Safety and outcome of three-dimensional transoral videolaryngoscopic surgery.

BACKGROUND: Transoral videolaryngoscopic surgery (TOVS) is widely used in Japan, and conventional two-dimensional (2D) endoscopic methods have been established. Three-dimensional (3D) endoscopic surgery offers superior distance perception because it provides stereoscopic views. Recently, we have developed 3D endoscopy for TOVS (3D TOVS). METHODS: This study included 46 patients with pharyngeal cancer who underwent 3D TOVS. The perioperative complications and survival curves were retrospectively analyzed. RESULTS: One patient with oropharyngeal cancer who underwent neck dissection and transoral resection simultaneously experienced postoperative hemorrhage of the neck. Another patient with oropharyngeal cancer underwent hemostasis for postoperative pharyngeal hemorrhage. There was one case of aspiration pneumonia. One patient developed cervical lymph node recurrence; however, there was no local recurrence or primary mortality. The 2-year overall survival, disease-specific survival, local control rates, locoregional control rate, and invasive disease-free survival were 90.9%, 100%, 100%, 97.4%, and 79.9%, respectively. CONCLUSIONS: Three-dimensional endoscopy can be safely applied to TOVS.

The gut microbiota-induced kynurenic acid recruits GPR35-positive macrophages to promote experimental encephalitis.

The intricate interplay between gut microbes and the onset of experimental autoimmune encephalomyelitis (EAE) remains poorly understood. Here, we uncover remarkable similarities between CD4+ T cells in the spinal cord and their counterparts in the small intestine. Furthermore, we unveil a synergistic relationship between the microbiota, particularly enriched with the tryptophan metabolism gene EC:1.13.11.11, and intestinal cells. This symbiotic collaboration results in the biosynthesis of kynurenic acid (KYNA), which modulates the recruitment and aggregation of GPR35-positive macrophages. Subsequently, a robust T helper 17 (Th17) immune response is activated, ultimately triggering the onset of EAE. Conversely, modulating the KYNA-mediated GPR35 signaling in Cx3cr1+ macrophages leads to a remarkable amelioration of EAE. These findings shed light on the crucial role of microbial-derived tryptophan metabolites in regulating immune responses within extraintestinal tissues.

Fusobacterium nucleatum infection activates the noncanonical inflammasome and exacerbates inflammatory response in DSS-induced colitis.

Caspase activation results in pyroptosis, an inflammatory cell death that contributes to several inflammatory diseases by releasing inflammatory cytokines and cellular contents. Fusobacterium nucleatum is a periodontal pathogen frequently detected in human cancer and inflammatory bowel diseases. Studies have reported that F. nucleatum infection leads to NLRP3 activation and pyroptosis, but the precise activation process and disease association remain poorly understood. This study demonstrated that F. nucleatum infection exacerbates acute colitis in mice and activates pyroptosis through caspase-11-mediated gasdermin D cleavage in macrophages. Furthermore, F. nucleatum infection in colitis mice induces the enhancement of IL-1⍺ secretion from the colon, affecting weight loss and severe disease activities. Neutralization of IL-1⍺ protects F. nucleatum infected mice from severe colitis. Therefore, F. nucleatum infection facilitates inflammation in acute colitis with IL-1⍺ from colon tissue by activating noncanonical inflammasome through gasdermin D cleavage.

Effect of low oxygen concentration on activation of inflammation by Helicobacter pylori.

The gastrointestinal tract of the human body is characterized by a highly unique oxygenation profile, where the oxygen concentration decreases toward the lower tract, not found in other organs. The epithelial cells lining the mucosa where Helicobacter pylori resides exist in a relatively low oxygen environment with a partial pressure of oxygen (pO2) below 58 mm Hg. However, the contribution of hypoxia to H. pylori-induced host immune responses remains elusive. In this study, we investigated the inflammasome activation induced by H. pylori under hypoxic, compared with normoxic, conditions. Our results indicated that the activation of caspase-1 and the subsequent secretion of IL-1ß were significantly enhanced in infected macrophages under 1% oxygen, compared with those under a normal 20% oxygen concentration. The proliferation of H. pylori under aerobic conditions was 3-fold higher than under microaerophilic conditions, and the bacterial growth was more dependent on CO2 than on oxygen. Also, we observed that hypoxia-induced cytokine production as well as HIF-1α accumulation were both decreased when murine macrophages were treated with an HIF-1α inhibitor, KC7F2. Furthermore, hypoxia enhanced the phagocytosis of H. pylori in an HIF-1α-dependent manner. IL-1ß production was also affected by the HIF-1α inhibitor in a mouse infection model, suggesting the important role of HIF-1α in the host defense system during infection with H. pylori. Our findings provide new insights into the intersection of low oxygen, H. pylori, and inflammation and disclosed how H. pylori under low oxygen tension can aggravate IL-1ß secretion.

A bacterial small RNA regulates the adaptation of Helicobacter pylori to the host environment.

Long-term infection of the stomach with Helicobacter pylori can cause gastric cancer. However, the mechanisms by which the bacteria adapt to the stomach environment are poorly understood. Here, we show that a small non-coding RNA of H. pylori (HPnc4160, also known as IsoB or NikS) regulates the pathogen's adaptation to the host environment as well as bacterial oncoprotein production. In a rodent model of H. pylori infection, the genomes of bacteria isolated from the stomach possess an increased number of T-repeats upstream of the HPnc4160-coding region, and this leads to reduced HPnc4160 expression. We use RNA-seq and iTRAQ analyses to identify eight targets of HPnc4160, including genes encoding outer membrane proteins and oncoprotein CagA. Mutant strains with HPnc4160 deficiency display increased colonization ability of the mouse stomach, in comparison with the wild-type strain. Furthermore, HPnc4160 expression is lower in clinical isolates from gastric cancer patients than in isolates derived from non-cancer patients, while the expression of HPnc4160's targets is higher in the isolates from gastric cancer patients. Therefore, the small RNA HPnc4160 regulates H. pylori adaptation to the host environment and, potentially, gastric carcinogenesis.

Inflammasome Activation Induced by Perfringolysin O of Clostridium perfringens and Its Involvement in the Progression of Gas Gangrene.

Clostridium perfringens (C. perfringens) is Gram-positive anaerobic, spore-forming rod-shaped bacterial pathogen that is widely distributed in nature. This bacterium is known as the causative agent of a foodborne illness and of gas gangrene. While the major virulence factors are the α-toxin and perfringolysin O (PFO) produced by type A strains of C. perfringens, the precise mechanisms of how these toxins induce the development of gas gangrene are still not well understood. In this study, we analyzed the host responses to these toxins, including inflammasome activation, using mouse bone marrow-derived macrophages (BMDMs). Our results demonstrated, for the first time, that C. perfringens triggers the activation of caspase-1 and release of IL-1ß through PFO-mediated inflammasome activation via a receptor of the Nod-like receptor (NLR) family, pyrin-domain containing 3 protein (NLRP3). The PFO-mediated inflammasome activation was not induced in the cultured myocytes. We further analyzed the functional roles of the toxins in inducing myonecrosis in a mouse model of gas gangrene. Although the myonecrosis was found to be largely dependent on the α-toxin, PFO also induced myonecrosis to a lesser extent, again through the mediation of NLRP3. These results suggest that C. perfringens triggers inflammatory responses via PFO-mediated inflammasome activation via NLRP3, and that this axis contributes in part to the progression of gas gangrene. Our findings provide a novel insight into the molecular mechanisms underlying the pathogenesis of gas gangrene caused by C. perfringens.

Gut pathobionts underlie intestinal barrier dysfunction and liver T helper 17 cell immune response in primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a chronic inflammatory liver disease and its frequent complication with ulcerative colitis highlights the pathogenic role of epithelial barrier dysfunction. Intestinal barrier dysfunction has been implicated in the pathogenesis of PSC, yet its underlying mechanism remains unknown. Here, we identify Klebsiella pneumonia in the microbiota of patients with PSC and demonstrate that K. pneumoniae disrupts the epithelial barrier to initiate bacterial translocation and liver inflammatory responses. Gnotobiotic mice inoculated with PSC-derived microbiota exhibited T helper 17 (TH17) cell responses in the liver and increased susceptibility to hepatobiliary injuries. Bacterial culture of mesenteric lymph nodes in these mice isolated K. pneumoniae, Proteus mirabilis and Enterococcus gallinarum, which were prevalently detected in patients with PSC. A bacterial-organoid co-culture system visualized the epithelial-damaging effect of PSC-derived K. pneumoniae that was associated with bacterial translocation and susceptibility to TH17-mediated hepatobiliary injuries. We also show that antibiotic treatment ameliorated the TH17 immune response induced by PSC-derived microbiota. These results highlight the role of pathobionts in intestinal barrier dysfunction and liver inflammation, providing insights into therapeutic strategies for PSC.

Porphyromonas gingivalis triggers NLRP3-mediated inflammasome activation in macrophages in a bacterial gingipains-independent manner.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that has been considered to be one of the bacteria associated with progression of human periodontitis. Subgingival biofilms formed by bacteria, including P. gingivalis, induce chronic inflammation, and activation of inflammasome in the gingival tissue. However, the mechanisms of P. gingivalis-triggering inflammasome activation and the role of bacteria-host interactions are controversial. In this study, we investigated the potential of P. gingivalis for triggering inflammasome activation in human cells and mouse models. We demonstrated that secreted or released factors from bacteria are involved in triggering NLR family, pyrin-domain containing 3 protein (NLRP3) inflammasome in a gingipain-independent manner. Our data indicated that released active caspase-1 and mature IL-1ß are eliminated by proteolytic activity of secreted gingipains. These results elucidate the molecular bases for the mechanisms underlying P. gingivalis-triggered inflammasome activation.

Shigella IpaH7.8 E3 ubiquitin ligase targets glomulin and activates inflammasomes to demolish macrophages.

When nucleotide-binding oligomerization domain-like receptors (NLRs) sense cytosolic-invading bacteria, they induce the formation of inflammasomes and initiate an innate immune response. In quiescent cells, inflammasome activity is tightly regulated to prevent excess inflammation and cell death. Many bacterial pathogens provoke inflammasome activity and induce inflammatory responses, including cell death, by delivering type III secreted effectors, the rod component flagellin, and toxins. Recent studies indicated that Shigella deploy multiple mechanisms to stimulate NLR inflammasomes through type III secretion during infection. Here, we show that Shigella induces rapid macrophage cell death by delivering the invasion plasmid antigen H7.8 (IpaH7.8) enzyme 3 (E3) ubiquitin ligase effector via the type III secretion system, thereby activating the NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing 4 (NLRC4) inflammasomes and caspase-1 and leading to macrophage cell death in an IpaH7.8 E3 ligase-dependent manner. Mice infected with Shigella possessing IpaH7.8, but not with Shigella possessing an IpaH7.8 E3 ligase-null mutant, exhibited enhanced bacterial multiplication. We defined glomulin/flagellar-associated protein 68 (GLMN) as an IpaH7.8 target involved in IpaH7.8 E3 ligase-dependent inflammasome activation. This protein originally was identified through its association with glomuvenous malformations and more recently was described as a member of a Cullin ring ligase inhibitor. Modifying GLMN levels through overexpression or knockdown led to reduced or augmented inflammasome activation, respectively. Macrophages stimulated with lipopolysaccharide/ATP induced GLMN puncta that localized with the active form of caspase-1. Macrophages from GLMN(+/-) mice were more responsive to inflammasome activation than those from GLMN(+/+) mice. Together, these results highlight a unique bacterial adaptation that hijacks inflammasome activation via interactions between IpaH7.8 and GLMN.

Helmholtz illusion makes you look fit only when you are already fit, but not for everyone.

A square filled with horizontal stripes is perceived as thinner than one with vertical stripes (Helmholtz illusion). This is not consistent with a common belief that horizontally striped clothing makes a person look fatter, and studies on this problem have shown inconsistent results. Here, we demonstrate three factors that could have complicated the issue. First, the Helmholtz effect is stronger for a thin figure than for a fat one, with possible reversal for the latter. Second, we found large variability across participants, suggesting dependence on features to attend. Third, there was strong hysteresis as to the order of testing fat and thin figures, suggesting the effect of surrounding people in daily life. There can be yet other factors, but we should note that this apparently simple case of application of a geometrical illusion in daily perception should be taken as a rather complex phenomenon.

Low-level laser therapy for spinal cord injury in rats: effects of polarization.

The effects of laser polarization on the efficacy of near-infrared low-level laser therapy for spinal cord injury (SCI) are presented. Rat spinal cords were injured with a weight-drop device, and the lesion sites were directly irradiated with a linearly polarized 808-nm diode laser positioned either perpendicular or parallel to the spine immediately after the injury and daily for five consecutive days. Functional recovery was assessed daily by an open-field test. Regardless of the polarization direction, functional scores of SCI rats that were treated with the 808-nm laser irradiation were significantly higher than those of SCI alone group (Group 1) from day 5 after injury. The locomotive function of SCI rats irradiated parallel to the spinal column (Group 3) was significantly improved from day 10 after injury, compared to SCI rats treated with the linear polarization perpendicular to the spinal column (Group 2). There were no significant differences in ATP contents in the injured tissue among the three groups. We speculate that the higher efficacy with parallel irradiation is attributable to the deeper light penetration into tissue with anisotropic scattering.

Shigella IpaH0722 E3 ubiquitin ligase effector targets TRAF2 to inhibit PKC-NF-κB activity in invaded epithelial cells.

NF-κB plays a central role in modulating innate immune responses to bacterial infections. Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation. The invasion of and subsequent replication of Shigella within epithelial cells is recognized by various pathogen recognition receptors as pathogen-associated molecular patterns. These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway. Here, we show the inhibition of the NF-κB activation by the delivery of the IpaH E3 ubiquitin ligase family member IpaH0722 using Shigella's type III secretion system. IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation.

The Shigella OspC3 effector inhibits caspase-4, antagonizes inflammatory cell death, and promotes epithelial infection.

Caspase-mediated inflammatory cell death acts as an intrinsic defense mechanism against infection. Bacterial pathogens deploy countermeasures against inflammatory cell death, but the mechanisms by which they do this remain largely unclear. In a screen for Shigella flexneri effectors that regulate cell death during infection, we discovered that Shigella infection induced acute inflammatory, caspase-4-dependent epithelial cell death, which is counteracted by the bacterial OspC3 effector. OspC3 interacts with the caspase-4-p19 subunit and inhibits its activation by preventing caspase-4-p19 and caspase-4-p10 heterodimerization by depositing the conserved OspC3 X1-Y-X2-D-X3 motif at the putative catalytic pocket of caspase-4. Infection of guinea pigs with a Shigella ospC3-deficient mutant resulted in enhanced inflammatory cell death and associated symptoms, correlating with decreased bacterial burdens. Salmonella Typhimurium and enteropathogenic Escherichia coli infection also induced caspase-4-dependent epithelial death. These findings highlight the importance of caspase-4-dependent innate immune responses and demonstrate that Shigella delivers a caspase-4-specific inhibitor to delay epithelial cell death and promote infection.

Diffuse light reflectance signals as potential indicators of loss of viability in brain tissue due to hypoxia: charge-coupled-device-based imaging and fiber-based measurement.

Brain tissue is highly vulnerable to ischemia/hypoxia, and real-time monitoring of its viability is important. By fiber-based measurements for rat brain, we previously observed a unique triphasic reflectance change (TRC) after a certain period of time after hypoxia. After TRC, rats could not be rescued, suggesting that TRC can be used as an indicator of loss of brain tissue viability. In this study, we investigated this diffuse-reflectance change due to hypoxia in three parts. First, we developed and validated a theoretical method to quantify changes in the absorption and reduced scattering coefficients involved in TRC. Second, we performed charge-coupled-device-based reflectance imaging of the rat brain during hypoxia followed by reoxygenation to examine spatiotemporal characteristics of the reflectance and its correlation with reversibility of brain tissue damage. Third, we made simultaneous imaging and fiber-based measurement of the reflectance for the rat to compare signals obtained by these two modalities. We observed a nontriphasic reflectance change by the imaging, and it was associated with brain tissue viability. We found that TRC measured by the fibers preceded the reflectance-signal change captured by the imaging. This time difference is attributable to the different observation depths in the brain with these two methods.

The Shigella flexneri effector OspI deamidates UBC13 to dampen the inflammatory response.

Many bacterial pathogens can enter various host cells and then survive intracellularly, transiently evade humoral immunity, and further disseminate to other cells and tissues. When bacteria enter host cells and replicate intracellularly, the host cells sense the invading bacteria as damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) by way of various pattern recognition receptors. As a result, the host cells induce alarm signals that activate the innate immune system. Therefore, bacteria must modulate host inflammatory signalling and dampen these alarm signals. How pathogens do this after invading epithelial cells remains unclear, however. Here we show that OspI, a Shigella flexneri effector encoded by ORF169b on the large plasmid and delivered by the type ΙΙΙ secretion system, dampens acute inflammatory responses during bacterial invasion by suppressing the tumour-necrosis factor (TNF)-receptor-associated factor 6 (TRAF6)-mediated signalling pathway. OspI is a glutamine deamidase that selectively deamidates the glutamine residue at position 100 in UBC13 to a glutamic acid residue. Consequently, the E2 ubiquitin-conjugating activity required for TRAF6 activation is inhibited, allowing S. flexneri OspI to modulate the diacylglycerol-CBM (CARD-BCL10-MALT1) complex-TRAF6-nuclear-factor-κB signalling pathway. We determined the 2.0 Å crystal structure of OspI, which contains a putative cysteine-histidine-aspartic acid catalytic triad. A mutational analysis showed this catalytic triad to be essential for the deamidation of UBC13. Our results suggest that S. flexneri inhibits acute inflammatory responses in the initial stage of infection by targeting the UBC13-TRAF6 complex.

Bacteria and host interactions in the gut epithelial barrier.

The gut mucosa acts as a barrier against microbial invaders, whereas resident commensal and foreign invading bacteria interact intimately with the gut epithelium and influence the host cellular and immune systems. The epithelial barrier serves as an infectious foothold for many bacterial pathogens and as an entry port for pathogens to disseminate into deeper tissues. Enteric bacterial pathogens can efficiently infect the gut mucosa using highly sophisticated virulence mechanisms that allow bacteria to circumvent the defense barriers in the gut. We provide an overview of the components of the mucosal barrier and discuss the bacterial stratagems that circumvent these barriers with particular emphasis on the roles of bacterial effector proteins.

Shigella are versatile mucosal pathogens that circumvent the host innate immune system.

The intestinal mucosa is equipped with multiple innate immune defense systems that sense bacterial infection, transmit alarm signals to the immune system, defeat intruding bacteria, and renew damaged and aging epithelial cells. Nevertheless, mucosal bacterial pathogens have versatile pathogenic mechanisms that modulate the host inflammatory and immune responses, manipulate host cell death and survival signal pathways, and renovate the injured epithelium. These properties enable pathogens to adapt to the intestinal mucosal environment, exploit cellular and immune functions, and facilitate infection. Here we review current topics on host defense mechanisms against bacterial infection and the countermeasures that Shigella use to evade the innate immune system.

BabA-mediated adherence is a potentiator of the Helicobacter pylori type IV secretion system activity.

Chronic infection of Helicobacter pylori in the stomach mucosa with translocation of the bacterial cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV secretion system (TFSS) into host epithelial cells are major risk factors for gastritis, gastric ulcers, and cancer. The blood group antigen-binding adhesin BabA mediates the adherence of H. pylori to ABO/Lewis b (Le(b)) blood group antigens in the gastric pit region of the human stomach mucosa. Here, we show both in vitro and in vivo that BabA-mediated binding of H. pylori to Le(b) on the epithelial surface augments TFSS-dependent H. pylori pathogenicity by triggering the production of proinflammatory cytokines and precancer-related factors. We successfully generated Le(b)-positive cell lineages by transfecting Le(b)-negative cells with several glycosyltransferase genes. Using these established cell lines, we found increased mRNA levels of proinflammatory cytokines (CCL5 and IL-8) as well as precancer-related factors (CDX2 and MUC2) after the infection of Le(b)-positive cells with WT H. pylori but not with babA or TFSS deletion mutants. This increased mRNA expression was abrogated when Le(b)-negative cells were infected with WT H. pylori. Thus, H. pylori can exploit BabA-Le(b) binding to trigger TFSS-dependent host cell signaling to induce the transcription of genes that enhance inflammation, development of intestinal metaplasia, and associated precancerous transformations.

Shigella deploy multiple countermeasures against host innate immune responses.

Although the intestinal epithelium is equipped with multiple defense systems that sense bacterial components, transmit alarms to the immune system, clear the bacteria, and renew the injured epithelial lining, mucosal bacterial pathogens are capable of efficiently colonizing the intestinal epithelium, because they have evolved systems that modulate the inflammatory and immune responses of the host and exploit the harmful environments as replicative niches. In this review we highlight current topics concerning Shigella's tactics that interfere with the innate immune systems.

Assessment of tissue alteration in skin after interaction with photomechanical waves used for gene transfection.

BACKGROUND AND OBJECTIVE: We previously delivered a therapeutic gene to skin grafts of rats by using photomechanical waves (PMWs), also called laser-induced stress waves (LISWs), with the objective of enhancing adhesion of grafted tissue. The objective of this study was to evaluate tissue alterations that are possibly caused by PMWs used for gene delivery on the basis of immunohistochemistry and electron microscopy. MATERIALS AND METHODS: PMWs were generated by irradiating an elastic laser target (rubber disk) with 532 nm nanosecond laser pulses from a (of) Q-switched Nd:YAG laser. Tissue alterations were evaluated by histological analysis using hematoxylin and eosin (H&E) staining and immunohistochemical stainings, including anti-rat CD68 antibody staining to identify macrophages for detection of inflammation and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining for assessment of apoptosis. Morphological changes of cell membranes and organelles were evaluated by transmission electron microscopy. RESULTS: Skin exposed to PMWs that were generated at a laser fluence of 1.2 J/cm(2) (42 MPa in peak pressure), which is the optimum laser fluence (pressure) for therapeutic gene delivery to skin graft, showed no noticeable damage. At fluences higher than 1.8 J/cm(2) (>51 MPa), fragmentation of nuclei was observed and the number of CD68-positive cells increased remarkably. No significant increases in the numbers of TUNEL-positive keratinocytes and fibroblasts were observed at 1.2 J/cm(2). At fluences higher than 1.8 J/cm(2), the averaged ratio of TUNEL-positive cells also increased. The results of electron microscopy revealed that PMWs generated at 1.2 J/cm(2) caused neither damage to the cell membrane, nuclear membrane, or organelles. CONCLUSION: We observed no noticeable tissue alteration under the optimum laser irradiation conditions used for therapeutic gene delivery to a skin graft, demonstrating low invasiveness of our PMW-based gene transfection.