. As the ICOS-enhancing potentials of IL-12 and IL-23 were maintained for several days after stimulation, both on Th1 and Th2 cells, we propose the concept that local regulation of ICOS expression on activated Th cells by IL-12 and/or IL-23 may provide a powerful means to amplify effector T cell responses in peripheral tissues, independently of the polarized state of the Th cell

Continuous IL-23 stimulation drives ILC3 depletion in the upper GI tract and, in combination with TNFα, induces robust activation and a phenotypic switch of ILC3

<div><p>Mutations in the Interleukin (IL)-23/IL-23 receptor loci are associated with increased inflammatory bowel disease (IBD) susceptibility, and IL-23 neutralization has shown efficacy in early clinical trials. To better understand how an excess of IL-23 affects the gastrointestinal tract, we investigated chronic systemic IL-23 exposure in healthy wildtype mice. As expected, IL-23 exposure resulted in early activation of intestinal type 3 innate lymphoid cells (ILC3), followed by infiltration of activated RORγt+ T helper cells. Surprisingly, however, sustained IL-23 stimulus also dramatically reduced classical ILC3 populations within the proximal small intestine, and a phenotypically distinct T-bet expressing ILC3 population emerged. TNFα neutralization, a widely used IBD therapy, reduced several aspects of the IL-23 driven ILC3 response, suggesting a synergy between IL-23 and TNFα in ILC3 activation. <i>In vitro</i> studies supported these findings, revealing previously unappreciated effects of IL-23 and TNFα within the intestine.</p></div

Both CCR6+ and NCR+ ILC3 subsets are reduced following IL-23 stimulation, and remaining ILC3 are phenotypically distinct.

<p>Mice were injected with sham mc (open symbols) or IL-23 mc (filled symbols), and flow cytometry was performed on LP cells from the proximal SI. (A) Representative staining shows the frequency of ILC3 amongst total ILC at 3 days and 2 weeks post mc injection, with compiled geometric MFI of RORγt expression by ILC3 shown. (B) Representative staining shows the frequency of ILC3 subsets amongst total ILC3 at 3 days (top) and 2 weeks (bottom) post mc injection with compiled absolute numbers. (C) Representative staining shows the frequency of IL-23 mc elicited ILC3 (ILC3²³; IL-7R- T-bet+) amongst CCR6- NCR- ILC3 at 3 days post mc injection, with compiled absolute numbers of these cells shown at 3 days and 2 weeks post mc injection. (D) Representative staining shows the frequency of Ki67+ cells amongst ILC3 subsets, with compiled data at 3 days post mc injection. Scatter plots show means ± SEM for all mice from one of at least 3 similar experiments, 4–5 mice per group, with each symbol representative of a single mouse.</p

ILC3 are rapidly activated and depleted from the proximal small intestine of IL-23 mc injected mice.

<p>Mice were injected with sham mc (open symbols) or IL-23 mc (filled symbols), and flow cytometry was performed on LP cells from the proximal SI. (A) The absolute number of ILC3 (defined as CD45+ CD90+ CD11b- CD11c- F4/80- Gr-1- Ter119- B220- CD3e- RORγt+ cells; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182841#pone.0182841.s001" target="_blank">S1B Fig</a> for typical gating) is shown. (B) Top: representative staining shows protein expression at 24 and 48 hours post mc injection. Bottom: compiled staining intensities are depicted as geometric MFI at 72 hours post mc injection. (C) Representative staining shows cytokine expression three days and 2 weeks post mc injection (left) along with compiled data (right). (D) Expression of cell death associated receptors by ILC3 is shown three days post mc injection. Representative and compiled staining intensities are shown. Scatter plots show means ± SEM for all mice from one of 2–4 similar experiments, 4–5 mice per group, with each symbol representative of a single mouse.</p

TNFα promotes cytokine production by ILC3 and ILC3²³ formation <i>in vivo</i>, and synergizes with IL-23 to directly activate ILC3 <i>in vitro</i>.

<p>(A-C) Mice were injected with sham mc (open symbols) or IL-23 mc (filled symbols), with some groups receiving anti-TNFα (or PBS) as indicated. (A) SI length 2 weeks post mc injection (left), and cytokine secretion from SI explants harvested 3 days post mc injection (right) are shown. (B) Flow cytometry was performed on LP cells from the proximal SI three days post mc injection. Absolute numbers of cytokine producing lymphocytes are shown. (C) Flow cytometry was performed on LP cells from the proximal SI 2 weeks post mc injection. Top: absolute numbers are shown. Bottom: representative staining shows the frequency of ILC3²³ amongst CCR6- NCR- ILC3. (D-E) FACS purified ILCs enriched for ILC3 (~90% RORγt+) were sorted from the proximal SI LP of RAG KO mice and cultured overnight with the indicated cytokines prior to flow cytometry. (D) Protein expression by ILC3 is shown for triplicate wells with staining intensity depicted as geometric MFI (top), and representative IL-7R and T-bet staining is shown for total ILC3 (bottom). (E) Cytokine expression by ILC3 is shown for triplicate wells treated with protein inhibitors for the last 3 hours of culture. Scatter plots show means ± SEM for all mice from one of 2–3 similar experiments, 4–5 mice per group, with each symbol representative of a single mouse. Bar graphs show means ± SEM for triplicate wells from one of two similar experiments.</p

Chronic IL-23 elevation results in intestinal inflammation within the upper GI tract of wildtype B6 mice.

<p>Mice were injected with sham mc (open symbols) or IL-23 mc (filled symbols). (A) IHC (top) and H+E staining (bottom) of duodenal tissue. For IHC, CD3e+ staining (T cells) is black, and IBA-1 staining (macrophages) is red. (B) SI length 2 weeks post mc injection. (B-C) Flow cytometry was performed on LP cells from the proximal SI and the absolute numbers of (B) hematopoietic and (C) cytokine producing ILC3 and RORγt+ T cells were quantified (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182841#pone.0182841.s001" target="_blank">S1B Fig</a> for typical gating strategy). Scatter plots show means ± SEM for all mice from one of 2–4 similar experiments, 4–5 mice per group, with each symbol representative of a single mouse.</p

ICOS-mediated signaling regulates cytokine production by human T cells and provides a unique signal to selectively control the clonal expansion of Th2 helper cells

The CD28 homologue inducible costimulator (ICOS) has been demonstrated to regulate a number of T cell-dependent immune responses in vivo. However, the expression and functional importance of ICOS during APC-Th cell interaction in the human is not fully understood. Here, we demonstrate that ICOS-mediated signaling plays an important role in the production of selective cytokines during both primary and subsequent Th cell responses upon allospecific or superantigen activation. In contrast, ICOS does not play a role in the differentiation of naive cells into Th1 or Th2 effector cells, nor does it determine the type of effector function of memory cells upon subsequent allogeneic challenge. In addition, our data demonstrate that ICOS provides a novel and unique role in regulating DC-mediated Th2, but not Th1 cell clonal expansion. These data suggest that ICOS-mediated signaling plays a discrete role in the regulation of human T helper cell response