Utility of Next-Generation Sequencing for Deciphering Intratumor Heterogeneity in Prostate Cancer

Prostate cancer has long been considered a disease with a heterogeneous phenotype. The intratumor heterogeneity (ITH) may affect diverse phenotypes such as treatment response, drug resistance, and overall clinical outcomes. Despite technical advances over the past decade, we have an incomplete understanding of the extent and effects of ITH in prostate cancer progression. We present here a comprehensive review of the various studies dealing with ITH in prostate cancer. We discuss the advanced next-generation sequencing analyses including single cell sequencing and circulating cell-free DNA sequencing that have the impact of heterogeneity on clinical decision making.

Autologous blood derived cell therapy in maxillofacial bone graft surgery

Tissue regeneration is one of the ultimate goals of maxillofacial surgery and various types of tissue engineering technologies have been utilized in clinics. Healthy resources of host cells and growth factors are essential for the tissue engineering, therefore autologous blood-derived cell therapy was introduced. In this article, clinical applications of the autologous platelet concentrates and stem cell separation therapy will be summarized and evaluated for their efficacy and feasibility in the current maxillofacial clinics.

Utility of Next-Generation Sequencing for Deciphering Intratumor Heterogeneity in Prostate Cancer

Prostate cancer has long been considered a disease with a heterogeneous phenotype. The intratumor heterogeneity (ITH) may affect diverse phenotypes such as treatment response, drug resistance, and overall clinical outcomes. Despite technical advances over the past decade, we have an incomplete understanding of the extent and effects of ITH in prostate cancer progression. We present here a comprehensive review of the various studies dealing with ITH in prostate cancer. We discuss the advanced next-generation sequencing analyses including single cell sequencing and circulating cell-free DNA sequencing that have the impact of heterogeneity on clinical decision making.

Efficacy of arthrocentesis and lavage for treatment of post-traumatic arthritis in temporomandibular joints

Objectives@#Joint injuries frequently lead to progressive joint degeneration that causes articular disc derangement, joint inflammation, and osteoarthritis. Such arthropathies that arise after trauma are defined as post-traumatic arthritis (PTA). Although PTA is well recognized in knee and elbow joints, PTA in the temporomandibular joint (TMJ) has not been clearly defined. Interestingly, patients experiencing head and neck trauma without direct jaw fracture have displayed TMJ disease symptoms; however, definitive diagnosis and treatment options are not available. This study will analyze clinical aspects of PTA in TMJ and their treatment outcomes after joint arthrocentesis and lavage. @*Materials and Methods@#Twenty patients with history of trauma to the head and neck especially without jaw fracture were retrospectively studied. Those patients developed TMJ disease symptoms and were diagnosed by computed tomography or magnetic resonance imaging. To decrease TMJ discomfort, arthrocentesis and lavage with or without conservative therapy were applied, and efficacy was evaluated by amount of mouth opening and pain scale. Statistical differences between pre- and post-treatment values were evaluated by Wilcoxon signed-rank test. @*Results@#Patient age varied widely between 20 and 80 years, and causes of trauma were diverse. Duration of disease onset was measured as 508 posttrauma days, and 85% of the patients sought clinic visit within 2 years after trauma. In addition, 85% of the patients showed TMJ disc derangement without reduction, and osteoarthritis was accompanied at the traumatized side or at both sides in 40% of the patients. After arthrocentesis or lavage, maximal mouth opening was significantly increased (28-44 mm on average, P<0.001) and pain scale was dramatically decreased (7.8-3.5 of 10, P<0.001); however, concomitant conservative therapy showed no difference in treatment outcome. @*Conclusion@#The results of this study clarify the disease identity of PTA in TMJ and suggest early diagnosis and treatment options to manage PTA in TMJ.

A novel technique of submandibular intubation with a camera cable drape: a case report

Submental or submandibular intubation has been reported to cause fewer complications than tracheostomy. However, the risk of infection is always inherent because oral wounds are exposed to microbial flora and bacteria in the oral cavity. A novel technique of submandibular intubation was devised to reduce infection and injury to the soft tissues. We would like to report a novel safe technique that can be performed in patients requiring submental or submandibular intubation. This is the first report of submandibular intubation using a sterile disposable camera cable drape. This novel technique of submandibular intubation is safer, more sterile, easier, and less invasive than conventional submandibular intubation.

CD24⁺ Cell Depletion Permits Effective Enrichment of Thymic iNKT Cells While Preserving Their Subset Composition

Invariant NKT (iNKT) cells are a small subset of thymus-generated T cells that produce cytokines to control both innate and adaptive immunity. Because of their very low frequency in the thymus, in-depth characterization of iNKT cells can be facilitated by their enrichment from total thymocytes. Magnetic-activated cell sorting (MACS) of glycolipid antigen-loaded CD1d-tetramer-binding cells is a commonly used method to enrich iNKT cells. Surprisingly, we found that this procedure also dramatically altered the subset composition of enriched iNKT cells. As such, NKT2 lineage cells that express large amounts of the transcription factor promyelocytic leukemia zinc finger were markedly over-represented, while NKT1 lineage cells expressing the transcription factor T-bet were significantly reduced. To overcome this limitation, here, we tested magnetic-activated depletion of CD24⁺ immature thymocytes as an alternative method to enrich iNKT cells. We found that the overall recovery in iNKT cell numbers did not differ between these 2 methods. However, enrichment by CD24⁺ cell depletion preserved the subset composition of iNKT cells in the thymus, and thus permitted accurate and reproducible analysis of thymic iNKT cells in further detail.

Immunogenicity of a bivalent killed thimerosal-free oral cholera vaccine, Euvichol, in an animal model

PURPOSE: An oral cholera vaccine (OCV), Euvichol, with thimerosal (TM) as preservative, was prequalified by the World Health Organization (WHO) in 2015. In recent years, public health services and regulatory bodies recommended to eliminate TM in vaccines due to theoretical safety concerns. In this study, we examined whether TM-free Euvichol induces comparable immunogenicity to its TM-containing formulation in animal model. MATERIALS AND METHODS: To evaluate and compare the immunogenicity of the two variations of OCV, mice were immunized with TM-free or TM-containing Euvichol twice at 2-week interval by intranasal or oral route. One week after the last immunization, mice were challenged with Vibrio cholerae O1 and daily monitored to examine the protective immunity against cholera infection. In addition, serum samples were obtained from mice to measure vibriocidal activity and vaccine-specific IgG, IgM, and IgA antibodies using vibriocidal assay and enzyme-linked immunosorbent assay, respectively. RESULTS: No significant difference in immunogenicity, including vibriocidal activity and vaccine-specific IgG, IgM, and IgA in serum, was observed between mice groups administered with TM-free and -containing Euvichol, regardless of immunization route. However, intranasally immunized mice elicited higher levels of serum antibodies than those immunized via oral route. Moreover, intranasal immunization completely protected mice against V. cholerae challenge but not oral immunization. There was no significant difference in protection between two Euvichol variations. CONCLUSION: These results suggested that TM-free Euvichol could provide comparable immunogenicity to the WHO prequalified Euvichol containing TM as it was later confirmed in a clinical study. The pulmonary mouse cholera model can be considered useful to examine in vivo the potency of OCVs.

Out-sourcing for Trans-presentation: Assessing T Cell Intrinsic and Extrinsic IL-15 Expression with Il15 Gene Reporter Mice

IL-15 is a cytokine of the common γ-chain family that is critical for natural killer (NK), invariant natural killer T (iNKT), and CD8 memory T cell development and homeostasis. The role of IL-15 in regulating effector T cell subsets, however, remains incompletely understood. IL-15 is mostly expressed by stromal cells, myeloid cells, and dendritic cells (DCs). Whether T cells themselves can express IL-15, and if so, whether such T cell-derived IL-15 could play an autocrine role in T cells are interesting questions that were previously addressed but answered with mixed results. Recently, three independent studies described the generation of IL-15 reporter mice which facilitated the identification of IL-15-producing cells and helped to clarify the role of IL-15 both in vitro and in vivo. Here, we review the findings of these studies and place them in context of recent reports that examined T cell-intrinsic IL-15 expression during CD4 effector T cell differentiation.

LPS Sensing Mechanism of Human Astrocytes: Evidence of Functional TLR4 Expression and Requirement of Soluble CD14

Among a myriad of pathogen-associated molecular pattern-sensing receptors, toll-like receptors (TLRs) are the principal core sensors of the host. Despite intensive studies for the expression of TLRs and their roles in the central nervous system, controversies remain regarding the expression and the function of TLR4 in human astrocytes. In order to clarify this issue, we attempted to verify functional expression of TLR4 in human astrocytes. Using Reverse transcription-polymerase chain reaction (RT-PCR), we confirmed that the human astrocytes express the TLR4 constitutively. To determine the function of TLR4, astrocytes were treated with TLR4 ligand or lipopolysaccharide (LPS), and then inflammatory cytokines expressions were checked using RT-PCR and enzyme-linked immunosorbent assay. Nuclear factor (NF)-κB activation was checked using electrophoretic mobility shift assay. Treatment of astrocytes with LPS increased tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 expression and induced NF-κB activation. Neutralizing anti-TLR4 antibody blocked the effect of LPS on cytokine production and NF-κB activation in astrocytes. The effect of LPS on cytokine production and NF-κB activation was shown in the presence of serum but not in the absence of serum. Therefore, we investigated the sensing mechanism of LPS in human astrocytes. Human astrocytes were treated with LPS following neutralizing anti-CD14 antibody treatment in the presence of serum. Neutralizing anti-CD14 antibody treatment abolished the effect of LPS on cytokine expression and NF-κB activation. Additionally, supplement of recombinant CD14 in serum-free media induced LPS effect on cytokine production and NF-κB activation. In these results, we showed that human astrocytes constitutively express functional TLR4 and require soluble CD14 to recognize LPS.

Proteus vulgaris and Proteus mirabilis Decrease Candida albicans Biofilm Formation by Suppressing Morphological Transition to Its Hyphal Form

PURPOSE: Candida albicans (C. albicans) and Proteus species are causative agents in a variety of opportunistic nosocomial infections, and their ability to form biofilms is known to be a virulence factor. In this study, the influence of co-cultivation with Proteus vulgaris (P. vulgaris) and Proteus mirabilis (P. mirabilis) on C. albicans biofilm formation and its underlying mechanisms were examined. MATERIALS AND METHODS: XTT reduction assays were adopted to measure biofilm formation, and viable colony counts were performed to quantify yeast growth. Real-time reverse transcriptase polymerase chain reaction was used to evaluate the expression of yeast-specific genes (rhd1 and rbe1), filament formation inhibiting genes (tup1 and nrg1), and hyphae-related genes (als3, ece1, hwp1, and sap5). RESULTS: Candida biofilm formation was markedly inhibited by treatment with either living or heat-killed P. vulgaris and P. mirabilis. Proteus-cultured supernatant also inhibited Candida biofilm formation. Likewise, treatment with live P. vulgaris or P. mirabilis or with Proteus-cultured supernatant decreased expression of hyphae-related C. albicans genes, while the expression of yeast-specific genes and the filament formation inhibiting genes of C. albicans were increased. Heat-killed P. vulgaris and P. mirabilis treatment, however, did not affect the expression of C. albicans morphology-related genes. CONCLUSION: These results suggest that secretory products from P. vulgaris and P. mirabilis regulate the expression of genes related to morphologic changes in C. albicans such that transition from the yeast form to the hyphal form can be inhibited.

The Influence of Urinary Catheter Materials on Forming Biofilms of Microorganisms

Biofilms are commonly associated with an increased risk of catheter-associated infection. To study the efficacy of materials designed to reduce biofilm formation, microbial biofilms on clinically used urinary catheter were examined. We performed 2, 3-bis (2-methyoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay to determine of biofilm formation ability and observed with scanning electron microscopy (SEM) to analyze biofilm architecture. Additionally, we calculated relative cell surface hydrophobicity (CSH) to measure hydrophobicity of microorganisms. On SEM, catheter surfaces made of latex or anti-infective (IC)-latex were rough but those of silicone, hydrogel-coated silicone (HCS), or silver-alloy-coated silicone (SCS) were relatively smoother. According to XTT reduction assay, biofilm formation was reduced on the surface of smooth silicone-based catheters compared to rough latex-based catheters. The greatest to lowest formation of microbial biofilm were as follows for these material types: silicone-elastomer-coated (SEC) latex > latex > silicone > IC-latex > HCS > SCS. Catheter materials can affect the microbial biofilm formations. First, rougher surfaces on the catheter made the microbial attachment easier and a greater amount of biofilm was formed. Second, when chemicals that inhibit growth and attachment of microorganisms on the inner and outer surfaces of the catheters were applied, the biofilm formation was inhibited. SCS was found to be the most effective in reducing the microbial biofilm formation. These results indicate that microbial biofilm formation may be closely related to the surface roughness and microbial CSH.

Effects of Reactive Oxygen Species and Nitrogen Species on the Excitability of Spinal Substantia Gelatinosa Neurons

Reactive oxygen species (ROS) and nitrogen species (RNS) are both important signaling molecules involved in pain transmission in the dorsal horn of the spinal cord. Xanthine oxidase (XO) is a well-known enzyme for the generation of superoxide anions (O₂˙⁻), while S-nitroso-N-acetyl-DL-penicillamine (SNAP) is a representative nitric oxide (NO) donor. In this study, we used patch clamp recording in spinal slices of rats to investigate the effects of O₂˙⁻ and NO on the excitability of substantia gelatinosa (SG) neurons. We also used confocal scanning laser microscopy to measure XO- and SNAP-induced ROS and RNS production in live slices. We observed that the ROS level increased during the perfusion of xanthine and xanthine oxidase (X/XO) compound and SNAP after the loading of 2',7'-dichlorofluorescin diacetate (H₂DCF-DA), which is an indicator of intracellular ROS and RNS. Application of ROS donors such as X/XO, β-nicotinamide adenine dinucleotide phosphate (NADPH), and 3-morpholinosydnomimine (SIN-1) induced a membrane depolarization and inward currents. SNAP, an RNS donor, also induced membrane depolarization and inward currents. X/XO-induced inward currents were significantly decreased by pretreatment with phenyl N-tert-butylnitrone (PBN; nonspecific ROS and RNS scavenger) and manganese(III) tetrakis(4-benzoic acid) porphyrin (MnTBAP; superoxide dismutase mimetics). Nitro-L-arginine methyl ester (NAME; NO scavenger) also slightly decreased X/XO-induced inward currents, suggesting that X/XO-induced responses can be involved in the generation of peroxynitrite (ONOO⁻). Our data suggest that elevated ROS, especially O₂˙⁻, NO and ONOO⁻, in the spinal cord can increase the excitability of the SG neurons related to pain transmission.

Analysis of the Baroreceptor and Vestibular Receptor Inputs in the Rostral Ventrolateral Medulla following Hypotension in Conscious Rats

Input signals originating from baroreceptors and vestibular receptors are integrated in the rostral ventrolateral medulla (RVLM) to maintain blood pressure during postural movement. The contribution of baroreceptors and vestibular receptors in the maintenance of blood pressure following hypotension were quantitatively analyzed by measuring phosphorylated extracellular regulated protein kinase (pERK) expression and glutamate release in the RVLM. The expression of pERK and glutamate release in the RVLM were measured in conscious rats that had undergone bilateral labyrinthectomy (BL) and/or sinoaortic denervation (SAD) following hypotension induced by a sodium nitroprusside (SNP) infusion. The expression of pERK was significantly increased in the RVLM in the control group following SNP infusion, and expression peaked 10 min after SNP infusion. The number of pERK positive neurons increased following SNP infusion in BL, SAD, and BL+SAD groups, although the increase was smaller than seen in the control group. The SAD group showed a relatively higher reduction in pERK expression when compared with the BL group. The level of glutamate release was significantly increased in the RVLM in control, BL, SAD groups following SNP infusion, and this peaked 10 min after SNP infusion. The SAD group showed a relatively higher reduction in glutamate release when compared with the BL group. These results suggest that the baroreceptors are more powerful in pERK expression and glutamate release in the RVLM following hypotension than the vestibular receptors, but the vestibular receptors still have an important role in the RVLM.

Influence of Bacterial Presence on Biofilm Formation of Candida albicans

PURPOSE: Candida albicans is an opportunistic pathogen that is commonly found in human microflora. Biofilm formation (BF) is known as a major virulence factor of C. albicans. The aim of this study was to examine the influence of bacterial presence on biofilm formation of C. albicans. MATERIALS AND METHODS: The BF of Candida was investigated when it was co-cultured with C. albicans (C. albicans 53, a yeast with a low BF ability, and C. albicans 163, a yeast with high BF ability) and bacteria. BF was assessed with XTT reduction assay. A scanning electron microscope was used to determine the structure of the biofilm, and real-time reverse transcriptase polymerase chain reaction was used to amplify and quantify hyphae-associated genes. RESULTS: Co-culturing with two different types of bacteria increased the BF value. Co-culturing with C. albicans 53 and 163 also increased the BF value compared to the value that was obtained when the C. albicans was cultured individually. However, co-culturing with bacteria decreased the BF value of C. albicans, and the BF of C. albicans 163 was markedly inhibited. The expression of adherence and morphology transition related genes were significantly inhibited by co-culturing with live bacteria. CONCLUSION: Bacteria have a negative effect on the formation of biofilm by C. albicans. This mechanism is the result of the suppression of genes associated with the hyphae transition of C. albicans, and bacteria particles physically affected the biofilm architecture and biofilm formation.

Effect of Vestibulosympathetic Reflex and Baroreflex on Expression of pERK in the Nucleus Tractus Solitarius following Acute Hypotension in Conscious Rats

Control of blood pressure is maintained by the interaction between the arterial baroreflex and vestibulosympathetic reflex during postural changes. In this study, the contributions of vestibular receptors and baroreceptors to the maintenance of blood pressure following acute hypotension were compared in terms of phosphorylated extracellular regulated protein kinase (pERK) expression in the nucleus tractus solitaries (NTS). Expression of pERK in the NTS was measured in conscious rats that had undergone bilateral labyrinthectomy (BL) and/or sinoaortic denervation (SAD) 5, 10, 20, and 40 min following acute hypotension induced by sodium nitroprusside (SNP) infusion. Expression of pERK increased significantly in the NTS in the control group following SNP infusion, and the expression peaked at 10 min after SNP infusion. The number of pERK positive neurons increased following SNP infusion in BL, SAD, and BL+SAD groups, although the increase was smaller than in control group. The BL group showed a relatively higher reduction in pERK expression than the SAD group, and the pERK expression in the NTS was localized to the caudal portion of the nuclei in the BL and SAD groups. These results suggest that the vestibular receptors may play a key role in maintaining blood pressure following acute hypotension; thus, the vestibular system may contribute to compensate for orthostatic hypotension.

Determination of midsagittal plane for evaluation of facial asymmetry using three-dimensional computed tomography

PURPOSE: The aim of the present study was to investigate the disagreement of cephalometric analysis depending on the reference determination of midsagittal plane on three-dimensional computed tomography. MATERIALS AND METHODS: A total of 102 young women with class III dentofacial deformity were evaluated using three-dimensional computed tomography. The cranial and facial midsagittal planes were defined and the amounts of jaw deviation were calculated. The amounts of jaw deviation were compared with paired t-test (2-tailed) and Bland-Altman plot was drawn. RESULTS: The landmark tracing were reproducible (r> or =.978). The jaws relative to the cranial midsagittal plane were 10-17 times more significantly deviated than to the facial midsagittal plane (P<.001). Bland-Altman plot demonstrated that the differences between the amounts of jaw deviation from two midsagittal planes were not normally distributed versus the average of the amounts of jaw deviation from two midsagittal planes. CONCLUSION: The cephalometric analyses of facial asymmetry were significantly inconsistent depending on the reference determination of midsagittal plane. The reference for midsagittal plane should be carefully determined in three-dimensional cephalometric analysis of facial asymmetry of patients with class III dentofacial deformity.

Protective Effects of Immunization with Proteinase, Phospholipase and Cell Wall Proteins of Candida albicans in Mice Candidosis / 대한의진균학회지

BACKGROUND: The opportunistic fungus Candida albicans is a major pathogen especially to immunocompromised patients. OBJECTIVES: We examined the protective effect of the active and passive immunizations to evaluate the applicability for the treatment of candidosis in Candida-infected mice model. METHODS: Candida cell wall components were obtained by treatment of lyticase, proteinase K, and dithiothreitol. The proteinase was purified from the culture filtrates of C. albicans using a series of chromatographic steps consisting of DEAE-Sepharose FF, Sephacryl S-200 HR and size-exclusion high performance liquid chromatography. The phospholipase was purified from the culture supernatant of C. albicans with DEAE column chromatography, reverse phase column chromatography, revere phase HPLC and size-exclusion HPLC. Antibodies to cell wall protein components, proteinase and phospholipase were produced by immunization into mice of same strain. RESULTS: The mean survival times of active and passive immunized mice groups were longer than those of non-immunized groups. CONCLUSION: These results showed that immunization with proteinase and its antibody were the most effective to prolong survival time in Candida-infected mice.

Case Report: A Plunging Ranula Extended into the Parapharyngeal Space

Effect of Culture Conditions on the Protein Fibril Expression of Candida albicans

Candida albicans is an important human pathogen that causes systemic infections, predominantly among population with weakened immune system. Cell wall structures of C. albicans are important to adhere to host tissue and evade to host immune system. Among cell wall structure, the outermost fibrillar layer of C. albicans is of interest since it may play important roles in antigenicity, phagocytosis, and adherence. The expression of virulent factors could be affected by the growth conditions. The dynamic nature of the cell surface alters the physical properties of the fungal interface with host cells and thereby influences adhesion to the host and recognition by components of the host immune system. In this study, we investigated the effects of culture conditions on cell surface fibril expression of C. albicans by a transmitting electron microscopy and SDS-PAGE. The protein fibril of C. albicans was expressed in the presence of whole serum, however, the fibril expression was decreased in 25% serum and serum containing 1% glucose. Also, germ tube can be induced by serum, RMPI medium, N-acetyl glucosamine, and 39 degrees C culture condition, hence, the fibrillar structure of C. albicans was detected only in serum-induced germ tube. The expression of fibril layer and the major fibril proteins of 66, 47, 30 kDa were reduced as increasing cell concentration of intial inoculum from 2x10(7) cells/ml to 8x10(7) cells/ml. The fibrillar layer of C. albicans was expressed in serum early within 10 min, and the thickness of fibril layer was increased according to the increase of culture time. When the fibrillar proteins were analysed by SDS-PAGE, major protein of 30 kDa was maintained continuously during over night culture although expression of the other proteins were various. These results suggest that expression of serum induced protein fibril is influenced by culture conditions and is not related to hyphal transition of C. albicans.

The Expression Pattern of Toll-like Receptor (TLR) and Cytokine Production to TLR Agonists in Human Retinal Pigment Epithelial Cells

Retinal pigment epithelium (RPE) constituting the outer blood-retina barrier plays an important role in ocular defense mechanism. Many studies reported that RPE participates in ongoing immune responses in the retina. However, the exact mechanism is still uncertain. Toll-like receptors (TLRs) participate in the recognition of pathogen-associated molecular patterns (PAMP), such as LPS, zymosan, lipoprotein, and dsRNA. The expression and function of TLRs in human RPE have not been established. In this study, we investigated TLRs expression in human fetal RPE and their recognition of PAMP to determine how human RPE participates in ocular defense mechanism against microbial component. RT-PCR and real time PCR revealed that TLR1 through 5 were constitutively expressed in human fetal RPE, and their expressions were slightly increased by LPS. We determined the TNF-alpha, IL-6, and IL-8 expression in human fetal RPE after treatment with LPS, zymosan, petidoglycan, or poly I:C. RT-PCR demonstrated that LPS and poly I:C treatment increased the production of TNF-alpha, IL-6, and IL-8 in human fetal RPE. LPS showed more potent effects on TNF-alpha and IL-8 production. Peptidoglycan and zymosan did not induce the production of TNF-alpha. CD14, the co-receptor of LPS was weakly expressed and functioned in recognizing LPS in human fetal RPE. These results suggest that human RPE may participate in ocular defense mechanism against microbial component through toll-like receptors.