ABSTRACT
Improved survival for patients with head and neck cancers (HNC) with recurrent and metastatic disease warrants that cancer therapy is specific, with protected delivery of the therapeutic agent to primary and metastatic cancer cells. A further objective should be that downregulation of the intracellular therapy target leads to cell death without compensation by an alternate pathway. To address these goals, we report the utilization of a sub-50-nm tenfibgen (s50-TBG) nanocapsule that delivers RNAi oligonucleotides directed against the essential survival signal protein kinase CK2 (RNAi-CK2) in a cancer cell-specific manner. We have evaluated mechanism and efficacy of using s50-TBG-RNAi-CK2 nanocapsules for therapy of primary and metastatic head and neck squamous cell carcinoma (HNSCC). s50-TBG nanocapsules enter cancer cells via the lipid raft/caveolar pathway and deliver their cargo (RNAi-CK2) preferentially to malignant but not normal tissues in mice. Our data suggest that RNAi-CK2, a unique single-stranded oligonucleotide, co-opts the argonaute 2/RNA-induced silencing complex pathway to target the CK2αα' mRNAs. s50-TBG-RNAi-CK2 inhibited cell growth corresponding with reduced CK2 expression in targeted tumor cells. Treatment of three xenograft HNSCC models showed that primary tumors and metastases responded to s50-TBG-RNAi-CK2 therapy, with tumor shrinkage and 6-month host survival that was achieved at relatively low doses of the therapeutic agent without any adverse toxic effect in normal tissues in the mice. We suggest that our nanocapsule technology and anti-CK2 targeting combine into a therapeutic modality with a potential of significant translational promise.
Subject(s)
Carcinoma, Squamous Cell/therapy , Casein Kinase II/genetics , Head and Neck Neoplasms/therapy , Nanocapsules/chemistry , Peptide Fragments/chemistry , Splenic Neoplasms/therapy , Tenascin/chemistry , Animals , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Base Sequence , Carcinoma, Squamous Cell/secondary , Female , Gene Knockdown Techniques , Head and Neck Neoplasms/pathology , Humans , Membrane Microdomains/metabolism , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , RNA Interference , RNA, Small Interfering/genetics , Splenic Neoplasms/secondary , Tissue Distribution , TransfectionABSTRACT
The type III secretion system (T3SS) is a macromolecular 'injectisome' that allows bacterial pathogens to transport virulence proteins into the eukaryotic host cell. This macromolecular complex is composed of connected ring-like structures that span both bacterial membranes. The crystal structures of the periplasmic domain of the outer membrane secretin EscC and the inner membrane protein PrgH reveal the conservation of a modular fold among the three proteins that form the outer membrane and inner membrane rings of the T3SS. This leads to the hypothesis that this conserved fold provides a common ring-building motif that allows for the assembly of the variably sized outer membrane and inner membrane rings characteristic of the T3SS. Using an integrated structural and experimental approach, we generated ring models for the periplasmic domain of EscC and placed them in the context of the assembled T3SS, providing evidence for direct interaction between the outer membrane and inner membrane ring components and an unprecedented span of the outer membrane secretin.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Conserved Sequence , Periplasm/metabolism , Salmonella typhimurium/pathogenicity , Virulence Factors/chemistry , Virulence Factors/metabolism , Amino Acid Motifs , Bacterial Proteins/ultrastructure , Crystallography, X-Ray , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Models, Molecular , Protein Structure, Secondary , Protein Structure, Tertiary , Protein Transport , Secretin/chemistry , Secretin/metabolism , Structure-Activity RelationshipABSTRACT
OBJECTIVE: To investigate whether children with less than 48 hours of localized symptoms of deep neck infection are less likely to have an abscess on CT scan. STUDY DESIGN: Case series. SUBJECTS AND METHODS: The charts of children seen in a tertiary children's hospital for deep neck infections between 2000 and 2007 were reviewed. RESULTS: Of 179 children identified, 167 (93.3%) underwent a CT scan of the neck of which 102 (61.1%) were positive for abscess. There was no significant difference in the rate of abscess on CT between children with less than 48 hours of localizing symptoms and 48 or more hours of symptoms at 58.1 percent and 58.3 percent, respectively (P = 0.98). Furthermore, there was no significant difference in age, gender, C-reactive protein levels, disease location, or length of stay between children with and without abscess on CT. White blood cell counts were significantly higher in the abscess group (P = 0.01); however, the median white blood cell count in both groups was above normal. CONCLUSION: Because duration of symptoms does not predict finding of abscess on CT, it is appropriate to obtain a CT scan upon presentation in all children with symptoms concerning for neck abscess.
Subject(s)
Abscess/complications , Abscess/diagnostic imaging , Neck , Streptococcal Infections/complications , Streptococcal Infections/diagnostic imaging , Tomography, X-Ray Computed , Abscess/therapy , Adolescent , Child , Child, Preschool , Cohort Studies , Female , Humans , Infant , Male , Neck Pain/microbiology , Predictive Value of Tests , Retrospective Studies , Streptococcal Infections/therapy , Time FactorsABSTRACT
Type III secretion systems (TTSSs) are multi-protein macromolecular 'machines' that have a central function in the virulence of many Gram-negative pathogens by directly mediating the secretion and translocation of bacterial proteins (termed effectors) into the cytoplasm of eukaryotic cells. Most of the 20 unique structural components constituting this secretion apparatus are highly conserved among animal and plant pathogens and are also evolutionarily related to proteins in the flagellar-specific export system. Recent electron microscopy experiments have revealed the gross 'needle-shaped' morphology of the TTSS, yet a detailed understanding of the structural characteristics and organization of these protein components within the bacterial membranes is lacking. Here we report the 1.8-A crystal structure of EscJ from enteropathogenic Escherichia coli (EPEC), a member of the YscJ/PrgK family whose oligomerization represents one of the earliest events in TTSS assembly. Crystal packing analysis and molecular modelling indicate that EscJ could form a large 24-subunit 'ring' superstructure with extensive grooves, ridges and electrostatic features. Electron microscopy, labelling and mass spectrometry studies on the orthologous Salmonella typhimurium PrgK within the context of the assembled TTSS support the stoichiometry, membrane association and surface accessibility of the modelled ring. We propose that the YscJ/PrgK protein family functions as an essential molecular platform for TTSS assembly.
Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/chemistry , Escherichia coli/metabolism , Salmonella typhimurium/chemistry , Salmonella typhimurium/metabolism , Amino Acid Sequence , Biotinylation , Crystallization , Crystallography, X-Ray , Entropy , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Transport , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Static ElectricityABSTRACT
The type III secretion needle complex (NC) of Salmonella typhimurium is a complex secretory system that functions to translocate virulence proteins into eukaryotic cells. Evolutionarily it is related to bacterial flagella. Assembly of the NC occurs through ordered secretion, polymerization, and assembly, and requires the coordinated expression and association of over 20 different proteins. Recent progress in the understanding of the assembly and architecture of the NC is reviewed.