Despite advances in perfusion imaging, burn wound imaging technology continues to lag behind that of other fields. Quantification of blood flow is able to predict time for healing, but clear assessment of burn depth is still questionable. Active dynamic thermography (ADT) is a noncontact imaging modality capable of distinguishing tissue of different thermal conductivities. Utilizing the abnormal heat transfer properties of the burn zones, we examined whether ADT was useful in the determination of burn depth in a model of early burn wound evaluation. Duroc pigs (castrated male; n = 3) were anesthetized, and two burns were created with an aluminum billet at 3 and 12 seconds. These contact times resulted in superficial partial and deep partial thickness burn wounds, respectively. ADT and laser Doppler imaging (LDI) imaging were performed every 30 minutes postburn for a total of five imaging sessions ending 150 minutes postburn. For ADT, imaging excitation was performed for 42-120 seconds with dual quartz-infrared lamps, and subsequent infrared image capture was performed for 300 seconds. MATLAB-assisted image analysis was performed to determine burn zone region of interest thermal relaxation and characteristic patterns. LDI was performed with a moorLDI system, and biopsies were captured for histology following the 150-minute imaging session. Both ADT and LDI imaging modalities are able to detect different physical properties at 30, 60, 90 120, and 150 minutes postburn with statistical significance (P < 0.05). Resultant ADT cooling curves characterize greater differences with greater stimulation and a potentially more identifiable differential cooling characteristic. Histological analysis confirmed burn depth. This preliminary work confirms that ADT can measure burn depth and is deserving of further research either as a stand-alone imaging technology or in combination with a device to assess perfusion.
Mucus-invasive bacterial biofilms are identified on the colon mucosa of approximately 50% of colorectal cancer (CRC) patients and approximately 13% of healthy subjects. Here, we test the hypothesis that human colon biofilms comprise microbial communities that are carcinogenic in CRC mouse models. Homogenates of human biofilm-positive colon mucosa were prepared from tumor patients (tumor and paired normal tissues from surgical resections) or biofilm-positive biopsies from healthy individuals undergoing screening colonoscopy; homogenates of biofilm-negative colon biopsies from healthy individuals undergoing screening colonoscopy served as controls. After 12 weeks, biofilm-positive, but not biofilm-negative, human colon mucosal homogenates induced colon tumor formation in 3 mouse colon tumor models (germ-free ApcMinΔ850/+;Il10-/- or ApcMinΔ850/+ and specific pathogen-free ApcMinΔ716/+ mice). Remarkably, biofilm-positive communities from healthy colonoscopy biopsies induced colon inflammation and tumors similarly to biofilm-positive tumor tissues. By 1 week, biofilm-positive human tumor homogenates, but not healthy biopsies, displayed consistent bacterial mucus invasion and biofilm formation in mouse colons. 16S rRNA gene sequencing and RNA-Seq analyses identified compositional and functional microbiota differences between mice colonized with biofilm-positive and biofilm-negative communities. These results suggest human colon mucosal biofilms, whether from tumor hosts or healthy individuals undergoing screening colonoscopy, are carcinogenic in murine models of CRC.
Abstract G protein-coupled receptor (GPR)35 is highly expressed in the gastro-intestinal tract, predominantly in colon epithelial cells (CEC), and has been associated with inflammatory bowel diseases (IBD), suggesting a role in gastrointestinal inflammation. The enterotoxigenic Bacteroides fragilis (ETBF) toxin (BFT) is an important virulence factor causing gut inflammation in humans and animal models. We identified that BFT signals through GPR35. Blocking GPR35 function in CECs using the GPR35 antagonist ML145, in conjunction with shRNA knock-down and CRISPRcas-mediated knock-out, resulted in reduced CEC-response to BFT as measured by E-cadherin cleavage, beta-arrestin recruitment and IL-8 secretion. Importantly, GPR35 is required for the rapid onset of ETBF-induced colitis in mouse models. GPR35-deficient mice showed reduced death and disease severity compared to wild-type C57Bl6 mice. Our data support a role for GPR35 in the CEC and mucosal response to BFT and underscore the importance of this molecule for sensing ETBF in the colon.
We present the first, most compact, ultrahigh-resolution, high-speed, distal scanning optical coherence tomography (OCT) endoscope operating at 800 nm. Achieving high speed imaging while maintaining an ultrahigh axial resolution is one of the most significant challenges with endoscopic OCT at 800 nm. Maintaining an ultrahigh axial resolution requires preservation of the broad spectral bandwidth of the light source throughout the OCT system. To overcome this critical limitation we implemented a distal scanning endoscope with diffractive optics to minimize loss in spectral throughput. In this paper, we employed a customized miniature 900 µm diameter DC micromotor fitted with a micro reflector to scan the imaging beam. We integrated a customized diffractive microlens into the imaging optics to reduce chromatic focal shift over the broad spectral bandwidth of the Ti:Sapphire laser of an approximately 150 nm 3dB bandwidth, affording a measured axial resolution of 2.4 µm (in air). The imaging capability of this high-speed, ultrahigh-resolution distal scanning endoscope was validated by performing 3D volumetric imaging of mouse colon in vivo at 50 frames-per-second (limited only by the A-scan rate of linear CCD array in the spectral-domain OCT system and sampling requirements). The results demonstrated that fine microstructures of colon could be clearly visualized, including the boundary between the absorptive cell layer and colonic mucosa as well the crypt patterns. Furthermore, this endoscope was employed to visualize morphological changes in an enterotoxigenic Bacteriodes fragilis (ETBF) induced colon tumor model. We present the results of our feasibility studies and suggest the potential of this system for visualizing time dependent morphological changes associated with tumorigenesis on murine models in vivo.
Pro-carcinogenic bacteria have the potential to initiate and/or promote human colon cancer. However, the mechanism(s) by which a bacterium may trigger carcinogenesis remains unclear. Herein, we show Bacteroides fragilis toxin (BFT) secretion by the human pathobiont enterotoxigenic Bacteroides fragilis (ETBF) triggers in Min Apc /- mice a ETBF pro- carcinogenic inflammatory cascade requiring IL-17R, NF-κB, and Stat3 signaling in colonic epithelial cells (CEC). This signaling promotes the chemokine-dependent recruitment of pro-tumoral myeloid cells. Although necessary, Stat3 activation in CEC upon ETBF colonization is not sufficient to trigger colon tumorigenesis. Notably, IL-17-dependent NF-κB activation in CECs induces a proximal to distal mucosal gradient of C-X-C chemokines, including CXCL1 that mediates CXCR2-expressing polymorphonuclear immature myeloid cell recruitment with parallel onset of ETBF tumorigenesis in the distal colon, explaining the restricted localization of ETBF tumorigenesis. Inhibitors of pathway drivers linking specific microbial mediators to early colon tumorigenesis could disrupt a myeloid/epithelial pro-tumoral loop.
Colorectal cancer (CRC) remains the third most common cancer worldwide, with a growing incidence among young adults. Multiple studies have presented associations between the gut microbiome and CRC, suggesting a link with cancer risk. Although CRC microbiome studies continue to profile larger patient cohorts with increasingly economical and rapid DNA sequencing platforms, few common associations with CRC have been identified, in part due to limitations in taxonomic resolution and differences in analysis methodologies. Complementing these taxonomic studies is the newly recognized phenomenon that bacterial organization into biofilm structures in the mucus layer of the gut is a consistent feature of right-sided (proximal), but not left-sided (distal) colorectal cancer. In the present study, we performed 16S rRNA gene amplicon sequencing and biofilm quantification in a new cohort of patients from Malaysia, followed by a meta-analysis of eleven additional publicly available data sets on stool and tissue-based CRC microbiota using Resphera Insight, a high-resolution analytical tool for species-level characterization. Results from the Malaysian cohort and the expanded meta-analysis confirm that CRC tissues are enriched for invasive biofilms (particularly on right-sided tumors), a symbiont with capacity for tumorigenesis (Bacteroides fragilis), and oral pathogens including Fusobacterium nucleatum, Parvimonas micra, and Peptostreptococcus stomatis. Considered in aggregate, species from the Human Oral Microbiome Database are highly enriched in CRC. Although no detected microbial feature was universally present, their substantial overlap and combined prevalence supports a role for the gut microbiota in a significant percentage (>80%) of CRC cases.
Individuals with sporadic colorectal cancer (CRC) frequently harbor abnormalities in the composition of the gut microbiome; however, the microbiota associated with precancerous lesions in hereditary CRC remains largely unknown. We studied colonic mucosa of patients with familial adenomatous polyposis (FAP), who develop benign precursor lesions (polyps) early in life. We identified patchy bacterial biofilms composed predominately of
Bacteroides fragilis is an extensively studied anaerobic bacterium comprising the normal flora of the human gut. B. fragilis is known to be one of the most commonly isolated species from clinical samples and has been shown to cause a wide range of pathologies in humans [1, 2]. As an opportunistic pathogen B. fragilis can cause abscess formation and bacteremia [2]. Additionally in its enterotoxigenic form, B. fragilis is a known cause of diarrheal illness, is associated with inflammatory bowel disease, and has been recently characterized in patients with colon cancer [3 - 5]. As research in the field of the gut microbiome continues to expand at an ever increasing rate due to advances in the availability of next generation sequencing and analysis tools it is important to outline various molecular methods that can be employed in quickly detecting and isolating relevant strains of B. fragilis. This review outlines methods that are routinely employed in the isolation and detection of B. fragilis, with an emphasis on characterizing enterotoxigenic B. fragilis (ETBF) strains.
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