cd /home/daytona/workspace/attachments/48ed36fc-7412-4bc7-acbe-faa6b6fa02af/ && python3 -c " import pdfplumber files = [ 'ji2014.pdf', 'Bacterial invasion of epithelial cells and spreading in periodontal tissue - PMC.pdf' ] for f in files: print('='*80) print(f'FILE: {f}') print('='*80) with pdfplumber.open(f) as pdf: for i, page in enumerate(pdf.pages): text = page.extract_text() if text: print(f'--- Page {i+1} ---') print(text) " 2>&1 | head -600

cd /home/daytona/workspace/attachments/48ed36fc-7412-4bc7-acbe-faa6b6fa02af/ && python3 -c " import pdfplumber with pdfplumber.open('Bacterial invasion of epithelial cells and spreading in periodontal tissue - PMC.pdf') as pdf: for i, page in enumerate(pdf.pages): text = page.extract_text() if text: print(f'--- Page {i+1} ---') print(text) " 2>&1 | head -800

mkdir -p /home/daytona/workspace/perio-invasion && python3 << 'PYEOF' import matplotlib matplotlib.use('Agg') import matplotlib.pyplot as plt import matplotlib.patches as mpatches from matplotlib.patches import FancyArrowPatch, FancyBboxPatch import numpy as np # ───────────────────────────────────────────────────────────────── # FIGURE 1 – Overall Pathogenesis Model Flowchart # ───────────────────────────────────────────────────────────────── fig, ax = plt.subplots(figsize=(14, 20)) ax.set_xlim(0, 10) ax.set_ylim(0, 22) ax.axis('off') ax.set_facecolor('#f8f9fa') fig.patch.set_facecolor('#f8f9fa') def box(ax, x, y, w, h, text, color='#2c7bb6', textcolor='white', fontsize=9.5, style='round,pad=0.1'): bbox = FancyBboxPatch((x - w/2, y - h/2), w, h, boxstyle=style, linewidth=1.5, edgecolor='#1a1a2e', facecolor=color, zorder=3) ax.add_patch(bbox) ax.text(x, y, text, ha='center', va='center', fontsize=fontsize, color=textcolor, fontweight='bold', wrap=True, multialignment='center', zorder=4) def arrow(ax, x1, y1, x2, y2, color='#1a1a2e'): ax.annotate('', xy=(x2, y2), xytext=(x1, y1), arrowprops=dict(arrowstyle='->', color=color, lw=2), zorder=5) # Title ax.text(5, 21.5, 'PROPOSED MODEL FOR THE PATHOGENESIS OF PERIODONTITIS', ha='center', va='center', fontsize=12, fontweight='bold', color='#1a1a2e') ax.text(5, 21.0, '(Ji et al., 2014; Tribble & Lamont, 2010)', ha='center', va='center', fontsize=9, color='#555555', style='italic') # Boxes: (x, y, w, h, text, color) steps = [ (5, 20.0, 8.5, 0.7, 'CLINICALLY HEALTHY GINGIVAL SULCUS\n(~10³ bacteria, early colonizers, homeostasis maintained)', '#27ae60'), (5, 18.8, 8.5, 0.7, 'PLAQUE ACCUMULATION → SHIFT TO GRAM-NEGATIVE ANAEROBES\n(orange complex bridging colonizers → red complex late colonizers)', '#e67e22'), (5, 17.6, 8.5, 0.7, 'RED COMPLEX PATHOGENS COLONIZE\n(P. gingivalis, T. forsythia, T. denticola — "keystone pathogens")', '#c0392b'), (5, 16.4, 8.5, 0.7, 'PATHOGENS IMPAIR EPITHELIAL BARRIERS\n(disrupt tight junctions, degrade E-cadherin, ZO-1, occludin via gingipains)', '#8e44ad'), (5, 15.2, 8.5, 0.7, 'BACTERIAL INVASION OF GINGIVAL EPITHELIAL CELLS\n(FimA–β1 integrin, gingipains, SerB phosphatase, actin remodeling)', '#2980b9'), (5, 14.0, 8.5, 0.7, 'INTRACELLULAR SURVIVAL & REPLICATION\n(P. gingivalis escapes lysosomes → perinuclear area; resists phagocytosis)', '#16a085'), (5, 12.8, 8.5, 0.7, 'SUBVERSION OF INNATE IMMUNITY\n(IL-8 suppression, IL-12 inhibition, TLR2 subversion via CXCR4/CR3/C5aR)', '#d35400'), (5, 11.6, 8.5, 0.7, 'CELL-TO-CELL SPREAD & TISSUE INVASION\n(actin-based membranous projections → penetrate basement membrane → connective tissue)', '#1a5276'), (5, 10.4, 8.5, 0.7, 'BACTERIAL PERSISTENCE WITHIN HOST TISSUE\n(chronic intracellular reservoir; resistant to antibiotics & scaling)', '#7b241c'), (5, 9.2, 8.5, 0.7, 'INFLAMMATORY INFILTRATION\n(T cells, B cells, plasma cells, macrophages recruited; Th1/Th2 imbalance)', '#6c3483'), (5, 8.0, 8.5, 0.7, 'OSTEOCLASTOGENIC ACTIVATION\n(IL-17, RANKL upregulation → osteoclast differentiation)', '#b7950b'), (5, 6.8, 8.5, 0.7, 'TISSUE DESTRUCTION & ALVEOLAR BONE LOSS\n(collagen/elastin degraded; periodontal ligament destroyed → tooth loss)', '#922b21'), ] for (x, y, w, h, text, color) in steps: box(ax, x, y, w, h, text, color=color) # Arrows between consecutive boxes ys = [s[1] for s in steps] for i in range(len(ys) - 1): arrow(ax, 5, ys[i] - 0.35, 5, ys[i+1] + 0.35) # Risk factor side boxes rf_data = [ (1.2, 13.0, 2.2, 1.4, 'RISK FACTORS\n(Immunologic)\n• Neutropenia\n• Chediak-Higashi\n• Papillon-Lefèvre\n• Leukocyte adhesion\n deficiency', '#1a5276'), (8.8, 13.0, 2.2, 1.4, 'RISK FACTORS\n(Environmental)\n• Smoking (cotinine\n increases invasion)\n• Alcohol (disrupts\n tight junctions)\n• Poor oral hygiene', '#1a5276'), ] for (x, y, w, h, text, color) in rf_data: box(ax, x, y, w, h, text, color=color, fontsize=7.5) # arrow to main path if x < 5: ax.annotate('', xy=(1.35, 15.2), xytext=(2.3, 13.7), arrowprops=dict(arrowstyle='->', color='#1a5276', lw=1.5, linestyle='dashed')) else: ax.annotate('', xy=(8.65, 15.2), xytext=(7.7, 13.7), arrowprops=dict(arrowstyle='->', color='#1a5276', lw=1.5, linestyle='dashed')) plt.tight_layout() plt.savefig('/home/daytona/workspace/perio-invasion/fig1_pathogenesis_flowchart.png', dpi=180, bbox_inches='tight') plt.close() print("Fig 1 saved") PYEOF

python3 << 'PYEOF' import matplotlib matplotlib.use('Agg') import matplotlib.pyplot as plt import matplotlib.patches as mpatches from matplotlib.patches import FancyBboxPatch import numpy as np # ───────────────────────────────────────────────────────────────── # FIGURE 2 – P. gingivalis Invasion Mechanism (Molecular Detail) # ───────────────────────────────────────────────────────────────── fig, ax = plt.subplots(figsize=(16, 12)) ax.set_xlim(0, 16) ax.set_ylim(0, 12) ax.axis('off') fig.patch.set_facecolor('#ffffff') def rbox(ax, x, y, w, h, text, fc='#2980b9', ec='#1a252f', fs=8.5, tc='white'): p = FancyBboxPatch((x-w/2, y-h/2), w, h, boxstyle='round,pad=0.15', fc=fc, ec=ec, lw=1.5, zorder=3) ax.add_patch(p) ax.text(x, y, text, ha='center', va='center', fontsize=fs, color=tc, fontweight='bold', multialignment='center', zorder=4) def arr(ax, x1, y1, x2, y2, label='', lw=1.8, color='#2c3e50', style='->', ls='-'): ax.annotate('', xy=(x2, y2), xytext=(x1, y1), arrowprops=dict(arrowstyle=style, color=color, lw=lw, linestyle=ls), zorder=5) if label: mx, my = (x1+x2)/2, (y1+y2)/2 ax.text(mx+0.1, my, label, fontsize=7, color=color, style='italic') # Title ax.text(8, 11.6, 'MOLECULAR MECHANISM OF P. gingivalis INVASION INTO GINGIVAL EPITHELIAL CELLS', ha='center', va='center', fontsize=11, fontweight='bold') # --- Bacteria (top left) --- bact = FancyBboxPatch((0.5, 8.5), 3.5, 2.2, boxstyle='round,pad=0.2', fc='#e74c3c', ec='#922b21', lw=2, zorder=3) ax.add_patch(bact) ax.text(2.25, 9.9, 'P. gingivalis', ha='center', fontsize=10, fontweight='bold', color='white', zorder=4) ax.text(2.25, 9.45, '• Major fimbriae (FimA)', ha='center', fontsize=8, color='white', zorder=4) ax.text(2.25, 9.05, '• Gingipains (RgpA, RgpB, Kgp)', ha='center', fontsize=8, color='white', zorder=4) ax.text(2.25, 8.65, '• SerB serine phosphatase', ha='center', fontsize=8, color='white', zorder=4) # --- Host cell membrane --- membrane = plt.Rectangle((0, 6.5), 15.5, 0.3, fc='#f0d060', ec='#b8a000', lw=2, zorder=2) ax.add_patch(membrane) ax.text(7.75, 6.65, 'GINGIVAL EPITHELIAL CELL MEMBRANE', ha='center', fontsize=9, fontweight='bold', color='#5a4000', zorder=4) # Receptor boxes on membrane rbox(ax, 4.0, 7.2, 2.8, 0.8, 'β1 Integrin receptor\n(αvβ3 / α5β1)', fc='#27ae60') rbox(ax, 8.0, 7.2, 2.5, 0.8, 'CXCR4 / CR3\n(complement receptors)', fc='#8e44ad') rbox(ax, 12.0, 7.2, 2.8, 0.8, 'TLR2 (Toll-like\nreceptor 2)', fc='#e67e22') # Step boxes inside cell rbox(ax, 3.0, 5.5, 4.0, 0.8, 'FAK + Paxillin recruited →\nRho GTPase (Rac1) activation', fc='#2980b9') rbox(ax, 8.5, 5.5, 3.5, 0.8, 'PKA signaling pathway\n(cAMP-dependent)', fc='#8e44ad') rbox(ax, 13.0, 5.5, 3.5, 0.8, 'NF-κB INHIBITION\n(via SerB → IκB preservation)', fc='#e67e22') rbox(ax, 5.0, 4.2, 5.5, 0.8, 'ACTIN + MICROTUBULE REMODELING\n(cofilin activation by SerB; Ca²⁺ released)', fc='#16a085') rbox(ax, 11.5, 4.2, 3.5, 0.8, 'IL-8 SUPPRESSION\n(chemokine paralysis)', fc='#c0392b') rbox(ax, 5.0, 3.0, 5.5, 0.8, 'BACTERIAL INTERNALIZATION\n(~15 min; lipid raft-mediated endocytosis)', fc='#1a5276') rbox(ax, 5.0, 1.9, 5.5, 0.8, 'ESCAPE FROM ENDOSOME → FREE IN CYTOPLASM\n(Clp serine protease prevents lysosome fusion)', fc='#7b241c') rbox(ax, 5.0, 0.8, 5.5, 0.8, 'PERINUCLEAR LOCALIZATION & REPLICATION\n(~4 h replication; endoplasmic reticulum utilized as nutrient source)', fc='#1a5276') # Arrows arr(ax, 2.25, 8.5, 3.8, 7.6, 'FimA binds β1 integrin', color='#27ae60') arr(ax, 2.25, 8.5, 7.8, 7.6, 'Fimbriae to CXCR4/CR3', color='#8e44ad') arr(ax, 2.25, 8.5, 11.9, 7.6, 'Gingipains activate C5a→C5aR', color='#e67e22') arr(ax, 4.0, 6.8, 3.2, 5.9) arr(ax, 8.0, 6.8, 8.4, 5.9) arr(ax, 12.0, 6.8, 12.9, 5.9) arr(ax, 3.2, 5.1, 4.8, 4.6) arr(ax, 8.5, 5.1, 6.5, 4.6) arr(ax, 8.5, 5.1, 11.4, 4.6) arr(ax, 5.0, 3.8, 5.0, 3.4) arr(ax, 5.0, 2.6, 5.0, 2.3) arr(ax, 5.0, 1.5, 5.0, 1.2) plt.tight_layout() plt.savefig('/home/daytona/workspace/perio-invasion/fig2_invasion_mechanism.png', dpi=180, bbox_inches='tight') plt.close() print("Fig 2 saved") PYEOF

python3 << 'PYEOF' import matplotlib matplotlib.use('Agg') import matplotlib.pyplot as plt from matplotlib.patches import FancyBboxPatch import matplotlib.patches as mpatches # ───────────────────────────────────────────────────────────────── # FIGURE 3 – Intracellular Survival Strategies & Spreading # ───────────────────────────────────────────────────────────────── fig, ax = plt.subplots(figsize=(16, 11)) ax.set_xlim(0, 16) ax.set_ylim(0, 11) ax.axis('off') fig.patch.set_facecolor('#fafafa') def rbox(ax, x, y, w, h, text, fc='#2980b9', ec='#1a252f', fs=8.5, tc='white'): p = FancyBboxPatch((x-w/2, y-h/2), w, h, boxstyle='round,pad=0.15', fc=fc, ec=ec, lw=1.5, zorder=3) ax.add_patch(p) ax.text(x, y, text, ha='center', va='center', fontsize=fs, color=tc, fontweight='bold', multialignment='center', zorder=4) def arr(ax, x1, y1, x2, y2, color='#2c3e50', label='', lw=1.8, ls='-'): ax.annotate('', xy=(x2, y2), xytext=(x1, y1), arrowprops=dict(arrowstyle='->', color=color, lw=lw, linestyle=ls), zorder=5) if label: mx, my = (x1+x2)/2+0.05, (y1+y2)/2 ax.text(mx, my, label, fontsize=7, color=color, style='italic') ax.text(8, 10.6, 'INTRACELLULAR SURVIVAL, PERSISTENCE AND SPREADING OF P. gingivalis', ha='center', fontsize=11, fontweight='bold') # Left column – SURVIVAL ax.text(3.5, 9.8, 'INTRACELLULAR SURVIVAL', ha='center', fontsize=9.5, fontweight='bold', color='#1a5276', bbox=dict(boxstyle='round', fc='#d6eaf8', ec='#1a5276')) rbox(ax, 3.5, 8.9, 6.5, 0.8, 'Bacteria enter endosome after internalization', fc='#2980b9') rbox(ax, 3.5, 7.9, 6.5, 0.8, 'Clp serine protease prevents lysosomal fusion\n(endoplasmic reticulum association)', fc='#16a085') rbox(ax, 3.5, 6.9, 6.5, 0.8, 'Escape to cytoplasm → free perinuclear replication', fc='#1a5276') rbox(ax, 3.5, 5.9, 6.5, 0.8, 'Downregulate FimA, gingipains, hemin-uptake systems\n(proteomic reprogramming: ~50% of proteome altered)', fc='#117a65') rbox(ax, 3.5, 4.9, 6.5, 0.8, 'Upregulate stress proteins: Clp, HtrA, peroxidases\n(adaptation to intracellular oxidative environment)', fc='#16a085') rbox(ax, 3.5, 3.9, 6.5, 0.8, 'Metabolic shift: asparagine→ATP pathway ↑\nPropionate production ↑ (less apoptotic than butyrate)', fc='#1a5276') rbox(ax, 3.5, 2.9, 6.5, 0.8, 'Anti-apoptotic program: Bcl-2 ↑, survivin ↑\nPI3K/Akt + JAK/STAT pathways activated\nATP hydrolysis → inhibits P2X₇ receptor apoptosis', fc='#8e44ad') rbox(ax, 3.5, 1.9, 6.5, 0.8, 'Cell cycle acceleration (S-phase progression ↑)\n→ maintains reservoir of infected cells', fc='#1a5276') # Right column – SPREADING ax.text(12.0, 9.8, 'CELL-TO-CELL SPREADING & TISSUE INVASION', ha='center', fontsize=9.5, fontweight='bold', color='#7b241c', bbox=dict(boxstyle='round', fc='#f9ebea', ec='#7b241c')) rbox(ax, 12.0, 8.9, 7.0, 0.8, 'After ~24 h intracellular residence:\nTransmission initiated', fc='#c0392b') rbox(ax, 12.0, 7.9, 7.0, 0.8, 'Route 1: Actin-based membranous projections\n(stealth spreading; host cell remains viable)', fc='#922b21') rbox(ax, 12.0, 6.9, 7.0, 0.8, 'Route 2: Endocytic recycling pathway\n(Rab11 + RalA recycling endosomes → exocytosis → re-invasion)', fc='#7b241c') rbox(ax, 12.0, 5.9, 7.0, 0.8, 'P. gingivalis spreads through upper epithelial layers\n(3-D organotypic model evidence)', fc='#c0392b') rbox(ax, 12.0, 4.9, 7.0, 0.8, 'Gingipains degrade basement membrane components:\nType IV collagen, laminin, fibronectin', fc='#922b21') rbox(ax, 12.0, 3.9, 7.0, 0.8, 'Penetration through basement membrane\n→ invasion of connective tissue', fc='#7b241c') rbox(ax, 12.0, 2.9, 7.0, 0.8, 'Cooperative invasion with F. nucleatum\n(T. forsythia, P. aeruginosa also facilitated)', fc='#c0392b') rbox(ax, 12.0, 1.9, 7.0, 0.8, 'Access to alveolar bone environment\n→ osteoclast activation → BONE LOSS', fc='#922b21') # Arrows within columns ys_l = [8.9, 7.9, 6.9, 5.9, 4.9, 3.9, 2.9] for i in range(len(ys_l)-1): arr(ax, 3.5, ys_l[i]-0.4, 3.5, ys_l[i+1]+0.4) ys_r = [8.9, 7.9, 6.9, 5.9, 4.9, 3.9, 2.9] for i in range(len(ys_r)-1): arr(ax, 12.0, ys_r[i]-0.4, 12.0, ys_r[i+1]+0.4, color='#7b241c') # Bridge arrow arr(ax, 6.8, 4.0, 8.5, 4.9, color='#555555', label='bacteria exit\nhost cell', ls='--') plt.tight_layout() plt.savefig('/home/daytona/workspace/perio-invasion/fig3_survival_spreading.png', dpi=180, bbox_inches='tight') plt.close() print("Fig 3 saved") PYEOF

python3 << 'PYEOF' import matplotlib matplotlib.use('Agg') import matplotlib.pyplot as plt from matplotlib.patches import FancyBboxPatch import matplotlib.patches as mpatches # ───────────────────────────────────────────────────────────────── # FIGURE 4 – Risk Factors Impairing Epithelial Barriers # ───────────────────────────────────────────────────────────────── fig, ax = plt.subplots(figsize=(16, 10)) ax.set_xlim(0, 16) ax.set_ylim(0, 10) ax.axis('off') fig.patch.set_facecolor('#fefefe') def rbox(ax, x, y, w, h, text, fc='#2980b9', ec='#1a252f', fs=8.5, tc='white'): p = FancyBboxPatch((x-w/2, y-h/2), w, h, boxstyle='round,pad=0.12', fc=fc, ec=ec, lw=1.5, zorder=3) ax.add_patch(p) ax.text(x, y, text, ha='center', va='center', fontsize=fs, color=tc, fontweight='bold', multialignment='center', zorder=4) def arr(ax, x1, y1, x2, y2, color='#2c3e50', lw=1.8): ax.annotate('', xy=(x2, y2), xytext=(x1, y1), arrowprops=dict(arrowstyle='->', color=color, lw=lw), zorder=5) ax.text(8, 9.6, 'RISK FACTORS IMPAIRING EPITHELIAL BARRIERS IN PERIODONTITIS', ha='center', fontsize=11.5, fontweight='bold') # Three barrier types # ── Immunologic barrier (left) ax.text(2.8, 8.9, 'IMMUNOLOGIC BARRIER', ha='center', fontsize=10, fontweight='bold', color='white', bbox=dict(boxstyle='round', fc='#1a5276', ec='#0d2e4a', pad=0.4)) rbox(ax, 2.8, 8.1, 5.2, 0.65, 'Neutrophils = primary immunologic barrier\n(form wall between plaque & epithelium)', fc='#1a5276', fs=8) rbox(ax, 2.8, 7.2, 5.2, 0.65, 'Agranulocytosis / Neutropenia\n(Kostmann syndrome — 1st reported genetic cause)', fc='#2980b9', fs=8) rbox(ax, 2.8, 6.3, 5.2, 0.65, 'Chediak-Higashi syndrome\n(lysosomal trafficking regulator mutation)', fc='#2471a3', fs=8) rbox(ax, 2.8, 5.4, 5.2, 0.65, 'Papillon-Lefèvre syndrome\n(cathepsin C mutation → defective granule serine proteases)', fc='#1a5276', fs=8) rbox(ax, 2.8, 4.5, 5.2, 0.65, 'Leukocyte adhesion deficiency\n(β2-integrin / fMLP receptor mutation)', fc='#2980b9', fs=8) arr(ax, 2.8, 7.8, 2.8, 7.5) arr(ax, 2.8, 6.9, 2.8, 6.6) arr(ax, 2.8, 6.0, 2.8, 5.7) arr(ax, 2.8, 5.1, 2.8, 4.8) # ── Chemical barrier (middle) ax.text(8.0, 8.9, 'CHEMICAL BARRIER', ha='center', fontsize=10, fontweight='bold', color='white', bbox=dict(boxstyle='round', fc='#1e8449', ec='#0b5729', pad=0.4)) rbox(ax, 8.0, 8.1, 5.2, 0.65, 'Antimicrobial peptides (AMPs): α-defensins,\nβ-defensins (HBD-1/2/3), LL-37', fc='#1e8449', fs=8) rbox(ax, 8.0, 7.2, 5.2, 0.65, 'Kostmann syndrome — absolute neutropenia corrected\nbut LL-37 absent in saliva & neutrophils → severe periodontitis', fc='#27ae60', fs=8) rbox(ax, 8.0, 6.3, 5.2, 0.65, 'LL-37 increases cell stiffness, decreases\ntransepithelial permeability, prevents invasion', fc='#1e8449', fs=8) rbox(ax, 8.0, 5.4, 5.2, 0.65, 'Breach of chemical barrier → reduced LL-37\n→ facilitated bacterial invasion', fc='#27ae60', fs=8) arr(ax, 8.0, 7.8, 8.0, 7.5) arr(ax, 8.0, 6.9, 8.0, 6.6) arr(ax, 8.0, 6.0, 8.0, 5.7) # ── Physical barrier (right) ax.text(13.2, 8.9, 'PHYSICAL BARRIER', ha='center', fontsize=10, fontweight='bold', color='white', bbox=dict(boxstyle='round', fc='#922b21', ec='#6b1f19', pad=0.4)) rbox(ax, 13.2, 8.1, 5.4, 0.65, 'Tight junctions (ZO-1, occludin, JAM-A),\nadherens junctions (E-cadherin), β1-integrin', fc='#922b21', fs=8) rbox(ax, 13.2, 7.2, 5.4, 0.65, 'P. gingivalis gingipains degrade E-cadherin,\nZO-1, occludin → barrier disruption', fc='#c0392b', fs=8) rbox(ax, 13.2, 6.3, 5.4, 0.65, 'T. denticola chymotrypsin-like protease degrades\nfibronectin → increased epithelial permeability', fc='#922b21', fs=8) rbox(ax, 13.2, 5.4, 5.4, 0.65, 'SMOKING: cotinine ↑ bacterial invasion;\ncytoskeletal/tight junction disruption in vivo & in vitro', fc='#c0392b', fs=8) rbox(ax, 13.2, 4.5, 5.4, 0.65, 'ALCOHOL: TGF-β1 ↑ → tight junction disassembly\n→ ↑ paracellular permeability → bacterial invasion', fc='#922b21', fs=8) arr(ax, 13.2, 7.8, 13.2, 7.5) arr(ax, 13.2, 6.9, 13.2, 6.6) arr(ax, 13.2, 6.0, 13.2, 5.7) arr(ax, 13.2, 5.1, 13.2, 4.8) # Convergence arrow to common outcome rbox(ax, 8.0, 2.8, 12.0, 0.9, 'BREACH OF EPITHELIAL BARRIERS\n→ Increased bacterial invasion → Inflammatory infiltration → Alveolar Bone Loss\n(Aggressive periodontitis in immunologic disorders; Chronic periodontitis in environmental risk)', fc='#1a1a2e', fs=9) # Arrows converging arr(ax, 2.8, 4.15, 4.5, 3.25, color='#1a5276') arr(ax, 8.0, 5.05, 8.0, 3.25, color='#1e8449') arr(ax, 13.2, 4.15, 11.5, 3.25, color='#922b21') plt.tight_layout() plt.savefig('/home/daytona/workspace/perio-invasion/fig4_risk_factors.png', dpi=180, bbox_inches='tight') plt.close() print("Fig 4 saved") PYEOF

ls /home/daytona/skills/docx/scripts/office/ 2>/dev/null | head -10; ls /home/daytona/skills/docx/ | head -20

cat > /home/daytona/workspace/perio-invasion/build_doc.js << 'JSEOF' const { Document, Packer, Paragraph, TextRun, ImageRun, HeadingLevel, AlignmentType, Table, TableRow, TableCell, WidthType, BorderStyle, PageBreak, ShadingType, VerticalAlign } = require('docx'); const fs = require('fs'); const path = require('path'); const IMGDIR = '/home/daytona/workspace/perio-invasion/'; function img(fname, w, h) { const buf = fs.readFileSync(path.join(IMGDIR, fname)); return new Paragraph({ alignment: AlignmentType.CENTER, spacing: { before: 200, after: 200 }, children: [new ImageRun({ data: buf, transformation: { width: w, height: h } })] }); } function h1(text) { return new Paragraph({ heading: HeadingLevel.HEADING_1, spacing: { before: 360, after: 120 }, children: [new TextRun({ text, bold: true, size: 28, color: '1a1a2e' })] }); } function h2(text) { return new Paragraph({ heading: HeadingLevel.HEADING_2, spacing: { before: 280, after: 100 }, children: [new TextRun({ text, bold: true, size: 24, color: '1a5276' })] }); } function h3(text) { return new Paragraph({ heading: HeadingLevel.HEADING_3, spacing: { before: 200, after: 80 }, children: [new TextRun({ text, bold: true, size: 22, color: '2980b9' })] }); } function p(text, opts={}) { return new Paragraph({ alignment: AlignmentType.JUSTIFIED, spacing: { before: 80, after: 80, line: 360 }, children: [new TextRun({ text, size: 22, ...opts })] }); } function bullet(text) { return new Paragraph({ bullet: { level: 0 }, spacing: { before: 40, after: 40 }, children: [new TextRun({ text, size: 21 })] }); } function subbullet(text) { return new Paragraph({ bullet: { level: 1 }, spacing: { before: 30, after: 30 }, children: [new TextRun({ text, size: 20, italics: true })] }); } function figcap(text) { return new Paragraph({ alignment: AlignmentType.CENTER, spacing: { before: 60, after: 240 }, children: [new TextRun({ text, size: 19, italics: true, color: '555555' })] }); } function rule() { return new Paragraph({ border: { bottom: { color: '2980b9', space: 1, value: BorderStyle.SINGLE, size: 6 } }, spacing: { before: 100, after: 100 }, children: [] }); } const doc = new Document({ styles: { paragraphStyles: [] }, sections: [{ properties: { page: { margin: { top: 720, bottom: 720, left: 900, right: 900 } } }, children: [ // ════ TITLE ════ new Paragraph({ alignment: AlignmentType.CENTER, spacing: { before: 0, after: 120 }, children: [new TextRun({ text: 'BACTERIAL INVASION AND ITS ROLE IN THE PATHOGENESIS OF PERIODONTAL DISEASE', bold: true, size: 32, color: '1a1a2e' })] }), new Paragraph({ alignment: AlignmentType.CENTER, spacing: { before: 0, after: 60 }, children: [new TextRun({ text: 'A Comprehensive Long Note for Postgraduate Examination', italics: true, size: 24, color: '555555' })] }), new Paragraph({ alignment: AlignmentType.CENTER, spacing: { before: 0, after: 80 }, children: [new TextRun({ text: 'Sources: Ji S, Choi YS, Choi Y. J Periodont Res 2014; Tribble GD, Lamont RJ. Periodontol 2000, 2010;52:68–83', italics: true, size: 19, color: '777777' })] }), rule(), // ════ 1. INTRODUCTION ════ h1('1. INTRODUCTION'), p('Periodontitis is the chronic inflammation of the periodontium caused by the host\'s inflammatory response to plaque biofilm, which destroys tooth-supporting soft and hard tissues. It is a complex disease that involves microbial components, environmental factors and host genetic variations in its development.'), p('It is widely accepted that the conversion from periodontal health to disease accompanies a shift in the indigenous flora of the plaque biofilm from gram-positive facultative to gram-negative anaerobic motile microorganisms. For decades, periodontitis has been regarded as the result of hyperimmune or hyperinflammatory responses to plaque bacteria. However, recent studies indicate that periodontal pathogens are rather poor activators and/or suppressors of the host immune response, raising the question of how they cause inflammation.'), p('To resolve this issue, the authors (Ji et al., 2014) propose that invasion of bacteria into gingival tissues and the persistence of periodontal pathogens are major events leading to chronic inflammation. According to Tribble & Lamont (2010), the ability to invade and persist in host cells is evidently an important factor in the overall disease process, and P. gingivalis strains isolated from disease sites possess greater invasion capabilities in vitro than strains from healthy sites.'), // ════ 2. HOST–MICROBE INTERACTIONS IN THE GINGIVAL SULCUS ════ h1('2. HOST–MICROBE INTERACTIONS IN THE GINGIVAL SULCUS'), p('The gingival sulcus is a unique anatomic site surrounded by hard tissue at one side and soft tissue at the other. At the interface where the gingiva meets the tooth surface, up to 700 bacterial species can colonize the gingival sulcus in varying amounts, from approximately 10³ bacteria in healthy sulci to >10⁸ bacteria in pathologic pockets. Some of the bacteria exist in harmony with the host; however, certain bacteria can disrupt this bacteria-host homeostasis.'), h2('2.1 Socransky Classification of Plaque Bacteria'), p('According to Socransky\'s classification, plaque bacteria are classified into six related complexes – yellow, green, violet, orange, red and Actinomyces. The diverse plaque bacteria colonize in a certain order through cell-to-cell coaggregation:'), bullet('Primary colonizers (early colonizers): Streptococcus gordonii, S. mitis, S. oralis, S. sanguinis. They form the acquired pellicle receptors.'), bullet('Secondary colonizers: Actinomyces naeslundii, Capnocytophaga ochracea, Eikenella corrodens, Haemophilus parainfluenzae, Veillonella atypica. These belong to yellow, green, and violet complexes.'), bullet('Bridging colonizers (orange complex): Fusobacterium nucleatum (the best example), Campylobacter gracilis, Eubacterium nodatum, F. periodonticum, Peptostreptococcus micros, Prevotella intermedia, P. nigrescens, Streptococcus constellatus. F. nucleatum coaggregates with early and late colonizers.'), bullet('Late colonizers (red complex – PERIODONTAL PATHOGENS): Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia. Detected only in the presence of the orange complex; strongly associated with periodontal destruction.'), h2('2.2 Host Defense in the Gingival Sulcus'), p('To defend against the colonizing subgingival bacteria, the host exhibits a unique innate immune system. Two major defense mechanisms are present in the gingival sulcus:'), bullet('Neutrophils: guided into the sulcus from capillary beds of connective tissue through junctional epithelia by specific chemoattractants such as IL-8, C3a, C5a and N-formyl peptides. Neutrophils build a wall between the plaque and the underlying epithelium and actively phagocytose adjacent bacteria.'), bullet('Antimicrobial Peptides (AMPs): cationic peptides with an amphipathic structure, referred to as endogenously produced antibiotics. The major AMPs are α-defensins, β-defensins and LL-37, produced by neutrophils, epithelial cells and both, respectively. AMPs effectively kill a broad range of bacteria, viruses and fungi. Additionally, LL-37 increases cell stiffness, decreases transepithelial permeability and prevents epithelial invasion.'), h2('2.3 Host–Microbe Interaction Profile of Each Complex'), p('Based on in-vitro studies, the outcome of the host–microbe interaction may be characterized as:'), bullet('Early colonizers (S. sanguinis, S. gordonii, A. naeslundii, V. atypica, E. corrodens): Homeostatic co-existence. Intermediate- to high-level susceptibility to AMPs and phagocytosis by neutrophils. Induce HBD-3 but not IL-8 from gingival epithelial cells.'), bullet('Bridging colonizers (F. nucleatum, P. nigrescens, P. intermedia, P. micros): Efficient clearance by host defense system. High susceptibility to phagocytosis and AMPs. F. nucleatum and P. intermedia induce HBD-2, HBD-3, LL-37 and IL-8 most efficiently, yet are highly susceptible to HBD-3 and LL-37.'), bullet('Late-colonizing periodontal pathogens (P. gingivalis, T. forsythia, T. denticola): Dysbiosis. Resistant to LL-37 and phagocytosis by neutrophils. Poor inducers of inflammatory mediators. Function as \'keystone pathogens\'.'), h2('2.4 Keystone Pathogen Concept'), p('P. gingivalis, at very low colonization levels (<0.01% of total microbiota), triggered increases in the number of total oral bacteria and changes in the composition of the oral commensal microbiota, leading to inflammatory periodontal bone loss in mice (Hajishengallis et al.). The term "keystone pathogen" was coined to describe a bacterial species with such a modulating ability. P. gingivalis and T. denticola are resistant to LL-37, phagocytosis and suppress IL-8 and N-formyl-methionyl-leucyl-phenylalanine-stimulated neutrophil chemotaxis, thus facilitating their initial colonization and generating an environment favorable for their own growth as well as that of other organisms.'), // ════ 3. INDUCTION OF INFLAMMATORY MEDIATORS ════ h1('3. INDUCTION OF INFLAMMATORY MEDIATORS FROM EPITHELIAL CELLS AND NEUTROPHILS'), p('In contrast to the prevalent concept that periodontal pathogens induce hyperinflammatory responses, a number of studies have shown that periodontal pathogens induce lower levels of inflammatory cytokines and chemokines than do nonpathogenic bacteria:'), bullet('F. nucleatum induced IL-6 from KB cells; however, P. gingivalis, T. forsythia and T. denticola did not affect or inhibit IL-6 levels.'), bullet('P. gingivalis biofilms significantly inhibited the production of Gro-1α, IL-1α, IL-6, TGF-α, fractalkine and IP-10 from OKF4 epithelial cells, whilst F. nucleatum and A. naeslundii biofilms induced elevated IL-6 and IP-10.'), bullet('P. gingivalis did not elicit IL-1β, TNF-α and IL-6 in a primary gingival epithelial cell multilayer model, whereas S. gordonii and F. nucleatum induced production of these proinflammatory cytokines.'), bullet('P. gingivalis, T. forsythia and T. denticola commonly have poor abilities to activate TLR2.'), bullet('P. gingivalis, T. forsythia and T. denticola induced much lower amounts of tissue-destructive molecules (reactive oxygen species, MMP-8, IL-1β) from neutrophils than did S. sanguinis and F. nucleatum.'), p('This raises the question: why do the late-colonizing periodontal pathogens — but not F. nucleatum or the early colonizers — cause chronic inflammation in gingival tissues? The answer lies in their ability to invade gingival tissue.'), // ════ 4. BACTERIAL INVASION ════ h1('4. BACTERIAL INVASION'), img('fig1_pathogenesis_flowchart.png', 580, 800), figcap('Figure 1. Proposed model for the pathogenesis of periodontitis (after Ji et al., 2014). The flowchart shows the sequence from healthy sulcus through plaque accumulation, keystone pathogen colonization, barrier disruption, invasion, intracellular survival, immune subversion and ultimately chronic tissue destruction. Risk factors (immunologic and environmental) that amplify each step are shown on either side.'), p('Although periodontal pathogens are poor inducers of inflammatory mediators, their ability to invade gingival tissue may enable them to cause inflammation within the tissue. Periodontal pathogens, such as P. gingivalis, T. forsythia and T. denticola, have the ability to invade gingival epithelial cells.'), h2('4.1 Invasion Mechanisms of Periodontal Pathogens into Epithelial Cells'), p('The initial interaction with epithelial cells via bacterial surface ligands causes rearrangement of the cellular machinery, which mediates pathogen entry into these nonphagocytic host cells. The most intensively studied of the invasive oral bacteria is P. gingivalis.'), h3('4.1.1 Porphyromonas gingivalis'), p('The mechanisms of P. gingivalis adhesion to, and invasion of, epithelial cells are multifaceted and involve a number of effector molecules:'), bullet('Major fimbriae (FimA): Composed of the FimA structural subunit protein along with minor proteins FimC, D and E. The FimA subunit directly engages αvβ3 and α5β1 integrins on the epithelial surface. There are at least six alleles of the fimA gene (fimA I, Ib, II, III, IV and V). Fimbriae composed of type Ib, II or IV are more commonly associated with periodontal disease.'), bullet('Intracellular invasion initiation: The major fimbriae engage β1 integrin receptors, resulting in a signaling cascade that remodels the host cytoskeleton to allow bacterial entry.'), bullet('Role of SerB (serine phosphatase): Optimal invasion of P. gingivalis requires activation of cofilin, one of the actin filament-rearranging proteins, which is mediated by P. gingivalis SerB, a bacterial serine phosphatase. SerB is secreted by P. gingivalis upon encountering the epithelial cell environment; it enters host cells, dephosphorylates target proteins including MAP kinase family members, prevents NF-κB activation and remodels microfilament and microtubule cytoskeletal architecture. A SerB mutant of P. gingivalis is defective in actin remodeling and in internalization.'), bullet('Gingipains (RgpA, RgpB, Kgp): A family of three arginine/lysine-specific proteases found in the outer membrane and also secreted. Gingipain protease activity improves P. gingivalis binding by modifying matrix proteins and revealing epithelial surface cryptitopes. RgpA and Kgp gingipains possess hemagglutinin/adhesin domains involved in attachment. Invasion occurs in the absence of FimA, albeit less efficiently — gingipains serve as alternative invasins.'), bullet('FAK and Paxillin: Focal adhesion adaptor and signaling proteins recruited to sites of P. gingivalis attachment. The resulting protein-protein interactions among integrin, FAK and paxillin produce a phosphorylation-regulated signaling scaffold that activates Rho-family GTPases (Rac1), which play a central role in initiating downstream signaling cascades and regulating cytoskeletal dynamics.'), bullet('Calcium signaling: Invasion by P. gingivalis induces a transient increase in epithelial cell cytosolic calcium concentrations. Calcium signaling regulates cytokine expression and modulates intracellular trafficking and cytoskeletal activities. Over-expression of the calcium-binding protein calprotectin in gingival epithelial cells inhibits invasion.'), bullet('Timeline: Invasion is completed in approximately 15 min; once inside the cell, the bacteria replicate during the first 4 h. Intracellular P. gingivalis accumulates in the perinuclear area, where they remain viable and can ultimately spread into adjacent cells or tissue.'), img('fig2_invasion_mechanism.png', 620, 460), figcap('Figure 2. Molecular mechanism of P. gingivalis invasion into gingival epithelial cells. The FimA–integrin–FAK–Rac1 signaling axis drives actin/microtubule remodeling and bacterial uptake. SerB phosphatase inhibits NF-κB and IL-8, while CXCR4/CR3 activation via C5a suppresses intracellular killing.'), h3('4.1.2 Tannerella forsythia'), p('Epithelial cell attachment and invasion by T. forsythia is dependent on the BspA protein (a leucine-rich surface protein), and its invasion of oral epithelial cells requires phosphoinositide 3-kinase activation and clathrin-mediated endocytosis.'), h3('4.1.3 Treponema denticola'), p('The invasion mechanism of T. denticola is not fully known. T. denticola survived for many hours within gingival epithelial cells by resisting targeting to the endolysosomal degradation pathway. T. denticola substantially suppresses the expression of IL-8 and HBD-1, -2 and -3 from gingival epithelial cells. Furthermore, T. denticola hijacks the F. nucleatum-driven induction of HBDs and IL-8 in gingival epithelial cells by interrupting endolysosomal maturation and reactive oxygen species-dependent TLR activation. The major outer sheath protein of T. denticola inhibits neutrophil polarization and chemotaxis in response to N-formyl-methionyl-leucyl-phenylalanine peptide in vitro.'), h2('4.2 Invasion of Orange Complex Bacteria and Non-periodontopathic Bacteria'), p('F. nucleatum and P. intermedia, members of the orange complex that are significantly increased in periodontal lesions but are not as virulent as periodontal pathogens, also presented substantial invasive ability. These two species are also able to invade both human gingival fibroblasts and endothelial cells. F. nucleatum rapidly fused with lysosomes and was degraded within epithelial cells. Bacterial invasion and subsequent trafficking to the endo-lysosomal compartment play important roles in the induction of IL-8 from gingival epithelial cells.'), p('Non-periodontopathic bacteria, such as S. sanguinis and V. atypica, rarely invade epithelial cells.'), h2('4.3 Cooperative (Polymicrobial) Invasion'), p('Bacteria can cooperate with one another to facilitate invasion — consistent with the multispecies etiology of periodontal disease:'), bullet('F. nucleatum can transport noninvasive Streptococcus cristatus into host cells via a combination of co-aggregation and invasion mechanisms.'), bullet('F. nucleatum can facilitate invasion of human gingival epithelial and endothelial cells by P. gingivalis (the synergistic effect results from F. nucleatum\'s interaction with host cells).'), bullet('Candida albicans enhances the invasion of epithelial cells by P. gingivalis.'), bullet('P. gingivalis can enhance the invasion of T. forsythia and Pseudomonas aeruginosa into epithelial cells.'), bullet('This co-operation among bacteria may cause a multispecies invasion into epithelial cells. For example, buccal epithelial cells observed ex vivo contained a polymicrobial intracellular microbiota, including streptococci.'), h2('4.4 Invasion Through the Intercellular (Paracellular) Route'), p('As was shown using motile and immotile T. denticola and three-dimensional cultured gingival epithelial cells, bacteria can also invade gingival tissue through a paracellular route. Gingival epithelial cells are adjoined by tight junction-related structures and adherent junctions. The increased expression levels of ZO-1 (tight junction protein 1), JAM-A and occludin in the sulcular/junctional epithelium compared with the oral epithelium support the important role of a tight junction-related structure in the absence of a keratinized layer.'), p('P. gingivalis and T. denticola, periodontal pathogens with powerful proteases, degrade epithelial junctional proteins such as E-cadherin, occludin and ZO-1. Therefore, the proteinases of periodontal pathogens may facilitate the intercellular invasion of bacteria by impairing the tight junction-related structure.'), // ════ 5. SURVIVAL & TRANSMISSION IN EPITHELIAL CELLS ════ h1('5. SURVIVAL AND TRANSMISSION OF PERIODONTAL PATHOGENS IN EPITHELIAL CELLS'), h2('5.1 Intracellular Survival Strategies of P. gingivalis'), p('Importantly, P. gingivalis has been shown to survive within the cytosol of epithelial cells and to spread to neighboring epithelial cells. The intracellular trafficking of P. gingivalis in epithelial cells was verified using bacteria genetically engineered to express the nontoxic green flavin mononucleotide-based fluorescent protein (FbFP).'), bullet('Quantitative co-localization analyses showed that intracellular P. gingivalis–FbFP was significantly associated with the endoplasmic reticulum network, whereas the majority of Clp serine protease-deficient mutants trafficked into the lysosomes. This indicated that the endoplasmic reticulum structure is utilized for the persistent survival of P. gingivalis and that the bacterial Clp serine protease is critical for optimal adaptation of the organism to intracellular life and survival in oral epithelial cells.'), bullet('In epithelial cells: P. gingivalis escapes from the initial membrane-bound vacuole and survives unbound in the cytoplasm. It ultimately localizes to the perinuclear region (densely packed with endoplasmic reticulum — hypothesized to act as a nutrient source for these proteolytic bacteria).'), bullet('In endothelial cells: P. gingivalis ultimately traffics to autophagosomes (double-membraned vacuoles). Once in these membrane compartments, the bacteria block fusion with lysosomes and probably use protein debris trafficked through the autophagic pathway as their nutrient source.'), h2('5.2 Molecular Adaptation to the Intracellular Environment'), p('Approximately 50% of the expressed proteome is differentially regulated by intracellular P. gingivalis. Key adaptations include:'), bullet('Virulence factor downregulation: FimA, RgpA/B and Kgp show decreased expression in internalized P. gingivalis. FimA is superfluous once inside; tight control of gingipain production prevents excessive damage to the host cell interior.'), bullet('Stress protein upregulation: Clp family proteins, HtrA, peroxidases. Deletion of clpB or clpP genes has a negative impact on bacterial survival in gingival epithelial cells. Mutation of htrA results in increased sensitivity to hydrogen peroxide and decreased survival in animal infection models.'), bullet('Metabolic shift: Increased abundance of proteins comprising the energy pathway from asparagine/aspartate to ATP. The pathway producing propionate shows increased abundance, while tendency towards decreased abundance is observed for the butyrate-producing pathway. As propionate is a less potent inducer of apoptosis than butyrate, this metabolic shift minimizes damage to host cells.'), bullet('Translational machinery: Significant increase in expression of ribosomal proteins and transfer RNA synthetases. The intracellular environment is energy-rich for P. gingivalis.'), h2('5.3 Anti-apoptotic Mechanisms'), p('The phenotype of colonized cells depends on the characteristics of the invading P. gingivalis strain, total bacterial burden and host cell type:'), bullet('High-protease strains or high bacterial numbers: cell rounding and loss of attachment due to gingipain cleavage of cadherins and integrins; gingipains can penetrate the host cell and activate pro-apoptotic molecules (caspase-3, caspase-8, caspase-9, Bid, Bax). Additional damage results from activation of matrix metalloproteases by gingipains.'), bullet('Low-protease strains or lower bacterial numbers (anti-apoptotic phenotype): Activation of PI3K/Akt and JAK/STAT pathways; up-regulation of anti-apoptosis genes Bcl-2 and survivin; inhibition of cytochrome c release and caspase-3 activity. P. gingivalis also secretes an ATP-hydrolyzing enzyme (homolog of nucleoside diphosphate kinase) that inhibits apoptosis by preventing ATP ligation of P2X₇ purinergic receptors.'), bullet('Cell cycle effects: Invasion with P. gingivalis results in increased proliferation associated with accelerated progression through the S-phase. Upregulating cell division may maintain a reservoir of bacterially infected cells; in disease states, loss of cell cycle control could impact wound healing in the periodontal pocket, facilitating bacterial penetration.'), h2('5.4 Transmission to Adjacent Cells (Cell-to-Cell Spread)'), p('The transmission of P. gingivalis into adjacent cells is mediated in two ways:'), bullet('Route 1 — Actin-based membranous projections: Transmission via membranous projections with a structural scaffold composed of actin filaments. This allows intercellular dissemination without exposure to the intercellular space — a "stealth" mechanism. Initiation of the spreading mechanism occurs at the highest frequency after 24 h of invasion.'), bullet('Route 2 — Endocytic recycling pathway: A considerable number of intracellular P. gingivalis are sorted to Rab11- and RalA-positive recycling endosomes, followed by bacterial exit from the cells. Exited bacteria can re-enter new cells.'), p('Importantly, this intercellular transmission does not appear to affect host-cell viability. These colonized epithelial cells are not necrotic or apoptotic, but remain viable.'), img('fig3_survival_spreading.png', 640, 440), figcap('Figure 3. Intracellular survival strategies (left column) and cell-to-cell spreading mechanisms (right column) of P. gingivalis. Note the dual routes of spreading: actin-based projections and endocytic recycling. The metabolic and proteomic reprogramming ensures long-term intracellular persistence.'), // ════ 6. SPREADING BEYOND EPITHELIA ════ h1('6. SPREADING BEYOND EPITHELIA'), p('The ability to disseminate beyond the initial site of infection is a characteristic of pathogenic bacteria in general. The continuous shedding and turnover of epithelial cells plays an important role in the protection against invading bacteria. However, the relatively slow turnover rate of the gingiva (41–57 d) compared with the doubling time of bacteria may provide enough time for invasive bacteria to spread beyond the epithelia.'), p('In a three-dimensional cellular model of bacterial dissemination, P. gingivalis was found to spread through the upper layers of gingival epithelial cells and to penetrate the basement membrane into the connective tissues. P. gingivalis gingipains are able to cleave the constituents of periodontal tissues, such as basement membranes and the structural proteins collagen and elastin. The T. denticola chymotrypsin-like protease may also play an important role in invasion and destruction of the basement membrane — under conditions where chymotrypsin-like protease activity was increased, more spirochetes migrated through a reconstituted basement membrane (Matrigel), and the purified enzyme degraded basement membrane components such as type IV collagen, laminin and fibronectin.'), p('Depending on the strain, F. nucleatum can also penetrate the basement membrane barrier. F. nucleatum (AHN 9508) was able to pass through the epithelial/basement membrane barrier and invade the collagen matrix in organotypic culture, whereas the invasiveness of F. nucleatum biofilm (ATCC 25586) was limited to the epithelium.'), p('A P. gingivalis gingipain mutant was able to invade the upper gingival layers but was unable to access the connective tissue layer. This confirms the gingipain-dependent nature of basement membrane penetration.'), // ════ 7. DETECTION OF BACTERIA EX VIVO ════ h1('7. DETECTION OF BACTERIA EX VIVO — EVIDENCE FOR BACTERIAL INVASION IN VIVO'), p('Bacterial invasion of the pocket epithelium and the underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods:'), bullet('Electron microscopy (1970s–early 1980s): First observation of the presence of bacteria within gingival tissues with periodontitis.'), bullet('Immunohistochemistry/immunofluorescence: Presence of P. gingivalis, Aggregatibacter actinomycetemcomitans, Capnocytophaga gingivalis and T. denticola in diseased gingival tissues visualized using bacteria-specific antibodies.'), bullet('In-situ hybridization (ISH): Using fluorescence- or digoxigenin-labeled probes targeting bacterial 16S rRNA. Increased numbers of intracellular P. gingivalis, T. forsythia and T. denticola within epithelial cells from periodontal pockets demonstrated by fluorescence ISH and confocal microscopy.'), bullet('Fluorescence in situ hybridization (FISH) + confocal microscopy: Established that buccal epithelial cells from healthy individuals contain a polymicrobial intracellular microbiota that includes P. gingivalis, T. forsythia, A. actinomycetemcomitans, F. nucleatum, P. intermedia, E. corrodens and T. denticola. These colonized cells are not necrotic or apoptotic but remain viable.'), p('Bacteria were observed in the pocket epithelium, in the lamina propria just beneath the basal lamina and also in the deep connective tissue. The presence of bacteria in deep connective tissue was more prevalent in the lesions than in the healthy sites of patients with periodontitis. These results imply that bacteria can penetrate through the epithelia into the connective tissue, particularly in periodontal lesions.'), h2('7.1 Bacterial Invasion and Inflammatory Infiltration in Periodontitis'), p('The combination of in-situ hybridization and immunohistochemical detection of the digoxigenin-labeled probe allowed appreciation of the relationship between the presence of bacteria and inflammatory infiltrate in the gingival tissues from patients with chronic periodontitis:'), bullet('At low magnification: P. gingivalis was detected at the highest levels directly below the junctional epithelium, where both B- and T cells were heavily infiltrated.'), bullet('At increased magnification: P. gingivalis was observed throughout the gingival connective tissue — higher levels at loose connective tissue areas (where inflammatory cells were observed) than at dense connective tissue areas.'), bullet('At highest magnification: The positive signal appeared to be dispersed within host cells or represented the discrete shape of the bacteria. In the dense connective tissue area, aggregates of several bacteria in line were examined.'), p('Using a mouse model: The number of T cells within the tissue had a strong, positive correlation with the number of bacterial invasion sites and was positively associated with alveolar bone loss. These results support the claim that bacteria within the tissue recruit inflammatory cells, including T cells.'), // ════ 8. BACTERIAL PERSISTENCE & IMMUNE EVASION ════ h1('8. PERSISTENCE OF PERIODONTAL PATHOGENS AND IMMUNE EVASION'), p('In addition to invasion and survival in epithelial cells, P. gingivalis can invade both human gingival fibroblasts and endothelial cells. T. denticola is also able to adhere to and invade endothelial cells. P. gingivalis can also manipulate the cell cycle of host cells to favor bacterial survival.'), h2('8.1 Survival Strategy in Macrophages — TLR2 Subversion'), p('P. gingivalis readily takes intracellular refuge in phagocytic cells, such as macrophages. The survival strategy of P. gingivalis in macrophages depends on the subversion of TLR2 activation through cross-talk between TLR2 and other immune receptors:'), bullet('P. gingivalis uses its fimbriae to bind to CXCR4 and CR3.'), bullet('Gingipains of P. gingivalis degrade C5, generating active C5a that activates C5aR.'), bullet('Activation of either CXCR4 or C5aR triggers cyclic AMP-dependent protein kinase A (PKA) signaling, which suppresses TLR2-mediated production of inducible nitric oxide synthase (iNOS). As a result, nitric oxide-dependent killing of P. gingivalis is impaired in vitro and in vivo.'), bullet('CR3 deficiency results in a dramatic reduction (by a factor of 1000) in the intracellular survival of P. gingivalis, indicating that CR3 is exploited by the pathogen as a relatively safe portal of entry.'), h2('8.2 Evasion of Cell-Mediated Immunity — Th1/Th2 Imbalance'), p('The ability of P. gingivalis to survive within the host cells warrants T-helper (Th)1 cell-mediated immunity to clear infection with this persistent pathogen. Remarkably, P. gingivalis proactively and selectively inhibits IL-12 expression in macrophages to escape intracellular killing by cell-mediated immunity:'), bullet('Both C5aR and CR3 are involved in the inhibition of TLR2-induced IL-12 production through the activation of ERK1/2.'), bullet('Lipopolysaccharide from P. gingivalis does not induce IL-12p70 from dendritic cells.'), bullet('Impaired IL-12 production leads to the suppression of Th1-cell differentiation, polarizing the immune response toward the Th2-cell response.'), bullet('The induction of a distinct T-cell response is dependent on P. gingivalis capsular serotypes K1–K5. K1- and K2-primed dendritic cells produced higher levels of Th1/Th17 cytokines; K3, K4 and K5 induced higher levels of Th2 cytokines.'), bullet('Td92, a surface protein of T. denticola, induced IFN-γ but inhibited IL-4 secretion; however, patients with chronic periodontitis showed a reduced IFN-γ/IL-4 cytokine balance, and Td92-induced IFN-γ levels were negatively associated with periodontal destruction.'), h2('8.3 Roles of Th17 and Regulatory T Cells'), p('Elevated levels of IL-17 were found in the gingival crevicular fluid of patients with severe periodontitis. The number of Th17 cells was significantly higher in periodontal lesions than in healthy sites. Th17 is a key osteoclastogenic helper T-cell subset that links T-cell activation and bone destruction. IL-17 plays a critical role in bone destruction.'), p('FoxP3-positive Treg cells have been characterized in the inflammatory infiltrate of gingival tissues, and an increased number of Treg cells has been found in periodontitis lesions compared with healthy sites or gingivitis sites. Application of the Treg-recruiting chemokine CCL22 reduced inflammation and alveolar bone loss, whilst inhibition of Treg function increased alveolar bone loss and inflammatory cell migration, suggesting Treg cells play a greater role in the suppression of inflammation.'), h2('8.4 Impact on Innate Immune Surveillance — "Stealth Pathogen" Concept'), p('P. gingivalis often suppresses or evades various components of innate immunity:'), bullet('Gingival epithelial cells do not express CD14 (a co-receptor for TLR2) on the surface and thus respond poorly to P. gingivalis FimA, which may limit inflammatory responses to fimbriated, invasive P. gingivalis.'), bullet('P. gingivalis suppresses transcription of the IL-8 gene in gingival epithelial cells, thus inhibiting expression of this chemokine. Moreover, P. gingivalis can antagonize IL-8 secretion following stimulation with other common plaque constituents — a phenomenon known as "localized chemokine paralysis".'), bullet('Mechanistically, invasive P. gingivalis inhibit NF-κB activity through the SerB-mediated disruption of signaling pathways. P. gingivalis proteases also degrade cytokines, chemokines and their receptors.'), bullet('A reduction in IL-8 levels, along with the down-regulation of ICAM-1, will impair neutrophil infiltration of gingival tissues and consequently debilitate local innate immunity.'), // ════ 9. PROPOSED PATHOGENESIS MODEL ════ h1('9. PROPOSED MODEL FOR THE PATHOGENESIS OF PERIODONTITIS'), p('Based on all the above evidence, the following model is proposed (Ji et al., 2014):'), bullet('Clinically healthy sites are colonized with a small number of subgingival bacteria composed mostly of early colonizers. Although a small number of bacteria may invade the gingival tissues, the invaded bacteria are soon cleared either within the gingival epithelial cells or by recruited inflammatory cells, without leading to tissue destruction.'), bullet('The accumulation of dental plaque as a result of poor oral hygiene results in the preferential increase of periodontal pathogens that not only have high invasive ability but also have strong proteases that impair the epithelial physical barriers.'), bullet('Subsequently, substantial amounts of bacteria invade the gingival tissues, recruiting an increased number of inflammatory cells into the sites.'), bullet('However, periodontal pathogens survive intracellularly by the subversion of innate immunity and by inducing a skewed Th1/Th2 cell balance.'), bullet('In addition, dental plaque provides a source of continuously invading bacteria as a reservoir.'), bullet('Such persistent infection leads to chronic inflammation and tissue destruction.'), bullet('Immunologic disorders such as neutrophil dysfunction and Kostmann syndrome contribute to aggressive periodontitis by breaching the immunologic or chemical barrier functions of gingival epithelia.'), bullet('Two major environmental risk factors — smoking and alcohol — may contribute to the development of periodontitis by weakening the epithelial physical barriers.'), // ════ 10. RISK FACTORS ════ h1('10. RISK FACTORS THAT IMPAIR EPITHELIAL BARRIERS'), p('The gingival epithelium forms barriers between plaque bacteria and gingival tissue, providing the first line of defense against invading bacteria. The epithelial barrier consists of physical, chemical and immunologic barriers.'), img('fig4_risk_factors.png', 620, 380), figcap('Figure 4. Risk factors that impair epithelial barriers in periodontitis. Immunologic disorders (left), chemical barrier defects (center), and physical barrier disruption by pathogens and environmental factors (right) all converge to increase bacterial invasion and promote aggressive or chronic periodontitis.'), h2('10.1 Risk Factors That Impair Epithelial Immunologic Barriers'), p('Neutrophils are the predominant cell type in the subgingival sulcus and gingival crevicular fluid. The importance of neutrophils as an immunologic barrier in the maintenance of periodontal health is evident from aggressive periodontitis associated with various defects in the number or function of neutrophils:'), bullet('Agranulocytosis/Neutropenia: The first immunologic disorder reported to be associated with severe periodontal destruction. All neutropenic conditions, either congenital (such as Kostmann syndrome) or drug-induced, are associated with aggressive periodontitis.'), bullet('Leukocyte adhesion deficiency (LAD) Type I syndrome: Caused by mutation in the beta-2 integrin gene and defective polymorphonuclear leukocyte formyl peptide receptor — reported as the causative genetic variation for the migration defect.'), bullet('Chédiak-Higashi syndrome: A lysosomal trafficking disorder. Severe periodontal destruction in adolescent patients. Mutations in the lysosomal trafficking regulator gene cause defects in the granule morphogenesis of neutrophils, resulting in neutropenia and defective bactericidal activity.'), bullet('Papillon-Lefèvre syndrome: A rare genetic disorder caused by mutations in the cathepsin C gene, resulting in palmoplantar keratosis and premature loss of both deciduous and permanent teeth from severe periodontitis. Cathepsin C plays an essential role in the activation of granule serine proteases required for the phagocytic destruction of bacteria; neutrophils demonstrate significantly decreased microbicidal activity.'), p('Along with the severe periodontal destruction, massive bacterial invasion of the epithelial cells and connective tissues has been reported in aggressive periodontitis in association with neutropenia, Chédiak-Higashi syndrome or Papillon-Lefèvre syndrome. Therefore, bacterial invasion into gingival tissue by breaching the immunological barrier seems to underlie the aggressive periodontitis associated with neutrophil dysfunction.'), h2('10.2 Risk Factors That Impair Epithelial Chemical Barriers'), p('The importance of epithelial chemical barriers in periodontal health has been shown in patients with Kostmann syndrome who maintain normal absolute neutrophil counts by therapy with granulocyte colony-stimulating factor. Although they no longer experience life-threatening bacterial infections, they frequently develop severe periodontitis, often starting in young childhood. This was caused by the lack of LL-37 in their saliva and neutrophils.'), p('In addition to killing microorganisms, LL-37 can increase cell stiffness, which prevents epithelial invasion by bacteria. LL-37 increased lung epithelial cell stiffness, decreased transepithelial permeability and prevented epithelial invasion with Pseudomonas aeruginosa. This suggests that the breach of epithelial chemical barriers may facilitate bacterial invasion into gingival tissue, leading to severe periodontitis.'), h2('10.3 Risk Factors That Impair Epithelial Physical Barriers'), p('Many pathogenic bacteria and viruses modulate epithelial physical barriers, particularly tight junctions, to enter host cells and/or tissues:'), bullet('P. gingivalis: Has the potential to disrupt epithelial integrity, contributing to the breakdown of the junctional epithelium. P. gingivalis can directly break the physical barriers of the epithelium by decreasing the expression of various proteins that form tight junctions (occludin, ZO-1 and JAM-1), adherens junctions (E-cadherin) and cell-extracellular matrix junctions (β1-integrin). Gingipains of P. gingivalis are responsible for the degradation of E-cadherin.'), bullet('T. denticola: A purified chymotrypsin-like proteinase induces the degradation of pericellular fibronectin and the loss of close contacts between the epithelial cells, resulting in the increased permeability of the infected epithelial multilayers.'), bullet('Smoking: Second to microbial dental plaque, smoking is the strongest modifiable environmental risk factor for periodontal disease. It can affect the progress of periodontitis in many ways, including a shift in subgingival flora, reduced microcirculation, dysfunction of neutrophils and increased levels of pathogenic T cells. Cotinine, the main derivative of cigarettes, significantly increased the association and invasion of epithelial cells by P. gingivalis. Cigarette smoke caused alterations in cytoskeletal and tight junction structure and function, resulting in increased macromolecular permeability in vitro and in vivo. Invasion of brain microvascular endothelial cells by E. coli was significantly enhanced by nicotine in a dose-dependent manner via actin cytoskeleton rearrangement.'), bullet('Alcohol: Alcohol consumption is a risk factor for periodontal disease, and its association with increased alveolar bone loss in a dose-dependent manner has been shown in humans and in animal experiments. Alcohol ingestion induced TGF-β1-dependent decreases in transepithelial resistance and increased paracellular dextran flux in alveolar epithelia. Ethanol also induced tight junction protein disassembly and an increase in paracellular permeability of the intestinal epithelial monolayer. Therefore, it can be inferred that alcohol may impair the integrity of the gingival epithelial barrier, thereby rendering the gingival surface susceptible to bacterial invasion.'), // ════ 11. THERAPEUTIC IMPLICATIONS ════ h1('11. IMPLICATIONS OF THE PROPOSED MODEL — THERAPEUTIC APPROACHES'), p('Knowledge of the correct pathogenesis of periodontitis is critical for developing novel preventive or therapeutic approaches. The proposed model suggests that the intervention of bacterial invasion may be the first critical step for both the prevention and treatment of periodontitis:'), bullet('Subgingival plaque control: Subgingival plaque provides a reservoir for the source of constant infection, justifying current periodontal therapy based on the mechanical removal of plaque (scaling and root planing).'), bullet('Maintaining early biofilm stage: Because periodontal pathogens can colonize only in the presence of bridging colonizers that colonize after the early colonizers do so, keeping the plaque at the early stage of biofilm maturation is recommended.'), bullet('Strengthening epithelial physical barriers: Preventing disruption of tight junctions and adherens junctions by targeting bacterial proteases (gingipains).'), bullet('Strengthening epithelial chemical barriers: LL-37-based therapeutic strategies to restore barrier integrity.'), bullet('Strengthening epithelial immunologic barriers: Granulocyte colony-stimulating factor therapy (as in Kostmann syndrome) to restore neutrophil counts.'), bullet('Targeting intracellular bacteria: Antibiotics that penetrate host cells and kill intracellular bacteria; scaling and root planing are less effective against intracellular populations (which constitute a reservoir for repopulation).'), bullet('Immunomodulation: Targeting Th1/Th2 imbalance, IL-12 restoration, TLR2 modulation, C5aR/CR3 blockade (abrogates P. gingivalis intracellular killing impairment in vitro and in vivo).'), bullet('Treg manipulation: CCL22 (Treg-recruiting chemokine) application reduced inflammation and alveolar bone loss in animal models.'), // ════ 12. SUMMARY ════ h1('12. SUMMARY'), p('Periodontitis is a complex infectious and immune-mediated disease. The pathogenesis, as proposed by Ji et al. (2014) and supported by Tribble & Lamont (2010), centers on bacterial invasion as the key initiating event:'), bullet('1. Plaque biofilm shifts from commensal early colonizers to virulent red-complex pathogens (keystone pathogens).'), bullet('2. These pathogens suppress local innate immunity (IL-8, IL-12, AMP downregulation) and impair epithelial barriers (tight junction degradation via gingipains and spirochete proteases).'), bullet('3. Substantial bacterial invasion of gingival epithelial cells occurs via integrin-mediated, gingipain-facilitated and SerB-driven mechanisms.'), bullet('4. Intracellular bacteria escape lysosomes, replicate in the perinuclear area, adopt anti-apoptotic strategies and spread cell-to-cell via actin projections — establishing a persistent intracellular reservoir resistant to mechanical therapy and antibiotics.'), bullet('5. Spreading beyond epithelia via basement membrane penetration recruits chronic inflammatory infiltrates (T cells, B cells, plasma cells) and induces a Th1/Th2 imbalance favoring persistence.'), bullet('6. Osteoclast activation via IL-17/RANKL leads to irreversible alveolar bone loss.'), bullet('7. Risk factors (neutropenia, Chediak-Higashi, Papillon-Lefèvre, Kostmann syndrome, smoking, alcohol) amplify bacterial invasion by breaching immunologic, chemical and physical epithelial barriers, explaining aggressive and chronic periodontitis respectively.'), p('Understanding this model opens avenues for novel therapies targeting invasion, intracellular persistence, immune evasion and epithelial barrier restoration — moving beyond conventional plaque-removal paradigms.'), rule(), new Paragraph({ alignment: AlignmentType.CENTER, spacing: { before: 80, after: 0 }, children: [new TextRun({ text: 'References: Ji S, Choi YS, Choi Y. J Periodont Res 2014; doi:10.1111/jre.12248 | Tribble GD, Lamont RJ. Periodontol 2000. 2010;52(1):68–83', italics: true, size: 18, color: '666666' })] }), ] }] }); Packer.toBuffer(doc).then(buf => { fs.writeFileSync('/home/daytona/workspace/perio-invasion/Bacterial_Invasion_Pathogenesis_Periodontitis.docx', buf); console.log('Document written successfully'); }).catch(e => console.error('Error:', e)); JSEOF cd /home/daytona/workspace/perio-invasion && node build_doc.js 2>&1