EFFICACY AND SAFETY OF A DEXAMETHASONE-BASED MOUTHWASH TO PREVENT CHEMOTHERAPY-INDUCED STOMATITIS IN WOMEN WITH BREAST CANCER: A MULTICENTRE, OPEN-LABEL, RANDOMISED PHASE 2 STUDY.

PURPOSE: No standard approach other than oral care is available for preventing chemotherapy-induced stomatitis in patients with breast cancer. In this randomized, controlled phase 2 trial, we aimed to assess the efficacy and safety of a dexamethasone-based mouthwash in preventing chemotherapy-induced stomatitis in patients with early breast cancer. BASIC PROCEDURES: Patients with breast cancer scheduled for epirubicin and cyclophosphamide (EC) or docetaxel and cyclophosphamide (TC) therapy were selected and allocated in a 1:1 ratio to the intervention and control groups. The intervention group received chemotherapy, oral care, and a dexamethasone-based mouthwash, whereas the control group received chemotherapy and oral care. The primary endpoint was the incidence of stomatitis. This was a phase 2 study, and the significance level for the analysis of the primary endpoint was set a priori at 0.2. MAIN FINDINGS: Data pertaining to 58 patients in the control group and 59 patients in the intervention group were analyzed. Stomatitis incidence was 55% and 38% in the control and intervention groups, respectively (risk ratio, 0.68; 80% confidence interval, 0.52-0.88; P = .052). Stomatitis severity was lower in the intervention group than in the control group (P = .03). The proportion of patients who adhered to the mouthwash regimen was 87% (interquartile range, 67.8%-95.3%). No severe oral infections were observed. PRINCIPAL CONCLUSIONS: The dexamethasone-based mouthwash safely reduced stomatitis incidence and severity in patients receiving chemotherapy for early breast cancer. Phase 3 clinical trials are warranted for validating our results.

Protocol for studying the efficiency of ChemoCalc software in helping patients to understand drug treatment costs for breast cancer: A multicenter, open-label, randomized phase 2 study.

Survival of patients with breast cancer can be prolonged by treatment with drugs, particularly new molecular-targeted drugs. However, these agents can be expensive and such treatments can be "an economic burden." In this ongoing trial, we aim to assess the usefulness of ChemoCalc, a software package for calculating drug costs, to help patients understand the financial outlays. In this multicenter, randomized controlled phase 2 trial, 106 patients with advanced breast cancer will be assigned to either the "ChemoCalc" or "Usual Explanation" group. Treatment using ChemoCalc will be discussed with patients in the ChemoCalc group, whereas standard treatments, without using ChemoCalc, will be discussed with patients in the Usual Explanation group. Subsequently, the participants will decide the treatment and complete a five-grade evaluation questionnaire; those in the Usual Explanation group will receive information about ChemoCalc. Investigators will report if patients subsequently decide to change treatments. The primary endpoint will be the scores of two key questions compared between the groups: "Did you understand the cost of treatment in today's discussion?" and "Do you think the cost of treatment is important in choosing a treatment?". The secondary endpoints will be to compare discrepancies between treatments recommended by physicians and those selected by patients, the time required for discussion, other questionnaire factors, and the relationship between Comprehensive Score for Financial Toxicity tool and treatment selection. This will be the first randomized controlled trial to assess the efficacy of software to help patients understand drug cost estimates and whether it subsequently affects treatment choice. This study will be conducted according to the CONSORT statement. All participants will sign a written consent form. The study protocol was reviewed and approved by the Clinical Research Review Board of Nagasaki University (19070801). The protocol (version 1) was designed and will be conducted in accordance with the Declaration of Helsinki (1964) and the Ethical Guidelines for Medical and Health Research Involving Human Subjects (2017). The findings will be disseminated through scientific and professional conferences, and in peer-reviewed journals. TRIAL REGISTRATION: UMIN Clinical Trials Registry, UMIN000039904. https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000041968.

Naringenin suppresses Toll-like receptor 2-mediated inflammatory responses through inhibition of receptor clustering on lipid rafts.

Toll-like receptors (TLRs) are important innate immune receptors that sometimes cause excessive inflammatory responses and a perpetuated inflammatory loop that can be involved in inflammatory and autoimmune diseases. TLR2 recognizes bacterial lipoproteins in association with TLR1 or TLR6, and triggers inflammatory responses through activation of the transcription factor NF-κB. Naringenin, a type of citrus flavonoid, has been shown to possess anti-inflammatory properties, but its detailed action against TLR2 remains to be fully elucidated. The present study was designed to determine whether naringenin affects the inflammatory responses triggered by TLR2. Naringenin inhibited proinflammatory cytokine production and attenuated NF-κB activation in cells stimulated with a synthetic triacylated-type lipopeptide known as a TLR2/TLR1 ligand, as well as a synthetic diacylated-type lipopeptide known as a TLR2/TLR6 ligand. Moreover, a similar inhibitory effect was observed in cells stimulated with a crude lipophilic fraction extracted from Staphylococcus aureus cell walls and in cells stimulated with S. aureus cells. Furthermore, we showed that such an effect is caused by inhibition of TLR2 clustering in lipid rafts on the cell membrane. These results suggest that naringenin suppresses the inflammatory responses induced by TLR2 signal transduction. Our findings indicate a novel anti-inflammatory property of naringenin, mediated through the regulation of cell surface TLR2 functioning.

Lipoprotein Extraction from Microbial Membrane and Lipoprotein/Lipopeptide Transfection into Mammalian Cells.

Microbial lipoproteins/lipopeptides are important virulence factors for periodontal diseases. The membrane lipoproteins from Mycoplasma salivarium or Tannerella forsythia can be easily extracted by exploiting a characteristic feature of Triton X-114: its aqueous nature at low temperatures (0-4 °C), which is absent at room temperature (25-37 °C). Transfection of these lipopeptides into macrophages was performed using the protein transfection reagent, PULSin.

Gasdermin D-independent release of interleukin-1ß by living macrophages in response to mycoplasmal lipoproteins and lipopeptides.

Interleukin-1ß (IL-1ß) plays pivotal roles in controlling bacterial infections and is produced after the processing of pro-IL-1ß by caspase-1, which is activated by the inflammasome. In addition, caspase-1 cleaves the cytosolic protein, gasdermin-D (GSDMD), whose N-terminal fragment subsequently forms a pore in the plasma membrane, leading to the pyroptic cell-death-mediated release of IL-1ß. Living cells can also release IL-1ß via GSDMD pores or other unconventional secretory pathways. However, the precise mechanisms are poorly defined. Here, we show that lipoproteins from Mycoplasma salivarium (MsLP) and Mycoplasma pneumoniae (MpLP) and an M. salivarium-derived lipopeptide (FSL-1), which are activators of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, induce IL-1ß release from mouse bone-marrow-derived macrophages (BMMs) without inducing cell death. The levels of IL-1ß release induced by MsLP, MpLP and FSL-1 were more than 100 times lower than those induced by the canonical NLRP3 activator nigericin. The IL-1ß release-inducing activities of MsLP, MpLP and FSL-1 were not attenuated in BMMs from GSDMD-deficient mice. Furthermore, both active caspase-1 and cleaved GSDMD were detected in response to transfection of FSL-1 into the cytosol of BMMs, but the release of IL-1ß was unaffected by GSDMD deficiency. Meanwhile, punicalagin, a membrane-stabilizing agent, drastically down-regulated the release of IL-1ß in response to FSL-1. These results suggest that mycoplasmal lipoprotein/lipopeptide-induced IL-1ß release by living macrophages is not mediated via GSDMD but rather through changes in membrane permeability.

Study protocol for efficacy and safety of steroid-containing mouthwash to prevent chemotherapy-induced stomatitis in women with breast cancer: a multicentre, open-label, randomised phase 2 study.

INTRODUCTION: Stomatitis is a frequent adverse event in patients undergoing chemotherapy for breast cancer. Stomatitis can hamper oral nutrition resulting in malnutrition, reduce quality of life and introduce the need for dose reductions and interruption of chemotherapy; however, there is currently no standard approach for preventing chemotherapy-induced stomatitis. We aimed to assess the safety and efficacy of a dexamethasone-based elixir mouthwash for preventing chemotherapy-induced stomatitis in patients with early breast cancer. METHODS AND ANALYSIS: In this multicenter, randomised, controlled phase 2 trial, we will randomly assign 120 women with early breast cancer undergoing chemotherapy to use of a dexamethasone-based elixir or standard oral care, to compare their preventive effects on chemotherapy-induced stomatitis. Patients will be assigned in a 1:1 ratio. Patients in the intervention group will receive chemotherapy, oral care and a dexamethasone-based elixir (10 mL 0.1 mg/mL; swish for 2 min and spit, four times daily for 9 weeks), and patients in the control group will receive chemotherapy and oral care. The primary endpoint is the difference in incidence of stomatitis between the two groups. The sample size allows for the detection of a minimum difference of 20% in the incidence of stomatitis between the two groups. Secondary endpoints are severity of stomatitis, duration of stomatitis, completion rate of chemotherapy and adverse events. ETHICS AND DISSEMINATION: All participants signed a written consent form, and the study protocol has been reviewed and approved by the Clinical Research Review Board of Nagasaki University (CRB7180001). TRIAL REGISTRATION NUMBER: UMIN Clinical Trials Registry (UMIN000030489).

Cephalic Vein Cut-down for Totally Implantable Central Venous Access Devices With Preoperative Ultrasonography by Surgical Residents.

BACKGROUND/AIM: Cephalic vein (CV) cut-down for totally implantable central venous access devices (TICVADs) is not frequently used due to its low success rate. We compared the outcomes of CV cut-down using preoperative ultrasonography (US) performed by experienced surgeons versus surgical residents. PATIENTS AND METHODS: From December 2015 to December 2017, 10 surgeons implanted 212 TICVADs using CV cut-down with preoperative US. The surgeons were divided into two groups of five each: surgical residents (Group A, n=124 procedures) and experienced surgeons (Group B, n=88 procedures). Duration of operation time, completion rate, and complications were retrospectively analyzed. RESULTS: The completion rate was significantly higher in Group A (98.4% versus 92.0%, p=0.04). Duration of operation time (45.2±14.5 versus 42.0±13.1 minutes, p=0.22), rates of early complications (1.6% versus 1.1%, p=0.77) and late complications (3.2% versus 2.3%, p=0.68) were equivalent between the two groups. No fatal complications occurred in either group. CONCLUSION: CV cut-down can be safely performed by surgical residents under the use of preoperative US.

MyD88 signaling causes autoimmune sialadenitis through formation of high endothelial venules and upregulation of LTß receptor-mediated signaling.

Autoimmune sialadenitis (AS), chronic inflammation of the salivary glands (SGs) with focal lymphocyte infiltration, appears in autoimmune diseases such as SjÓ§gren's syndrome. The pathological role of MyD88-dependent innate immune signaling in autoimmune diseases including AS has been studied using mouse models, such as NOD mice. Although AS development in NOD mice was reported to be suppressed by Myd88 deficiency, its specific role remains unclear. Here, we determined the potent suppressive effects of Myd88 deficiency on AS development in lupus-prone B6/lpr mice, which have lymphoproliferation abnormalities, and also in NOD mice, which have no lymphoproliferation abnormalities. This indicates that MyD88 signaling triggers AS through both lymphoproliferation-dependent and -independent mechanisms. To address the MyD88-dependent lymphoproliferation-independent AS manifestation, SGs from C57BL/6 mice were analyzed. Remarkable upregulation of Glycam1 and high endothelial venule (HEV)-associated changes were unexpectedly found in Myd88+/+ mice, compared with Myd88-/- mice. MyD88-dependent HEV-associated changes were also observed in NOD mice. Additionally, Lta, Ltb, and Ltbr in SGs of NOD mice were lowered by Myd88 deficiency. Interestingly, LTßR-induced HEV-associated gene expression in cultured cells was impaired by Myd88 deficiency. Our findings highlight novel roles for MyD88 in AS development, which imply the existence of MyD88-dependent HEV formation in ectopic lymphoid neogenesis.

Differences in interleukin-1ß release-inducing activity of Candida albicans toward dendritic cells and macrophages.

OBJECTIVE: The purpose of this study is to elucidate differences in the mechanism of the IL-1ß release-inducing activity of Candida albicans toward dendritic cells and macrophages because IL-1ß is one of the proinflammatory cytokines which is crucial in host defense against candidiasis. DESIGN: Two C. albicans strains were used in this study. One strain is uridine-auxotrophic (CAI4) that needs uridine to grow and form hyphae, and another is a strain without any specific auxotrophy (pACT1-GFP), which forms hyphae naturally by culturing with serum components. Murine macrophage and dendritic cell lines were primed with LPS and then stimulated with C. albicans CAI4 or pACT1-GFP. RESULTS: Both strains of C. albicans induced IL-1ß release from dendritic cells, and C. albicans pACT1-GFP induced IL-1ß release but CAI4 induced little amounts in macrophages. These differences were suggested to be due to the difference in the amount of extracellular ATP released in the cell culture supernatants induced by C. albicans CAI4 or pACT1-GFP. For induction of IL-1ß release from both macrophages and dendritic cells by C. albicans, direct contacts of the microbes with cells were required. In addition, macrophages required morphological change of C. albicans from yeast to hyphae for induction of IL-1ß release, whereas dendritic cells did not require it. Dead C. albicans could induce IL-1ß release from dendritic cells, but could not from macrophages. CONCLUSIONS: There are different mechanisms by which C. albicans induces IL-1ß release from dendritic cells and macrophages.

Validation of a Novel Diagnostic Kit Using the Semidry Dot-Blot Method to Detect Metastatic Lymph Nodes in Breast Cancer: Distinguishing Macrometastases From Nonmacrometastases.

BACKGROUND: The semidry dot-blot method is a diagnostic procedure for detecting lymph node (LN) metastases using the presence of cytokeratin (CK) in lavage fluid from sectioned LNs. We evaluated 2 novel kits that use newly developed anti-CK-19 antibodies to diagnose LN metastases in breast cancer. PATIENTS AND METHODS: We examined 159 LNs dissected that we sliced at 2-mm intervals and washed with phosphate-buffered saline. The suspended cells in the lavage were centrifuged and lysed to extract protein. This extracted protein was used with a low-power and a high-power kit to diagnose LN metastasis. Diagnoses on the basis of the kits were compared with pathological diagnoses. RESULTS: Of the 159 LNs, 68 were assessed as positive and 91 as negative in permanent section examination. Sensitivity, specificity, and accuracy of the low-power kit for detecting LN metastases was 83.8%, 100%, and 93.1%, respectively. Those of the high-power kit were 92.6%, 92.3%, and 92.5%, respectively. Combining the low- and high-power kit results, those for distinguishing macrometastases were 94.5%, 95.2%, and 95.0%, respectively. Diagnosis was achieved in approximately 20 minutes, at a cost of less than $30 USD. CONCLUSION: The kits were accurate, fast, and cost-effective in diagnosing LN metastases without the loss of LN tissue.

Activation of nucleotide-binding domain-like receptor containing protein 3 inflammasome in dendritic cells and macrophages by Streptococcus sanguinis.

Streptococcus sanguinis is frequently isolated from the blood of patients with infective endocarditis and contributes to the pathology of this disease through induction of interleukin (IL)-1ß responsible for the development of the disease. However, the mechanism of IL-1ß induction remains unknown. In this study, S. sanguinis activated a murine dendritic cell (DC) to induce IL-1ß and this activity was attenuated by silencing the mRNAs of nucleotide-binding domain-like receptor containing protein 3 (NLRP3) and caspase-1. S. sanguinis induced IL-1ß production in murine bone marrow-derived macrophage, but this activity was significantly reduced in bone marrow-derived macrophages from NLRP3-, apoptosis-associated speck-like protein containing a caspase-recruitment domain-, and caspase-1-deficient mice. DC phagocytosed S. sanguinis cells, followed by the release of adenosine triphosphate (ATP). The ATP-degradating enzyme attenuated the release of ATP and IL-1ß. The inhibitors for ATP receptor reduced IL-1ß release in DC. These results strongly suggest that S. sanguinis has the activity to induce IL-1ß through the NLRP3 inflammasome in macrophage and DC and interaction of purinergic receptors with ATP released is involved in expression of the activity.

Evaluation of totally implantable central venous access devices with the cephalic vein cut-down approach: Usefulness of preoperative ultrasonography.

BACKGROUND: The aims of this retrospective study, were to evaluate totally implantable central venous access device (TICVAD) implantation and to validate the efficacy of preoperative ultrasonography. METHODS: A total of 380 cases implanted with TICVADs were divided into four groups: cut-downs with ultrasonography (group A, n = 112); cut-downs without ultrasonography (group B, n = 37); venous puncture (group C, n = 122); and replacements using the existing catheter (group D, n = 109). Operation time, completion rate, and complications were compared. RESULTS: The average operating time was 41.7, 52.4, and 40.6 min in groups A, B (P < 0.01), and C, respectively. Group A and B experienced no postoperative pneumothorax, arterial puncture, or pinch-off syndrome. Completion rates were 93.7% in group A and 86.5% in group B. Preoperative ultrasonography identified the cephalic vein in 94.1% of subjects with an average diameter of 3.1 mm and depth of 10.2 mm. Identifying convergence of the cephalic vein and the axillary vein improved the completion rate. CONCLUSIONS: This study showed that the cephalic vein cut-down approach for TICVAD implantation reduced complications. Preoperative ultrasonography resulted in a shorter operating time and higher completion rate.

A novel diagnostic method targeting genomic instability in intracystic tumors of the breast.

BACKGROUND: Even after needle biopsy, the preoperative differential diagnoses of intracystic tumors of the breast are challenging because of their nonspecific radiological characteristics and subtle cytological and histological appearance. The aim of this study is to investigate a novel diagnostic method, targeting genomic instability (GIN) in intracystic tumors of the breast, using tumor DNA from samples obtained by fine-needle aspiration biopsy (FNAB). METHODS: Thirteen consecutive intracystic tumors of the breast, including five cancers and eight benign tumors, were studied. Three FNAB passages per tumor were used for array comparative genomic hybridization (aCGH) analysis to quantify GIN in each tumor. Tumor DNA from the main tumor, taken from formalin-fixed, paraffin-embedded (FFPE) blocks corresponding to FNAB samples, was also analyzed to compare cytogenetic profiles between these sample types. RESULTS: After three FNAB passages, an average of 7.09 µg (0.24-25.0 µg) of DNA was obtained. The quality of the DNA and the aCGH data was excellent, as judged by the mean derivative log ratio spread (DLRSpread) of 0.22 (0.15-0.29). The cytogenetic profiles of paired FNAB and main tumor FFPE samples were highly similar, with an average concordance rate of 97.7 % (81.2-100 %). aCGH analysis from FNAB samples showed significantly more GIN in cancers than in benign tumors, with mean frequencies of aberrant chromosomal regions of 17.5 and 0.34 %, respectively (Wilcoxon's rank sum test, P = 0.0016). CONCLUSIONS: Our novel diagnostic method, which targets GIN, can clearly distinguish cancers from benign tumors of breast intracystic lesions with minimal invasion, thereby avoiding the need for surgical excisional biopsy.

MyD88 deficiency alters expression of antimicrobial factors in mouse salivary glands.

The surfaces of oral mucosa are protected from infections by antimicrobial proteins and natural immunoglobulins that are constantly secreted in saliva, serving as principal innate immune defense in the oral cavity. MyD88 is an important adaptor protein for signal transduction downstream of Toll-like receptors and TACI, receptors for regulation of innate immunity and B cell responses, respectively. Although MyD88-mediated signaling has a regulatory role in the intestinal mucosal immunity, its specific role in the oral cavity has remained elusive. In the present study, we assessed the influence of MyD88 deficiency on the oral innate defense, particularly the expression of antimicrobial proteins in salivary glands and production of salivary basal immunoglobulins, in mice. Microarray analysis of the whole tissues of submandibular glands revealed that the expression of several genes encoding salivary antimicrobial proteins, such as secretory leukocyte peptidase inhibitor (SLPI), S100A8, and lactotransferrin, was reduced due to MyD88 deficiency. Histologically, SLPI-expressing acinar cells were evidently decreased in the glands from MyD88 deficient mice compared to wild-type mice. Flow cytometric analysis revealed that B cell populations, including B-1 cells and IgA+ plasma cells, residing in submandibular glands were increased by MyD88 deficiency. The level of salivary anti-phosphorylcholine IgA was elevated in MyD88 deficient mice compared to wild-type mice. Thus, this study provides a detailed description of the effect of MyD88 deficiency on expression of several salivary antimicrobial factors in mice, illustrating the role for MyD88-mediated signaling in the innate immune defense in the oral cavity.

Novel diagnostic procedure for determining metastasis to sentinel lymph nodes in breast cancer using a semi-dry dot-blot method.

We developed an easy, quick and cost-effective detection method for lymph node metastasis called the semi-dry dot-blot (SDB) method, which visualizes the presence of cancer cells with washing of sectioned lymph nodes by anti-pancytokeratin antibody, modifying dot-blot technology. We evaluated the validity and efficacy of the SDB method for the diagnosis of lymph node metastasis in a clinical setting (Trial 1). To evaluate the validity of the SDB method in clinical specimens, 180 dissected lymph nodes from 29 cases, including breast, gastric and colorectal cancer, were examined. Each lymph node was sliced at the maximum diameter and the sensitivity, specificity and accuracy of the SDB method were determined and compared with the final pathology report. Metastasis was detected in 32 lymph nodes (17.8%), and the sensitivity, specificity and accuracy of the SDB method were 100, 98.0 and 98.3%, respectively (Trial 2). To evaluate the efficacy of the SDB method in sentinel lymph node (SLN) biopsy, 174 SLNs from 100 cases of clinically node-negative breast cancer were analyzed. Each SLN was longitudinally sliced at 2-mm intervals and the sensitivity, specificity, accuracy and time required for the SDB method were determined and compared with the intraoperative pathology report. Metastasis was detected in 15 SLNs (8.6%), and the sensitivity, specificity, accuracy and mean required time of the SDB method were 93.3, 96.9, 96.6 and 43.3 min, respectively. The SDB method is a novel and reliable modality for the intraoperative diagnosis of SLN metastasis.

Differences in recognition of wild-type and lipoprotein-deficient strains of oral Streptococci in vitro and in vivo.

Whole cells of wild-type strains of Streptococcus gordonii and Streptococcus mutans induced Toll-like receptor 2 (TLR2)-mediated nuclear factor-κB (NF-κB) activation, whereas those of lipoprotein (LP)-deficient strains did not. All strains upregulated the proliferation of TLR2(+/+) splenocytes more strongly than TLR2(-/-) splenocytes. However, significant differences were not observed between the cytokine-inducing activities of wild-type and LP-deficient strains toward TLR2(+/+) and TLR2(-/-) splenocytes. Muramyl dipeptide as well as whole cells not only induced nucleotide-binding oligomerization domain 2 (NOD2)-mediated activation of NF-κB but also enhanced the proliferation of TLR2(-/-) as well as TLR2(+/+) splenocytes. Wild-type strains of these streptococci were more resistant to clearance from blood and organs (liver and spleen) in TLR2(+/+) but not TLR2(-/-) mice and induced production of larger amounts of blood TNF-α than the LP-deficient strains. Wild-type strains of both species adhered to human vascular endothelial cells more strongly than did the LP-deficient strains. Thus, this study suggested that LP plays an important role in the recognition of these streptococci by the host in vivo as well as in vitro and that these streptococci possess some components recognized by NOD2 and/or TLR2 that are involved in the mitogenic activity toward splenocytes.

Mycoplasma removal from cell culture using antimicrobial photodynamic therapy.

OBJECTIVE: The objective of this research was to determine the effectiveness of antimicrobial photodynamic therapy (aPDT) in the removal of mycoplasmas from contaminated cells. BACKGROUND DATA: Mycoplasmas often contaminate cell cultures. The cell-contaminating mycoplasmas are removed by antibiotics, but the use of antibiotics usually induces antibiotic-resistant bacteria. aPDT is expected to be a possible alternative to antibiotic treatments for suppressing infections. MATERIALS AND METHODS: Mycoplasma salivarium (Ms)-infected human embryonic kidney (HEK) 293 cells were irradiated using a red light-emitting diode (LED) in the presence of methylene blue (MB) as a photosensitizer. The Ms viable count was determined using culture on agar plates or using a mycoplasma detection kit. RESULTS: aPDT performed using red LED irradiation was effective in decreasing live Ms in the presence of MB without damaging the HEK293 cells. aPDT removed live Ms from the infected cells after washing the cells with sterilized phosphate-buffered saline (PBS) to decrease the initial number of live Ms before aPDT. CONCLUSIONS: This study suggests that aPDT could remove mycoplasmas from contaminated cells.

Chlamydophila pneumoniae in human immortal Jurkat cells and primary lymphocytes uncontrolled by interferon-γ.

Lymphocytes are a potential host cell for Chlamydophila pneumoniae, although why the bacteria must hide in lymphocytes remains unknown. Meanwhile, interferon (IFN)-γ is a crucial factor for eliminating chlamydiae from infected cells through indoleamine 2,3-dioxygenase (IDO) expression, resulting in depletion of tryptophan. We therefore assessed if lymphocytes could work as a shelter for the bacteria to escape IFN-γ. C. pneumoniae grew normally in human lymphoid Jurkat cells, even in the presence of IFN-γ or under stimulation with phorbol myristate acetate plus ionomycin. Although Jurkat cells expressed IFN-γ receptor CD119, their lack of IDO expression was confirmed by RT-PCR and western blotting. Also, C. pneumoniae survived in enriched human peripheral blood lymphocytes, even in the presence of IFN-γ. Furthermore, C. pneumoniae in spleen cells obtained from IFN-γ knockout mice with C57BL/6 background was maintained in a similar way to wild-type mice, supporting a minimal role of IFN-γ-related response for eliminating C. pneumoniae from lymphocytes. Thus, we concluded that IFN-γ did not remove C. pneumoniae from lymphocytes, possibly providing a shelter for C. pneumoniae to escape from the innate immune response, which has direct clinical significance.

Involvement of suppressor of cytokine signalling-1-mediated degradation of MyD88-adaptor-like protein in the suppression of Toll-like receptor 2-mediated signalling by the murine C-type lectin SIGNR1-mediated signalling.

Dendritic cells recognize pathogens through pattern recognition receptors such as Toll-like receptors and phagocytose and digest them by phagocytic receptors for antigen presentation. This study was designed to clarify the cross-talk between recognition and phagocytosis of microbes in dendritic cells. The murine dendritic cell line XS106 cells were stimulated with the murine C-type lectin SIGNR1 ligand lipoarabinomannan and the Toll-like receptor 2 ligand FSL-1. The co-stimulation significantly suppressed FSL-1-mediated activation of NF-κB as well as production of TNF-α, IL-6 and IL-12p40 in a dose-dependent manner. The suppression was significantly but not completely recovered by knock-down of SIGNR1. SIGNR1 was associated with Toll-like receptor 2 in XS106 cells. The co-stimulation upregulated the expression of suppressor of cytokine signalling-1 in XS106 cells, the knock-down of which almost completely recovered the suppression of the FSL-1-mediated cytokine production by lipoarabinomannan. In addition, it was found that the MyD88-adaptor-like protein in XS106 cells was degraded by co-stimulation with FSL-1 and lipoarabinomannan in the absence, but not the presence, of the proteasome inhibitor MG132 and the degradation was inhibited by knock-down of suppressor of cytokine signalling-1. This study suggests that Toll-like receptor 2-mediated signalling is negatively regulated by SIGNR1-mediated signalling in dendritic cells, possibly through suppressor of cytokine signalling-1-mediated degradation of the MyD88-adaptor-like protein.

Regulation of Toll-like receptor signaling by NDP52-mediated selective autophagy is normally inactivated by A20.

Toll-like receptor (TLR) signaling is linked to autophagy that facilitates elimination of intracellular pathogens. However, it is largely unknown whether autophagy controls TLR signaling. Here, we report that poly(I:C) stimulation induces selective autophagic degradation of the TLR adaptor molecule TRIF and the signaling molecule TRAF6, which is revealed by gene silencing of the ubiquitin-editing enzyme A20. This type of autophagy induced formation of autophagosomes and could be suppressed by an autophagy inhibitor and lysosomal inhibitors. However, this autophagy was not associated with canonical autophagic processes, including involvement of Beclin-1 and conversion of LC3-I to LC3-II. Through screening of TRIF-interacting 'autophagy receptors' in human cells, we identified that NDP52 mediated the selective autophagic degradation of TRIF and TRAF6 but not TRAF3. NDP52 was polyubiquitinated by TRAF6 and was involved in aggregation of TRAF6, which may result in the selective degradation. Intriguingly, only under the condition of A20 silencing, NDP52 could effectively suppress poly(I:C)-induced proinflammatory gene expression. Thus, this study clarifies a selective autophagic mechanism mediated by NDP52 that works downstream of TRIF-TRAF6. Furthermore, although A20 is known as a signaling fine-tuner to prevent excess TLR signaling, it paradoxically downregulates the fine-tuning effect of NDP52 on TLR signaling.