Facemask ventilation and vocal cord angle following neuromuscular blockade: a prospective observational study.

Numerous studies support the idea that neuromuscular blockade facilitates facemask ventilation after induction of anaesthesia. Although improved airway patency or pulmonary compliance and a resolution of laryngospasm have been suggested as possible causes, the exact mechanism remains unclear. We aimed to assess whether neuromuscular blockade improves facemask ventilation and to clarify whether this phenomenon is associated with the vocal cord angle. This prospective observational study included patients aged between 20 and 65 years scheduled for elective surgery under general anaesthesia. After induction of anaesthesia, patients' lungs were ventilated with pressure-controlled ventilation using a facemask. During facemask ventilation, a flexible bronchoscope was inserted through a self-sealing diaphragm at the elbow connector attached to the facemask and breathing circuit and positioned to allow a continuous view of the vocal cords. The mean tidal volume and vocal cord angle were measured before and after administration of neuromuscular blocking drugs. Of 108 patients, 100 completed the study. Mean (SD) tidal volume ((11.0 (3.9) ml.kg-1 vs. 13.6 (2.6) ml.kg-1 ; p < 0.001) and mean (SD) vocal cord angle (17° (10°) vs. 26° (5°); p < 0.001) increased significantly after neuromuscular blockade. The proportional increase in mean tidal volume after neuromuscular blockade was positively correlated with vocal cord angle (Spearman's ρ = 0.803; p < 0.001). In conclusion, neuromuscular blockade facilitated facemask ventilation, and the improvement was correlated with further opening of the vocal cords.

Diagnostic value of bronchoalveolar lavage and bronchial washing in sputum-scarce or smear-negative cases with suspected pulmonary tuberculosis: a randomized study.

OBJECTIVES: Bronchoalveolar lavage (BAL) and bronchial washing (BW) are two major methods used to obtain high-quality respiratory specimens from patients with suspected pulmonary tuberculosis (TB) but a sputum-scarce or smear-negative status. We aimed to compare the value of BAL and BW in the diagnosis of TB in such patients. METHODS: We enrolled patients with suspected pulmonary TB but with a sputum-scarce or smear-negative status who were referred for bronchoscopy between October 2013 and January 2016. Participants were randomized into the BAL and BW groups for evaluation. The primary outcome was the diagnostic yield for TB detection. Secondary outcomes included culture positivity, positivity of nucleic acid amplification tests (NAATs) for Mycobacterium tuberculosis and procedure-related complications. RESULTS: A total of 94 patients were assessed and 91 (43 in the BAL group, 48 in the BW group) were analysed. Twenty-one patients (48.8%) in the BAL group and 30 (62.5%) in the BW group had a final diagnosis of pulmonary TB. The detection rate of M. tuberculosis by culture or NAAT was significantly higher in BAL specimens than in BW specimens (85.7% vs 50.0%, p 0.009). The procedure-related complications were hypoxic events, 2/43 (4.7%) in the BAL group and 5/48 (10.4%) in the BW group; and post-bronchoscopic fever, 3/43 (7.0%) in the BAL group and 4/48 (8.3%) in the BW group. DISCUSSION: As long as it is tolerable, BAL rather than BW, should be used to obtain specimens for the diagnosis of pulmonary TB in sputum-scarce or smear-negative cases.

Predictors of responses to immune checkpoint blockade in advanced melanoma.

Immune checkpoint blockers (ICB) have become pivotal therapies in the clinical armamentarium against metastatic melanoma (MMel). Given the frequency of immune related adverse events and increasing use of ICB, predictors of response to CTLA-4 and/or PD-1 blockade represent unmet clinical needs. Using a systems biology-based approach to an assessment of 779 paired blood and tumor markers in 37 stage III MMel patients, we analyzed association between blood immune parameters and the functional immune reactivity of tumor-infiltrating cells after ex vivo exposure to ICB. Based on this assay, we retrospectively observed, in eight cohorts enrolling 190 MMel patients treated with ipilimumab, that PD-L1 expression on peripheral T cells was prognostic on overall and progression-free survival. Moreover, detectable CD137 on circulating CD8+ T cells was associated with the disease-free status of resected stage III MMel patients after adjuvant ipilimumab + nivolumab (but not nivolumab alone). These biomarkers should be validated in prospective trials in MMel.The clinical management of metastatic melanoma requires predictors of the response to checkpoint blockade. Here, the authors use immunological assays to identify potential prognostic/predictive biomarkers in circulating blood cells and in tumor-infiltrating lymphocytes from patients with resected stage III melanoma.

Changes of cortical activation in swallowing following high frequency repetitive transcranial magnetic stimulation in older adults.

BACKGROUND: This study explored whether high-frequency repetitive transcranial magnetic stimulation (rTMS) can induce positive changes in the cortical areas of older adults who do not have functional difficulties in swallowing. METHODS: Ten healthy, right-handed, elderly volunteers were subjected to 18F-labeled fluorodeoxyglucose positron emission tomography(FDG-PET) scans when at rest, swallowing before rTMS, and swallowing after rTMS. During the swallowing study, water was infused orally via a catheter at a rate of 600 mL/h. Subjects swallowed water every 20 seconds following a light flash for 30 minutes. During rest, the light source was active, but subjects were requested not to swallow. The rTMS consisted of 5 Hz applied to a pharyngeal motor hot spot in the right hemisphere for 10 minutes every weekday for 2 weeks. The intensity of the stimulation was set at 90% of the thenar motor threshold of the same hemisphere. The differences between each patient's active image and the control images (P<.05) on a voxel-by-voxel basis were examined to find significant increases in metabolism using statistical parametric mapping software. KEY RESULTS: The cortical areas activated by swallowing before rTMS included the bilateral sensorimotor cortex (Brodmann's areas 3 and 4) and showed symmetry. The cortical areas activated by swallowing after rTMS were the same as the areas before rTMS. There was no statistical difference between the two swallowing activation areas. CONCLUSIONS AND INFERENCES: Older adults displayed the symmetry of cortical control of swallowing function. High frequency rTMS did not affect the activation in the swallowing sensorimotor cortices of elderly people.

A case of isolated bilateral pulmonary arterial calcification diagnosed in utero.

We report a very rare case of isolated multiple pulmonary arterial calcification with severe bilateral peripheral pulmonary arterial stenosis diagnosed in utero. Despite treatment with bisphosphonate for 6 months, systolic right ventricular pressure increased persistently and surpassed left ventricular pressure. After successful bilateral pulmonary arterioplasty at 13 months of age, the patient showed decreased systolic right ventricular pressure with normal interventricular septal configuration. This is the first case report for an isolated pulmonary artery calcification without other arterial calcification proven by non-contrast computed tomography of a living patient.

H⁺-myo-inositol transporter SLC2A13 as a potential marker for cancer stem cells in an oral squamous cell carcinoma.

Cancer Stem Cells (CSCs) from tumors of different phenotypes possess a marked capacity for proliferation, self-renewal, and differentiation. They also play a critical role in cancer recurrence. Although CSC has been regarded as a new target for cancer therapy, the fundamental questions in the CSC study have not been resolved mainly due to the lack of proper CSC markers. To find new CSC markers for oral squamous cell carcinoma (OSCC), we cultured the primary tumor cells from OSCC patients the regular culture condition and the sphere-forming culture condition to enrich primary tumor cells and potential CSCs. We compared gene expression profiles between sphere-forming and non-forming cells, thus identifying that 23 membrane protein-coding genes were over-expressed in the sphere-forming cells. Among them, 8 belonged to the solute carrier (SLC) protein family. H⁺-myo-inositol transporter SLC2A13 and monocarbohydrate transporter SLC16A6 genes that were consistently increased in the sphere-forming cells in the primary cultures of OSCC samples. Confocal microscopy revealed that SLC2A13-expressing cells were embedded in the limited areas of tumor tissue as a cluster, while SLC16A6 was uniformly detected in hyperplastic epithelium. Moreover, SLC2A13 an expression was induced in human breast adenocarcinoma MCF7 cells after serum starvation. Taken together, our results suggest that SLC2A13 can be a potential markers for CSC in various tumors.

The tumour necrosis factor/TNF receptor superfamily: therapeutic targets in autoimmune diseases.

Autoimmune diseases are characterized by the body's ability to mount immune attacks on self. This results from recognition of self-proteins and leads to organ damage due to increased production of pathogenic inflammatory molecules and autoantibodies. Over the years, several new potential therapeutic targets have been identified in autoimmune diseases, notable among which are members of the tumour necrosis factor (TNF) superfamily. Here, we review the evidence that certain key members of this superfamily can augment/suppress autoimmune diseases.

Involvement of 4-1BB (CD137)-4-1BBligand interaction in the modulation of CD4 T cell-mediated inflammatory colitis.

4-1BB ligand (4-1BBL) expressed on antigen-presenting cells interacts with 4-1BB on activated T cells (especially CD8+ cells) and co-stimulates the latter to secrete cytokines and to proliferate. The role of 4-1BB-4-1BBL interaction was studied here in a model of colitis based on naive CD4+ T cell transfer to SCID mice, a disease model in which CD8 cells do not take part. We found that CD4+ T cells from 4-1BB-deficient mice, after transfer in SCID mice, proliferated more rapidly compared to wild-type CD4+ T cells. Mice reconstituted with naive CD4+ T cells from 4-1BB-deficient mice developed colitis, however, with a mixed Th1/Th2 response, in contrast to the Th1-type response in mice reconstituted with wild-type naive CD4+ T cells. Importantly, this altered cytokine response did not temper colitis severity. Although it has been reported previously that 4-1BB co-stimulation may contribute to regulatory T cell functioning, we found that CD4+CD25+ regulatory T cells from 4-1BB-deficient mice were perfectly able to prevent naive CD4+ T cell-induced colitis. In conclusion, our data provide evidence that 4-1BB-4-1BBL interaction modulates the effector CD4+ T cell-driven immune response and cytokine production in experimental colitis without affecting regulatory T cell function.

Blockade of 4-1BB (CD137)/4-1BB ligand interactions increases allograft survival.

We investigated the role of 4-1BB, a T cell co-stimulatory molecule, in alloimmune responses. In vivo mixed lymphocyte reactions showed that 4-1BB was preferentially expressed on actively dividing CD4(+) and CD8(+) T cells. Furthermore, following alloantigen challenge, the draining lymph nodes contained subpopulations of 4-1BB-expressing CD4(+) and CD8(+) T cells. 4-1BB-deficient C57BL/6 mice showed a delayed rejection of cardiac transplants mismatched for the major histocompatibility complex. Longer transplant survival was induced by blockade of 4-1BB/4-1BB ligand (4-1BBL) interactions using an anti-4-1BBL monoclonal antibody. Histological analysis showed that prolonged transplant survival in the 4-1BB-deficient and anti-4-1BBL-treated mice correlated with reduced lymphocytic infiltration and vasculitis in the donor heart tissue. Taken together, our data suggest that blockade of 4-1BB/4-1BBL interactions inhibited the expansion of alloreactive T cells and reduced CTL activity against host alloantigen, which in turn resulted in the prolongation of allograft survival. Blockade of the 4-1BB co-stimulatory pathway may be useful for preventing allograft rejection.

Cloning and characterization of GITR ligand.

The gene encoding the natural ligand of murine glucocorticoid-induced tumor necrosis factor receptor (GITR) was cloned and characterized. The putative GITR ligand (GITRL) is composed of 173 amino acids with features resembling those of type II membrane proteins and is 51% identical to the human activation-inducible TNF receptor (AITR) ligand, TL6. Expression of the GITRL is restricted to immature and mature splenic dendritic cells. GITRL binds GITR expressed on HEK 293 cells and triggers NF-kappaB activation. Functional studies reveal that soluble CD8-GITRL prevents CD4+CD25+ regulatory T-cell-mediated suppressive activities.

Molecular cloning of agonistic and antagonistic monoclonal antibodies against human 4-1BB.

Previously, we prepared two different monoclonal antibodies (mAbs) against human 4-1BB (CD137): an agonistic mAb BBK-1 and an antagonistic mAb BBK-2. In this paper, we describe the molecular cloning of these two mAbs and present comparisons of their amino acid sequences. cDNAs encoding the heavy (H) and light (L) chains of the two mAbs were cloned by screening of cDNA libraries constructed from hybridomas secreting these mAbs. Comparisons of amino acid sequences of the two mAbs showed that, while the constant regions of the H and L chains were identical between the two mAbs, the variable region showed 45% identity in H chains and 48% identity in L chains. This suggests that these two mAbs recognize different epitopes of 4-1BB and may have different effects on the activity of 4-1BB.

Tumor necrosis factor receptor superfamily 14 is involved in atherogenesis by inducing proinflammatory cytokines and matrix metalloproteinases.

Tumor necrosis factor (TNF) receptor superfamily 14 (TNFRSF14) is the cellular receptor for TNF superfamily 14 (LIGHT). Immunohistochemical staining of human carotid atherosclerotic plaques revealed a high level of expression of the TNFRSF14 in regions rich in macrophages/foam cells. To investigate the role of TNFRSF14 in the functioning of monocytes in relation to atherogenesis, we have analyzed TNFRSF14 expression levels and cellular events after stimulation of TNFRSF14 in peripheral blood monocytes or the human macrophage-like cell line, THP-1. A high level of expression of TNFRSF14 was detected in activated monocytes, in macrophages derived from monocytes, and in THP-1 cells. Concomitant activation of THP-1 cells with interferon-gamma and immobilized anti-TNFRSF14 monoclonal antibody resulted in synergistic induction of proatherogenic cytokines, such as TNF-alpha and interleukin-8. Activation of THP-1 cells with immobilized anti-TNFRSF14 monoclonal antibody induced expression of matrix metalloproteinase (MMP)-1, MMP-9, MMP-13, and tissue inhibitors of metalloproteinase-1 and -2. Furthermore, immunohistochemical staining of atherosclerotic plaques with severe infiltration of foam cells revealed that the expression patterns of TNFRSF14 and MMP-1, -9, and -13 overlapped. Treatment of THP-1 cells with soluble LIGHT also caused induction of MMP-9 and interleukin-8. These data suggest that TNFRSF14 is involved in atherosclerosis via the induction of proatherogenic cytokines and decreasing plaque stability by inducing extracellular matrix-degrading enzymes.

Murine keratocytes function as antigen-presenting cells.

Keratocytes express MHC class I molecules constitutively, and keratocytes stimulated with IFN-gamma express MHC class II molecules. Unstimulated keratocytes constitutively express B7-1 and ICAM-1, as well as low levels of CD40 and 4-1BBL. These findings indicate that keratocytes may deliver both antigen-specific and costimulatory signals to CD4(+) and CD8(+) T cells. To demonstrate that keratocytes expressing B7-1 provide a costimulatory signal to T cells, CD4(+) or CD8(+) mouse T cells were incubated with anti-CD3 mAb and irradiated keratocytes. Enhanced proliferation of both CD4(+) and CD8(+) T cells occurred, and could be inhibited by anti-B7-1 mAb, indicating T cell costimulatory activity by B7-1 on the keratocytes. To demonstrate that keratocytes can deliver an antigen-specific signal, CD4(+) and CD8(+) T cells from herpes-infected mice were incubated with HSV-1-infected, irradiated keratocytes. The resulting T cell proliferation and production of Th1 cytokines (IL-2, IFN-gamma) indicated T cell activation by antigens presented by the infected keratocytes. These results show that keratocytes in the corneal stroma of the mouse can function as antigen-presenting cells and, thus, may play a role in immune-mediated stromal inflammation such as herpetic stromal keratitis.

Osteoclastogenesis is enhanced by activated B cells but suppressed by activated CD8(+) T cells.

Host immune response is known to contribute to the progression of periodontitis, and alveolar bone destruction in periodontitis is associated with enhanced osteoclast activity. Therefore, we evaluated the roles of activated lymphocyte subsets in osteoclastogenesis. Osteoclast precursors were co-cultured with activated lymphocytes (B, CD4(+) T, CD8(+) T) in the presence of either macrophage colony-stimulating factor (M-CSF) alone or M-CSF plus soluble receptor activator of NF-kappaB ligand (sRANKL), and subsequent differentiation into active osteoclasts was evaluated by a resorption assay. The activated B and CD4(+) cells, but not CD8(+) T cells, induced osteoclast differentiation in the presence of M-CSF alone. In the presence of M-CSF and sRANKL, B cells induced the formation of small but highly active osteoclasts and increased resorption, while CD8(+) T cells profoundly suppressed osteoclastogenesis. Co-culture using an insert well or supernatant suggested that both B and CD8(+) T cells acted on osteoclasts mostly via soluble proteins. Activated B cells expressed many osteoclastogenic factors including RANKL, TNF-alpha, IL-6, MIP-1alpha, and MCP-3. CD8(+) T cells expressed a substantial amount of osteoprotegerin (OPG) along with RANKL. However, blocking antibody to OPG did not reverse the suppression by CD8(+) T cells, suggesting that other factor(s) are involved. Taken together, activated B cells promoted osteoclastogenesis, while CD8(+) T cells inhibited the osteoclast formation via direct interaction. The results imply the importance of lymphocyte subpopulations in the development of periodontitis.

Tumor necrosis factor receptor superfamily 12 may destabilize atherosclerotic plaques by inducing matrix metalloproteinases.

Immunohistochemical staining of human atherosclerotic plaques revealed expression of the tumor necrosis factor receptor superfamily (TNFRSF) 12 in regions rich in macrophage/foam cells. The role of TNFRSF12 in the functioning of monocytes in relation to atherogenesis was investigated by analysis of cellular events after stimulation of TNFRSF12 in a human macrophage-like cell line, THP-1. Activation of the THP-1 cells on plates coated with monoclonal antibody against TNFRSF12 induced the expression of matrix metalloproteinases (MMPs) -1, -9, and -13. Furthermore, the expression patterns of TNFRSF12 and the MMPs overlapped in atherosclerotic plaques. Signaling of TNFRSF12 may thus contribute to the induction of extracellular matrix degrading enzymes in macrophages.

Ligation of 4-1BB (CDw137) regulates graft-versus-host disease, graft-versus-leukemia, and graft rejection in allogeneic bone marrow transplant recipients.

4-1BB is expressed on activated CD4(+) and CD8(+) T cells; its ligand, 4-1BB ligand is expressed on APCs. Despite expression on both T cell subpopulations, 4-1BB has been reported to predominantly affect CD8(+) T cell responses. By quantifying graft-vs-host disease alloresponses in vivo, we demonstrate that both CD4(+) and CD8(+) T cell-mediated alloresponses are regulated by 4-1BB/4-1BB ligand interactions to approximately the same extent. 4-1BB receptor-facilitated CD4(+) T cell-mediated alloresponses were partly CD28 independent. In two distinct marrow graft rejection systems, host CD8(+) and CD4(+) T cells each separately contributed to host anti-donor T cell-mediated allograft rejection. alpha 4-1BB mAb increased the graft-vs-leukemia effect of a suboptimal number of donor splenocytes given later post bone marrow transplantation by bolstering allogeneic responses resulting in leukemia elimination. In summary, 4-1BB ligation is a potent regulator of CD4(+) and CD8(+) T cell-mediated allogeneic responses in vivo. Modifying the ligation of 4-1BB represents a new approach to altering the graft-vs-host disease and graft-vs-leukemia effects of allogeneic T cells post bone marrow transplantation.

A novel leucine-rich repeat protein (LRR-1): potential involvement in 4-1BB-mediated signal transduction.

4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is induced on CD4+ and CD8+ T cells upon engagement of the T cell receptor (TCR)/ CD3 complex with the antigen bound to MHC. 4-1BB plays an important role in transmitting costimulatory signal during T cell activation. However, 4-1BB-mediatded signal transduction pathways have remained elusive. We conducted the yeast two-hybrid screening to identify intracellular signaling molecules that associate with 4-1BB. A novel leucine-rich repeat (LRR)-containing protein, herein named LRR-1, was found to specifically interact with the cytoplasmic domain of 4-1BB. Overexpression of LRR-1 suppressed the activation of NF-KB induced by 4-1BB or TNF receptor-associated factor (TRAF) 2. In addition, LRR-1 down-regulated JNK1 activity was induced by 4-1BB. These results indicate that LRR-1 negatively regulates the 4-1BB-mediated signaling cascades which result in the activation of NF-kappaB and JNK1.

4-1BB: still in the midst of darkness.

4-1BB is a member of the tumor necrosis factor receptor superfamily. The receptor functions mainly as a costimulatory molecule in T lymphocytes. In addition, several lines of evidence have shown that interactions between 4-1BB and its ligand are involved in the antigen presentation process and the generation of cytotoxic T cells. Recent studies, however, have demonstrated that 4-1BB plays more diverse roles: Signals through 4-1BB are important for long-term survival of CD8+ T cells and the induction of helper T cell anergy. Clinically, there is great interest in 4-1BB, because T-cell activation induced by anti-4-1BB monoclonal antibodies is highly efficient in the eradication of established tumor cells in mice. Now, since mice deficient in 4-1BB or the 4-1BB ligand are available, subtle roles played by 4-1BB may be revealed in the near future.

Co-stimulation of antigen-specific CD4 T cells by 4-1BB ligand.

4-1BB is a member of the TNF receptor family predominantly expressed on activated T cells, and binds an inducible ligand found on B cells, macrophages and dendritic cells. Whereas ligation of 4-1BB has been shown to enhance response of purified CD8 T cells to mitogens, and to augment NK activity and generation of cytotoxic T lymphocytes in vivo, there are little direct data on 4-1BB action during CD4 responses. Using pigeon cytochrome c-presenting fibroblast antigen-presenting cells transfected with 4-1BB ligand (4-1BBL), we show that engaging 4-1BB on naive CD4 cells promotes proliferation, cell cycle progression and IL-2 secretion, and suppresses cell death, all to a similar extent as B7-1 engagement of CD28. In addition, 4-1BBL synergizes with B7 and ICAM to enhance naive CD4 proliferation when antigen is limiting. 4-1BBL alone, and to a greater extent with B7, also augmented IL-2 secretion resting antigen-experienced CD4 cells, as typified by T helper clones, whereas short-term effector cells showed similar levels of proliferation and cytokine secretion regardless of whether 4-1BB was engaged. A major role in augmenting IFN-gamma, IL-4 or IL-5 was not demonstrated. Blocking studies with activated B cells presenting antigen showed that 4-1BB participates in promoting IL-2 production by resting CD4 cells, confirming that 4-1BBL can play a role in antigen-specific CD4 T cell responses.