GNUV201, a novel human/mouse cross-reactive and low pH-selective anti-PD-1 monoclonal antibody for cancer immunotherapy.

BACKGROUND: Several PD-1 antibodies approved as anti-cancer therapies work by blocking the interaction of PD-1 with its ligand PD-L1, thus restoring anti-cancer T cell activities. These PD-1 antibodies lack inter-species cross-reactivity, necessitating surrogate antibodies for preclinical studies, which may limit the predictability and translatability of the studies. RESULTS: To overcome this limitation, we have developed an inter-species cross-reactive PD-1 antibody, GNUV201, by utilizing an enhanced diversity mouse platform (SHINE MOUSE™). GNUV201 equally binds to human PD-1 and mouse PD-1, equally inhibits the binding of human PD-1/PD-L1 and mouse PD-1/PD-L1, and effectively suppresses tumor growth in syngeneic mouse models. The epitope of GNUV201 mapped to the "FG loop" of hPD-1, distinct from those of Keytruda® ("C'D loop") and Opdivo® (N-term). Notably, the structural feature where the protruding epitope loop fits into GNUV201's binding pocket supports the enhanced binding affinity due to slower dissociation (8.7 times slower than Keytruda®). Furthermore, GNUV201 shows a stronger binding affinity at pH 6.0 (5.6 times strong than at pH 7.4), which mimics the hypoxic and acidic tumor microenvironment (TME). This phenomenon is not observed with marketed antibodies (Keytruda®, Opdivo®), implying that GNUV201 achieves more selective binding to and better occupancy on PD-1 in the TME. CONCLUSIONS: In summary, GNUV201 exhibited enhanced affinity for PD-1 with slow dissociation and preferential binding in TME-mimicking low pH. Human/monkey/mouse inter-species cross-reactivity of GNUV201 could enable more predictable and translatable efficacy and toxicity preclinical studies. These results suggest that GNUV201 could be an ideal antibody candidate for anti-cancer drug development.

Identification of dendritic cell precursor from the CD11c+ cells expressing high levels of MHC class II molecules in the culture of bone marrow with FLT3 ligand.

Dendritic cells (DCs) are readily generated from the culture of mouse bone marrow (BM) treated with either granulocyte macrophage-colony stimulating factor (GM-CSF) or FMS-like tyrosine kinase 3 ligand (FLT3L). CD11c+MHCII+ or CD11c+MHCIIhi cells are routinely isolated from those BM cultures and generally used as in vitro-generated DCs for a variety of experiments and therapies. Here, we examined CD11c+ cells in the BM culture with GM-CSF or FLT3L by staining with a monoclonal antibody 2A1 that is known to recognize mature or activated DCs. Most of the cells within the CD11c+MHCIIhi DC gate were 2A1+ in the BM culture with GM-CSF (GM-BM culture). In the BM culture with FLT3L (FL-BM culture), almost of all the CD11c+MHCIIhi cells were within the classical DC2 (cDC2) gate. The analysis of FL-BM culture revealed that a majority of cDC2-gated CD11c+MHCIIhi cells exhibited a 2A1-CD83-CD115+CX3CR1+ phenotype, and the others consisted of 2A1+CD83+CD115-CX3CR1- and 2A1-CD83-CD115-CX3CR1- cells. According to the antigen uptake and presentation, morphologies, and gene expression profiles, 2A1-CD83-CD115-CX3CR1- cells were immature cDC2s and 2A1+CD83+CD115-CX3CR1- cells were mature cDC2s. Unexpectedly, however, 2A1-CD83-CD115+CX3CR1+ cells, the most abundant cDC2-gated MHCIIhi cell subset in FL-BM culture, were non-DCs. Adoptive cell transfer experiments in the FL-BM culture confirmed that the cDC2-gated MHCIIhi non-DCs were precursors to cDC2s, i.e., MHCIIhi pre-cDC2s. MHCIIhi pre-cDC2s also expressed the higher level of DC-specific transcription factor Zbtb46 as similarly as immature cDC2s. Besides, MHCIIhi pre-cDC2s were generated only from pre-cDCs and common DC progenitor (CDP) cells but not from monocytes and common monocyte progenitor (cMoP) cells, verifying that MHCIIhi pre-cDC2s are close lineage to cDCs. All in all, our study identified and characterized a new cDC precursor, exhibiting a CD11c+MHCIIhiCD115+CX3CR1+ phenotype, in FL-BM culture.

Altered interictal serum histamine and immunoglobulin E but unchanged tryptase levels in individuals with episodic and chronic migraine.

BACKGROUND: Serum histamine, immunoglobulin E, and tryptase are markers of allergic diseases. Despite the reported association between migraine and allergic diseases, differences in these marker levels between episodic and chronic migraines remain unelucidated. METHODS: We investigated serum histamine, immunoglobulin E, and tryptase levels in 97 and 96 participants with episodic migraine and chronic migraine, respectively, and 56 controls according to the presence of allergic diseases. RESULTS: Serum histamine levels in episodic migraine (median and interquartile ranges, 0.78 [0.65-1.25] ng/mL, p < 0.001) and chronic migraine (0.89 [0.67-1.28] ng/mL, p < 0.001) participants were significantly lower than those in healthy controls (1.19 [0.81-2.08] ng/mL) among the 160 participants without allergic diseases. Serum immunoglobulin E levels in episodic migraine and chronic migraine participants with allergic diseases negatively correlated with headache frequency (correlation coefficient = -0.263, p = 0.017). Serum histamine levels in participants with allergic diseases and serum immunoglobulin E levels in participants without allergic diseases were not significantly different among episodic migraine, chronic migraine, and control groups. Serum tryptase levels did not significantly differ among episodic migraine, chronic migraine, and control participants with and without allergic diseases. CONCLUSIONS: Altered serum histamine and immunoglobulin E levels in episodic migraine and chronic migraine and different profiles concerning allergic diseases suggest the involvement of allergic mechanisms in migraine pathogenesis.

Distinct effects of different adjuvants in the mouse model of allergic airway inflammation.

BACKGROUND: Allergic asthma was typically considered as an inflammatory disease mediated by type 2 immunity. However, recent studies revealed that asthma is a complex disease displaying a variety of phenotypes and endotypes. OBJECTIVE: We examined cellular phenotypes in the mouse model of allergic asthma sensitized with different adjuvants. The aim of our study was to determine immunologic cellular characteristics in mouse asthma models induced by ovalbumin (OVA) and a variety of adjuvants. METHODS: Mice were sensitized intraperitoneally with the admixture of OVA and various adjuvants such as Alhydrogel (alum), papain, lipopolysaccharide (LPS), or CpG, and subsequently challenged with OVA intranasally. The cells in bronchoalveolar lavage (BAL) fluid, lung, and mediastinal lymph node (mLN) were examined by flow cytometric analyses. RESULTS: In the lung and BAL fluid, the highest eosinophil levels were observed in the alum group while the highest neutrophil levels were detected in the LPS group. Meanwhile, the LPS group exhibited the most elevated levels of both RORγt+ innate lymphoid cells (ILCs) and IL-17A+ Th cells in the lung and mediastinal lymph node. In the lung, the number of T-bet+ ILCs was highest in the papain group whereas the number of IFN-γ+ Th cells was highest in the CpG group. CONCLUSIONS: Notable variances are found in the composition of immune cells and expression of cytokines at the site of pathogenesis among the different mouse models of allergic asthma created by the sensitization with different adjuvants.

Global Gene Expression of T Cells Is Differentially Regulated by Peritoneal Dendritic Cell Subsets in an IL-2 Dependent Manner.

Dendritic cells (DCs) in peripheral tissues may have a unique role to regulate innate and adaptive immune responses to antigens that enter the tissues. Peritoneal cavity is the body compartment surrounding various tissues and organs and housing diverse immune cells. Here, we investigated the specialized features of classical DC (cDC) subsets following the intraperitoneal injection of a model antigen ovalbumin (OVA). Peritoneal cDC1s were superior to cDC2s in activating OVA-specific CD8 T cells, while both cDCs were similar in stimulating OVA-specific CD4 T cells. Each peritoneal cDC subset differentially regulated the homing properties of CD8 T cells. CD8 T cells stimulated by cDC1s displayed a higher level of lung-homing receptor CCR4, whereas those stimulated by cDC2s prominently expressed various homing receptors including gut-homing molecules CCR9 and α4ß7. Also, we found that cDC1s played a dominating role over cDC2s in controlling the overall gene expression of CD8 T cells. Soluble factor(s) emanating from CD8 T cells stimulated by peritoneal cDC1s were responsible for mediating this dominance of cDC1s, and we identified IL-2 as a soluble factor regulating the global gene expression of T cells. Collectively, our study indicates that different peritoneal cDC subsets effectively diversify T cell responses by altering the level of cytokines, such as IL-2, in the milieu.

Granulocyte Macrophage-Colony Stimulating Factor Produces a Splenic Subset of Monocyte-Derived Dendritic Cells That Efficiently Polarize T Helper Type 2 Cells in Response to Blood-Borne Antigen.

Dendritic cells (DCs) are key antigen-presenting cells that prime naive T cells and initiate adaptive immunity. Although the genetic deficiency and transgenic overexpression of granulocyte macrophage-colony stimulating factor (GM-CSF) signaling were reported to influence the homeostasis of DCs, the in vivo development of DC subsets following injection of GM-CSF has not been analyzed in detail. Among the treatment of mice with different hematopoietic cytokines, only GM-CSF generates a distinct subset of XCR1-33D1- DCs which make up the majority of DCs in the spleen after three daily injections. These GM-CSF-induced DCs (GMiDCs) are distinguished from classical DCs (cDCs) in the spleen by their expression of CD115 and CD301b and by their superior ability to present blood-borne antigen and thus to stimulate CD4+ T cells. Unlike cDCs in the spleen, GMiDCs are exceptionally effective to polarize and expand T helper type 2 (Th2) cells and able to induce allergic sensitization in response to blood-borne antigen. Single-cell RNA sequencing analysis and adoptive cell transfer assay reveal the sequential differentiation of classical monocytes into pre-GMiDCs and GMiDCs. Interestingly, mixed bone marrow chimeric mice of Csf2rb+/+ and Csf2rb-/- demonstrate that the generation of GMiDCs necessitates the cis expression of GM-CSF receptor. Besides the spleen, GMiDCs are generated in the CCR7-independent resident DCs of the LNs and in some peripheral tissues with GM-CSF treatment. Also, small but significant numbers of GMiDCs are generated in the spleen and other tissues during chronic allergic inflammation. Collectively, our present study identifies a splenic subset of CD115hiCD301b+ GMiDCs that possess a strong capacity to promote Th2 polarization and allergic sensitization against blood-borne antigen.

Two Distinct Subsets Are Identified from the Peritoneal Myeloid Mononuclear Cells Expressing both CD11c and CD115.

To this date, the criteria to distinguish peritoneal macrophages and dendritic cells (DCs) are not clear. Here we delineate the subsets of myeloid mononuclear cells in the mouse peritoneal cavity. Considering phenotypical, functional, and ontogenic features, peritoneal myeloid mononuclear cells are divided into 5 subsets: large peritoneal macrophages (LPMs), small peritoneal macrophages (SPMs), DCs, and 2 MHCII+CD11c+CD115+ subpopulations (i.e., MHCII+CD11c+CD115+CD14-CD206- and MHCII+CD11c+CD115+CD14+CD206+). Among them, 2 subsets of competent Ag presenting cells are demonstrated with distinct functional characteristics, one being DCs and the other being MHCII+CD11c+CD115+CD14-CD206- cells. DCs are able to promote fully activated T cells and superior in expanding cytokine producing inflammatory T cells, whereas MHCII+CD11c+CD115+CD14-CD206- cells generate partially activated T cells and possess a greater ability to induce Treg under TGF-ß and retinoic acid conditions. While the development of DCs and MHCII+CD11c+CD115+CD14-CD206- cells are responsive to the treatment of FLT3 ligand and GM-CSF, the number of LPMs, SPMs, and MHCII+CD11c+CD115+CD14+CD206+ cells are only influenced by the injection of GM-CSF. In addition, the analysis of gene expression profiles among MHCII+ peritoneal myeloid mononuclear cells reveals that MHCII+CD11c+CD115+CD14+CD206+ cells share high similarity with SPMs, whereas MHCII+CD11c+CD115+CD14-CD206- cells are related to peritoneal DC2s. Collectively, our study identifies 2 distinct subpopulations of MHCII+CD11c+CD115+ cells, 1) MHCII+CD11c+CD115+CD14-CD206- cells closely related to peritoneal DC2s and 2) MHCII+CD11c+CD115+CD14+CD206+ cells to SPMs.

Extended Culture of Bone Marrow with Granulocyte Macrophage-Colony Stimulating Factor Generates Immunosuppressive Cells.

Bone marrow-derived dendritic cells (BM-DCs) are generated from bone marrow (BM) cells cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) for a week. In this study we investigated the effect of duration on the BM culture with GM-CSF. Within several months, the cells in the BM culture gradually expressed homogeneous levels of CD11c and major histocompatibility complex II on surface, and they became unable to stimulate allogeneic naïve T cells in mixed lymphocyte reaction (MLR). In addition, when the BM culture were sustained for 32 wk or longer, the BM cells acquired ability to suppress the proliferation of allogeneic T cells in MLR as well as the response of ovalbumin-specific OT-I transgenic T cells in antigen-dependent manner. We found that, except for programmed death-ligand 1, most cell surface molecules were expressed lower in the BM cells cultured with GM-CSF for the extended duration. These results indicate that BM cells in the extended culture with GM-CSF undergo 2 distinct steps of functional change; first, they lose the immunostimulatory capacity; and next, they gain the immunosuppressive ability.

Skin-Specific CD301b+ Dermal Dendritic Cells Drive IL-17-Mediated Psoriasis-Like Immune Response in Mice.

Conventional dendritic cells (cDCs) are composed of heterogeneous subsets commonly arising from dendritic cell (DC)-committed progenitors. A population of CD301b-expressing DCs has recently been identified in non-lymphoid barrier tissues such as skin. However, whether CD301b+ DCs in the skin represent an ontogenetically unique subpopulation of migratory cDCs has not been fully addressed. Here, we demonstrated that CD301b+ dermal DCs were distinct subpopulation of FMS-like tyrosine kinase 3 ligand (FLT3L)-dependent CD11b+ cDC2 lineage, which required an additional GM-CSF cue for the adequate development. Although the majority of lymphoid-resident cDC2 lacked CD301b expression, dermal migratory cDC2 contained a substantial fraction of CD301b+ subset. Similar to CD301b- population, CD301b+ dermal DC development was closely regulated by FLT3 signaling, suggesting their common origin from FLT3L-responsive cDC progenitors. However, FLT3L-driven cDC progenitor culture was not sufficient, but additional GM-CSF treatment was required to produce CD301b+ cDC2. In vivo development of CD301b+ cDC2 was significantly augmented by exogenous GM-CSF, while the repopulation of CD301b+ dermal cDC2 was abrogated by GM-CSF neutralization. Functionally, CD301b+ cDC2 was capable of producing a high level of IL-23, and the depletion of CD301b+ cDC2 effectively prevented IL-17-mediated psoriasiform dermatitis. Therefore, our findings highlight the differentiation program of a distinct CD301b+ dermal cDC2 subset in the skin and its involvement in psoriatic inflammation.

Competent antigen-presenting cells are generated from the long-term culture of splenocytes with granulocyte-macrophage colony-stimulating factor.

Dendritic cells (DCs) are routinely produced from the culture of mouse bone marrow (BM) with granulocyte-macrophage colony-stimulating factor (GM-CSF) within a period of 10days. Although splenic extramedullary myelopoiesis was suggested to occur under the influence of GM-CSF, the hematopoietic outcome of splenic culture with GM-CSF has not been scrutinized. We have cultured mouse splenocytes with GM-CSF for an extended period of time, where we discovered that the CD11b⁺CD11c⁺ cells began to proliferate prominently after 10days and their number increased until the 4th week of the culture. In parallel experiments, FMS-like tyrosine kinase 3 (FLT3) and its ligand, FLT3L, were not found to influence the culture of splenocytes. Like DCs in the culture of BM with GM-CSF, a distinct population of CD11b⁺CD11c⁺MHC IIhi cells was readily identified as DCs in the long-term culture of splenocytes. After being isolated and plated overnight the CD11b⁺CD11c⁺MHC IIhi cells exhibited non-adherent dendritic morphology, while the other CD11b⁺CD11c⁺ cells became adherent. Besides, these CD11b⁺CD11c⁺MHC IIhi cells possessed relatively weak endocytic and phagocytic abilities but displayed strong antigen-presenting capacities, revealing DC-like characteristics; in contrast, the other CD11b⁺CD11c⁺ cells showed strong endocytosis and phagocytosis of antigens but were poor at antigen presentation, indicating macrophage-like traits. Therefore, we demonstrated that phenotypically as well as functionally genuine DCs are generated in the long-term culture of splenocytes with GM-CSF.

Reduced expression of granule proteins during extended survival of eosinophils in splenocyte culture with GM-CSF.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifaceted hematopoietic cytokine and the culture of mouse bone marrow with GM-CSF produces a variety of myeloid cells including granulocytes, macrophages, and dendritic cells. In the present study, we cultured mouse splenocytes with GM-CSF and examined the changes in hematopoietic cell populations over a week. Most of the splenic hematopoietic cells disappeared significantly from culture within 6days with or without the presence of GM-CSF. Among the splenic granulocyte populations, only eosinophils fully survived throughout the culture with GM-CSF for more than a week. During 10days of culture with GM-CSF, splenic eosinophils maintained their morphology as well as most of their surface molecules at high levels, including CCR3 and Siglec F. Meanwhile, the expression of mRNAs encoding major basic protein-1 (MBP-1) and eosinophil peroxidase (EPO), two major eosinophil-derived granule proteins, was diminished significantly from the cultured eosinophils. EPO assays also revealed that eosinophils in culture for more than 5days retained 30% or less EPO activity compared to those in uncultured splenocytes. In contrast, culture of splenocytes with GM-CSF did not change the capacity of eosinophils to migrate in response to eotaxin-1. Our results indicate that mouse splenic eosinophils are effectively cultured for lengthy periods while their expression of eosinophil-derived granule proteins is specifically suppressed. The relevance of these findings to eosinophilic inflammatory response is discussed.

TCF4-Targeting miR-124 is Differentially Expressed amongst Dendritic Cell Subsets.

Dendritic cells (DCs) are professional antigen-presenting cells that sample their environment and present antigens to naïve T lymphocytes for the subsequent antigen-specific immune responses. DCs exist in a range of distinct subpopulations including plasmacytoid DCs (pDCs) and classical DCs (cDCs), with the latter consisting of the cDC1 and cDC2 lineages. Although the roles of DC-specific transcription factors across the DC subsets have become understood, the posttranscriptional mechanisms that regulate DC development are yet to be elucidated. MicroRNAs (miRNAs) are pivotal posttranscriptional regulators of gene expression in a myriad of biological processes, but their contribution to the immune system is just beginning to surface. In this study, our in-house probe collection was screened to identify miRNAs possibly involved in DC development and function by targeting the transcripts of relevant mouse transcription factors. Examination of DC subsets from the culture of mouse bone marrow with Flt3 ligand identified high expression of miR-124 which was able to target the transcript of TCF4, a transcription factor critical for the development and homeostasis of pDCs. Further expression profiling of mouse DC subsets isolated from in vitro culture as well as via ex vivo purification demonstrated that miR-124 was outstandingly expressed in CD24(+) cDC1 cells compared to in pDCs and CD172α(+) cDC2 cells. These results imply that miR-124 is likely involved in the processes of DC subset development by posttranscriptional regulation of a transcription factor(s).

A lipophilic fluorescent LipidGreen1-based quantification method for high-throughput screening analysis of intracellular poly-3-hydroxybutyrate.

Poly-3-hydroxybutyrate (PHB), the most abundant type of polyhydroxyalkanoates (PHA) is synthesized inside a variety of microorganisms as a primary candidate for industrial PHB production. Lipophilic dyes such as Nile red and BODIPY have been used to quantify intracellular PHB, but their uses have often been limited in terms of sensitivity and accuracy. In this study, a newly developed lipophilic fluorescent dye LipidGreen1 was used to quantify intracellular PHB. LipidGreen1 stained viable colonies by adding the dye into the medium which enabled the effective selection of PHB-positive cells. Furthermore, the fluorescence intensity of LipidGreen1 maintained its fluorescence intensity much longer than that of Nile red. The fluorescence intensities of intracellular PHB stained by LipidGreen1 accurately agreed with PHB contents measured by gas chromatography. In addition, internalization of LipidGreen1 in Escherichia coli cell was not necessary to obtain quantitative measurements. PHB-synthase mutants were differentiated by fluorescence intensities with a good correlation to increased levels of PHB production. These results show that LipidGreen1 is sensitive and accurate in high-throughput screening of newly isolated and genetically modified bacteria with enhanced PHB production.

Globotriaosylceramide induces lysosomal degradation of endothelial KCa3.1 in fabry disease.

OBJECTIVE: Globotriaosylceramide (Gb3) induces KCa3.1 downregulation in Fabry disease (FD). We investigated whether Gb3 induces KCa3.1 endocytosis and degradation. APPROACH AND RESULTS: KCa3.1, especially plasma membrane-localized KCa3.1, was downregulated in both Gb3-treated mouse aortic endothelial cells (MAECs) and human umbilical vein endothelial cells. Gb3-induced KCa3.1 downregulation was prevented by lysosomal inhibitors but not by a proteosomal inhibitor. Endoplasmic reticulum stress-inducing agents did not induce KCa3.1 downregulation. Gb3 upregulated the protein levels of early endosome antigen 1 and lysosomal-associated membrane protein 2 in MAECs. Compared with MAECs from age-matched wild-type mice, those from aged α-galactosidase A (Gla)-knockout mice, an animal model of FD, showed downregulated KCa3.1 expression and upregulated early endosome antigen 1 and lysosomal-associated membrane protein 2 expression. In contrast, no significant difference was found in early endosome antigen 1 and lysosomal-associated membrane protein 2 expression between young Gla-knockout and wild-type MAECs. In aged Gla-knockout MAECs, clathrin was translocated close to the cell border and clathrin knockdown recovered KCa3.1 expression. Rab5, an effector of early endosome antigen 1, was upregulated, and Rab5 knockdown restored KCa3.1 expression, the current, and endothelium-dependent relaxation. CONCLUSIONS: -Gb3 accelerates the endocytosis and lysosomal degradation of endothelial KCa3.1 via a clathrin-dependent process, leading to endothelial dysfunction in FD.

Isolation and characterization of a metagenome-derived thermoalkaliphilic esterase with high stability over a broad pH range.

A novel alkaliphilic esterase (EstJ) was identified from a soil metagenome of Jeju Island, Korea, using a 96-well plate-based functional assay for determination of pH dependence of activity. The amino acid sequence of EstJ showed low similarity (32-45 %) to putative α/ß hydrolases derived from whole-genome sequencing studies. EstJ, although not belonging to any of the known families of bacterial lipolytic enzymes, however, it showed closest sequence identity to the family IV enzymes that are related to the mammalian hormone-sensitive lipases. The highly conserved motifs of family IV enzymes were found in EstJ, but the corresponding sequences of each motif in EstJ were unique; most particularly the -(F/Y)(F/Y/L)HGGG- motif was represented by -WMVSGG-. The purified EstJ was highly active from pH 8.5 to 10.5. More than 90 % of maximum activity was also retained over a wide pH range of 5.5-0.5 after prolonged incubation. EstJ was also moderately thermophilic with an optimum temperature of 55 °C. Therefore, EstJ is the first metagenome-derived bacterial family IV esterase possessing both highly alkaliphilic and moderately thermophilic properties.

Modified mandibulotomy technique to reduce postoperative complications: 5-year results.

PURPOSE: To review the 5-year outcomes of our modified mandibulotomy technique. Retrospective review of a tertiary level oral cancer center. MATERIALS AND METHODS: During a 5-year period, 30 patients who had a uniform surgical technique consisting of a lower lip-splitting, modified stair-step osteotomy with thin saw blade and osteotome after plate-precontouring and combination fixation with monocortical osteosynthesis (miniplate) and bicortical osteosynthesis (maxiplate and bicortical screws), with at least 14 months postoperative follow-up, were selected and reviewed retrospectively. RESULTS: There were 8 women and 22 men with an average age of 56.5 years. All the patients involved malignancies were squamous cell carcinoma. The main primary sites of the those who underwent a mandibulotomy were the tonsil, the base of tongue, the oral tongue, the retromolar pad area, and others. Others included buccal cheek, floor of mouth, and soft palate. 23 patients received postoperative radiation therapy, and among whom 8 patients also received chemotherapy. Total four (13%) mandibulotomy-related complications occurred, only two (6.7%) requiring additional operation under general anesthesia. CONCLUSION: Our modified mandibulotomy meets the criteria for an ideal mandibulotomy technique relatively well because it requires no intermaxillary fixation, can precise preserve the occlusion in a precise way, allows early function, requires no secondary procedures, and has few complications.

Contradictory Effects of Superoxide and Hydrogen Peroxide on KCa3.1 in Human Endothelial Cells.

Reactive oxygen species (ROS) are generated in various cells, including vascular smooth muscle and endothelial cells, and regulate ion channel functions. KCa3.1 plays an important role in endothelial functions. However, the effects of superoxide and hydrogen peroxide radicals on the expression of this ion channel in the endothelium remain unclear. In this study, we examined the effects of ROS donors on KCa3.1 expression and the K(+) current in primary cultured human umbilical vein endothelial cells (HUVECs). The hydrogen peroxide donor, tert-butyl hydroperoxide (TBHP), upregulated KCa3.1 expression, while the superoxide donors, xanthine/xanthine oxidase mixture (X/XO) and lysopho-sphatidylcholine (LPC), downregulated its expression, in a concentration-dependent manner. These ROS donor effects were prevented by antioxidants or superoxide dismustase. Phosphorylated extracellular signal-regulated kinase (pERK) was upregulated by TBHP and downregulated by X/XO. In addition, repressor element-1-silencing transcription factor (REST) was downregulated by TBHP, and upregulated by X/XO. Furthermore, KCa3.1 current, which was activated by clamping cells with 1 µM Ca(2+) and applying the KCa3.1 activator 1-ethyl-2-benzimidazolinone, was further augmented by TBHP, and inhibited by X/XO. These effects were prevented by antioxidants. The results suggest that hydrogen peroxide increases KCa3.1 expression by upregulating pERK and downregulating REST, and augments the K(+) current. On the other hand, superoxide reduces KCa3.1 expression by downregulating pERK and upregulating REST, and inhibits the K(+) current. ROS thereby play a key role in both physiological and pathological processes in endothelial cells by regulating KCa3.1 and endothelial function.

NADPH oxidase 2-derived superoxide downregulates endothelial KCa3.1 in preeclampsia.

Endothelial dysfunction is associated with KCa3.1 dysfunction and contributes to the development of hypertension in preeclampsia. However, evidence of endothelial KCa3.1 dysfunction in the vascular system from women with preeclampsia is still lacking. Therefore, we examined whether endothelial KCa3.1 dysfunction occurs in vessels from women with preeclampsia. We compared KCa3.1 and NADPH oxidase (NOX) expression in umbilical vessels and primary cultured human umbilical vein endothelial cells (HUVECs) from normal (NP; n=17) and preeclamptic pregnancy (PE; n=19) and examined the effects of plasma from NP or PE on KCa3.1 and NOX2 expression in primary cultured HUVECs from NP or human uterine microvascular endothelial cells. The endothelial KCa3.1 was downregulated, and NOX2 was upregulated, in umbilical vessels and HUVECs from PE, compared with those from NP. In addition, HUVECs from PE showed a significant decrease in KCa3.1 current. Plasma from PE induced KCa3.1 down regulation, NOX2 upregulation, phosphorylated-p38 mitogen-activated protein kinase downregulation, and superoxide generation, and these effects were prevented by antioxidants (tempol or tiron), NOX2 inhibition, or anti-lectin-like oxidized low-density lipoprotein (LDL) receptor 1 (LOX1) antibody. Oxidized LDL and the superoxide donor xanthine/xanthine oxidase mixture induced KCa3.1 downregulation. In contrast, plasma from PE did not generate hydrogen peroxide, and the hydrogen peroxide donor tert-butylhydroperoxide induced KCa3.1 upregulation. These results provide the first evidence that plasma from PE generates superoxide via a LOX1-NOX2-mediated pathway and downregulates endothelial KCa3.1, which may contribute to endothelial dysfunction and vasculopathy in preeclampsia. This suggests KCa3.1as a novel target for patients with preeclampsia.

Sclerosing polycystic adenosis of the parotid gland.

Sclerosing polycystic adenosis (SPA) is an uncommon salivary gland lesion similar to fibrocystic disease and adenosis of the mammary glands. To our knowledge, 51 cases of SPA have been reported in the literature to date. Sclerosing polycystic adenosis is well circumscribed, yet not encapsulated, and has sclerotic and irregularly defined lobules composed of abundant hyalinized collagen with ductal, acinar hyperplasia and areas of apocrine metaplasia. Focal cystic spaces within a dense fibrotic stroma are also characteristic features of this lesion. Most of the known cases occurred mainly in the parotid gland. In this article, we describe a case of SPA occurring in the parotid gland of a 47-year-old male patient.

TLR4-independent and PKR-dependent interleukin 1 receptor antagonist expression upon LPS stimulation.

Dendritic cells (DCs) induce innate immune responses by recognizing bacterial LPS through TLR4 receptor complexes. In this study, we compared gene expression profiles of TLR4 knockout (TLR4(neg)) DCs and wild type (TLR4(pos)) DCs after stimulating with LPS. We found that the expression of various inflammatory genes by LPS were TLR4-independent. Among them, interleukin 1 receptor antagonist (IL-1rn) was of particular interest since IL-1rn is a potent natural inhibitor of proinflammatory IL-1. Using RT-PCR, real-time PCR, immunoblotting and ELISA, we demonstrated that IL-1rn was induced by DCs stimulated with LPS in the absence of TLR4. 2-Aminopurine, a pharmacological PKR inhibitor, completely abrogated LPS-induced expression of IL-1rn in TLR4(neg) DCs, suggesting that LPS-induced TLR4-independent expression of IL-1rn might be mediated by PKR pathways. Considering that IL-1rn is a physiological inhibitor of IL-1, TLR4-independent and PKR-dependent pathways might be crucial in counter-balancing proinflammatory effector functions of DCs resulted from TLR4-dependent activation by LPS.