Higher fibular head is a risk for lateral hinge fracture in medial open wedge high tibial osteotomy.

PURPOSE: To examine the association between the hinge position, fibular head position, and type III lateral hinge fracture (LHF) in patients with knee osteoarthritis (OA) who underwent medial open wedge high tibial osteotomy (MOWHTO). METHODS: This retrospective study examined patients who underwent MOWHTO. Radiographically, the Kellgren-Lawrence (K/L) classification, distance between the articular surface and the tip of the fibular head (fibular head position), hinge point (hinge position), type of LHF, and safe zone (within the proximal tibiofibular joint) outlier were evaluated. To determine the cut-off value of the hinge position and fibular head position associated with type III LHF, a receiver operating characteristic (ROC) curve analysis was performed. The odds ratio (OR) was calculated from the obtained cut-off values using logistic regression, which was adjusted by age, gender, body mass index, and opening distance. RESULTS: Among 132 knees in 120 patients, the radiographic severity of knee OA was 19 (14%), 73 (55%), and 40 (30%) of K/L grades 2, 3, and 4, respectively. LHF was observed in 40 knees (30%), including types I, II, and III fractures in 21 (16%), 5 (4%), and 14 (11%) knees, respectively. Hinge and fibular head positions were 16 and 10 mm, respectively, with significant correlation. Safe zone outlier was observed in 38 knees (29%). The hinge and fibular head positions with type III LHF were significantly higher (more cranial) than those with no fracture or other LHF subtypes. The ROC curve revealed that the cut-off value for the hinge and fibular head positions was 13.3 and 8.6 mm, respectively. The OR of the hinge and fibular head positions was 22.42 and 13.86, respectively. CONCLUSIONS: A higher hinge position was a risk factor for type III LHF and was associated with a higher fibular head in patients with knee OA who underwent MOWHTO. The hinge position should be placed at a certain distance from the articular surface to avoid type III LHF, especially in participants with higher fibular head position, even if the hinge position is located in the safe zone. LEVEL OF EVIDENCE: Retrospective cohort study, Level III.

Effect of 10-Methacryloyloxydecyl Dihydrogen Phosphate Concentrations in Primers on Bonding Resin Cements to Zirconia.

PURPOSE: To investigate the effect of 10-methacryloyloxydecyl dihydrogen phosphate (MDP) concentration in primers on bonding of resin cements to zirconia. MATERIALS AND METHODS: Highly translucent partially stabilized zirconia plates were bonded to stainless steel rods using one of 3 commercially available resin cements without adhesive functional monomers and without air-abrasion. Ten specimens of each resin cement group were pretreated with one of the primers containing 0.5, 1.0, 2.0, 3.0, 4.0, or 5.0 wt% of MDP dissolved in ethanol, or one of 3 commercial ceramic primers containing MDP and silane coupling agents. Untreated specimens were used as controls. The tensile bond strength was measured after 24 hours (TC0) and after thermal cycling at 4 to 60°C (TC3,500). Data were statistically analyzed using 1- or 2-way ANOVA and Tukey-Kramer post-hoc tests (α = 0.05). RESULTS: The mean tensile bond strength in the unpretreated group was significantly lower compared to that in the pretreated groups with MDP-containing primers regardless of the resin cement used (p < 0.0001). The tensile bond strengths increased with an increase in MDP concentration from 0.5 wt% to 3.0 wt% or 4.0 wt%. Pretreated groups with 4.0-wt% MDP-containing primer showed significantly higher tensile bond strengths than pretreated groups with commercial ceramic primers regardless of the resin cement used and thermal cycling (p < 0.0001). CONCLUSION: Primers containing 4.0-wt% MDP achieved higher bond strengths of resin cements to zirconia compared to commercial ceramic primers containing MDP and silane coupling agent.

Increased Stiffness of Rotator Cuff Tendons in Frozen Shoulder on Shear Wave Elastography.

OBJECTIVES: To evaluate the stiffness and morphologic characteristics of the capsule, rotator cuff tendons and muscles, coracohumeral ligament (CHL), and long head of the biceps in patients with frozen shoulder using shear wave elastography (SWE) with B-mode ultrasound. METHODS: Thirty-two patients with frozen shoulder were divided into freezing and frozen phases. All patients had limitations of their range of motion without rotator cuff tears. Stiffness was measured by SWE in the supraspinatus (SSp) tendon, infraspinatus (ISp) tendon, SSp muscle, ISp muscle, teres minor muscle, upper and lower trapezius muscles, posterior capsule, CHL, and long head of the biceps. The posterior capsule and CHL thicknesses were also investigated with B-mode ultrasound. All values were compared in the affected and unaffected shoulders in each phase. RESULTS: The SWE values for the SSp and ISp tendons in the freezing phase and the CHL in the frozen phase were significantly greater on the affected side than the unaffected side (mean ± SD, 280.4 ± 125.3 versus 178.1 ± 73.3, 318.4 ± 110.7 versus 240.8 ± 91.5, and 287.2 ± 135.3 versus 214.1 ± 91.1 kPa, respectively; P < .05). The posterior capsule in both the freezing and frozen phases and the CHL in the frozen phase were significantly thicker on the affected side than the unaffected side (1.3 ± 0.2 versus 0.9 ± 0.3, 1.2 ± 0.4 versus 0.9 ± 0.3, and 4.4 ± 1.4 versus 3.3 ± 1.1 mm; P < .01). CONCLUSIONS: The SWE values of the both SSp and ISp tendons increased in the freezing phase, and that of the CHL also increased in the frozen phase. Not only the change in thickness of the capsule but also the change in stiffness of the rotator cuff may correlate with frozen shoulder.

Influence of cleaning methods on the bond strength of resin cement to saliva-contaminated lithium disilicate ceramic.

OBJECTIVES: The aim of the present study was to examine the influence of different cleaning methods on the bond strength of resin cement to saliva-contaminated lithium disilicate ceramic. MATERIALS AND METHODS: Lithium disilicate ceramic specimens (n = 8/group) were etched with 5% hydrofluoric acid (HF) to comprise the control group. After or before saliva contamination, specimens were cleaned with one of five methods: rinsing with water-spray (WS), K etchant GEL (PA), Ivoclean (IC), AD Gel (ADG), or application of a silane coupling agent before immersion in saliva (SCA). Stainless steel rods were bonded to the ceramic with resin cement. The tensile bond strength was measured after 24 h (TC0) and after thermal cycling at 4-60 °C (TC20000). Specimen surfaces were also evaluated using X-ray photoelectron spectroscopy. Data were statistically analyzed using two-way ANOVA and Tukey tests (α = 0.05). RESULTS: Both the cleaning method (p = 0.0001) and storage condition (p = 0.0001) significantly affected the bond strength results. Before or after TCs, no significant differences in bond strength were observed between the control group and the other cleaning groups except for the WS group, which had a significantly lower bond strength than did the PA, IC, ADG, or SCA groups (p < 0.05). The level of nitrogen in the ADG group was almost equal to that in the control group. CONCLUSIONS: WS cleaning did not restore the bond strength of resin cement to saliva-contaminated lithium disilicate ceramic etched with HF, while PA, IC, ADG, and SCA all benefited. CLINICAL RELEVANCE: Lithium disilicate ceramic restorations etched with HF should to be cleaned with ADG after saliva contamination.

Suppression of Non-Random Fertilization by MHC Class I Antigens.

Hermaphroditic invertebrates and plants have a self-recognition system on the cell surface of sperm and eggs, which prevents their self-fusion and enhances non-self-fusion, thereby contributing to genetic variation. However, the system of sperm-egg recognition in mammals is under debate. To address this issue, we explored the role of major histocompatibility complex class I (MHC class I, also known as histocompatibility 2-Kb or H2-Kb and H2-Db in mice) antigens by analyzing H2-Kb-/-H2-Db-/-ß2-microglobulin (ß2M)-/- triple-knockout (T-KO) male mice with full fertility. T-KO sperm exhibited an increased sperm number in the perivitelline space of wild-type (WT) eggs in vitro. Moreover, T-KO sperm showed multiple fusion with zona pellucida (ZP)-free WT eggs, implying that the ability of polyspermy block for sperm from T-KO males was weakened in WT eggs. When T-KO male mice were intercrossed with WT female mice, the percentage of females in progeny increased. We speculate that WT eggs prefer fusion with T-KO sperm, more specifically X-chromosome-bearing sperm (X sperm), suggesting the presence of preferential (non-random) fertilization in mammals, including humans.

Healing Process of Gastrocnemius Muscle Injury on Ultrasonography Using B-Mode Imaging, Power Doppler Imaging, and Shear Wave Elastography.

OBJECTIVES: Muscle injury often occurs in sports activity. To avoid reinjury, it is important to determine the appropriate period until return to play after injury. The purpose of this study was to evaluate characteristics of the healing process for gastrocnemius muscle injury by B-mode imaging, power Doppler (PD) imaging, and shear wave elastography (SWE). METHODS: Twenty patients with acute calf musculotendinous injury were enrolled. Scar thickness on B-mode imaging, new vessels according to PD grades, and SWE values in the muscle, musculotendinous junction, and tendon of the medial head of gastrocnemius were measured at 4, 8, and 12 weeks after injury. RESULTS: Scar thickness was significantly larger at 8 and 12 weeks compared with 4 weeks (P < .01 for both). Power Doppler grades at 4 and 8 weeks were significantly higher compared with 12 weeks (P < .01 for both). Shear wave elastographic values in the muscle on the injury side were significantly higher at 8 and 12 weeks compared with 4 weeks (P < .01 for both), whereas those in the musculotendinous junction on the injury side were significantly higher at 12 weeks compared with 4 and 8 weeks (P < .01; P = .01, respectively). CONCLUSIONS: B-mode imaging, PD imaging, and SWE can measure the healing process after musculotendinous injury of the gastrocnemius medial head.

Deletion of Eqtn in mice reduces male fertility and sperm-egg adhesion.

A number of sperm proteins are involved in the processes from gamete adhesion to fusion, but the underlying mechanism is still unclear. Here, we established a mouse mutant, the EQUATORIN-knockout (EQTN-KO, Eqtn - / - ) mouse model and found that the EQTN-KO males have reduced fertility and sperm-egg adhesion, while the EQTN-KO females are fertile. Eqtn - / - sperm were normal in morphology and motility. Eqtn - / - -Tg (Acr-Egfp) sperm, which were produced as the acrosome reporter by crossing Eqtn - / - with Eqtn +/+ -Tg(Acr-Egfp) mice, traveled to the oviduct ampulla and penetrated the egg zona pellucida of WT females. However, Eqtn - / - males mated with WT females showed significant reduction in both fertility and the number of sperm attached to the zona-free oocyte. Sperm IZUMO1 and egg CD9 behaved normally in Eqtn - / - sperm when they were fertilized with WT egg. Another acrosomal protein, SPESP1, behaved aberrantly in Eqtn - / - sperm during the acrosome reaction. The fertility impairment of EQTN/SPESP1-double KO males lacking Eqtn and Spesp1 (Eqtn/Spesp1 - / - ) was more severe compared with that of Eqtn - / - males. Eqtn - / - -Tg (Eqtn) males, which were generated to rescue Eqtn - / - males, restored the reduced fertility.

High affinity sugar ligands of C-type lectin receptor langerin.

BACKGROUND: Langerin, a C-type lectin receptor (CLR) expressed in a subset of dendritic cells (DCs), binds to glycan ligands for pathogen capture and clearance. Previous studies revealed that langerin has an unusual binding affinity toward 6-sulfated galactose (Gal), a structure primarily found in keratan sulfate (KS). However, details and biological outcomes of this interaction have not been characterized. Based on a recent discovery that the disaccharide L4, a KS component that contains 6-sulfo-Gal, exhibits anti-inflammatory activity in mouse lung, we hypothesized that L4-related compounds are useful tools for characterizing the langerin-ligand interactions and their therapeutic application. METHODS: We performed binding analysis between purified long and short forms of langerin and a series of KS disaccharide components. We also chemically synthesized oligomeric derivatives of L4 to develop a new high-affinity ligand of langerin. RESULTS: We show that the binding critically requires the 6-sulfation of Gal and that the long form of langerin displays higher affinity than the short form. The synthesized trimeric (also designated as triangle or Tri) and polymeric (pendant) L4 derivatives displayed over 1000-fold higher affinity toward langerin than monomeric L4. The pendant L4, but not the L4 monomer, was found to effectively transduce langerin signaling in a model cell system. CONCLUSIONS: L4 is a specific ligand for langerin. Oligomerization of L4 unit increased the affinity toward langerin. GENERAL SIGNIFICANCE: These results suggest that oligomeric L4 derivatives will be useful for clarifying the langerin functions and for the development of new glycan-based anti-inflammatory drugs.

Shear Wave Elastography Can Predict Passive Stiffness of Supraspinatus Musculotendinous Unit During Arthroscopic Rotator Cuff Repair for Presurgical Planning.

PURPOSE: To determine the feasibility of shear wave elastography (SWE) with B-mode ultrasound in predicting the stiffness of the rotator cuff muscle before arthroscopic rotator cuff repair to evaluate the difficulty of the surgical procedure, as well as to compare SWE with the Goutallier stage on magnetic resonance imaging (MRI). METHODS: Thirty-eight patients with a full-thickness supraspinatus tear requiring arthroscopic rotator cuff repair participated. The Goutallier stage of fatty infiltration on MRI was measured before surgery, as was the SWE modulus of the anterior superficial, anterior deep, posterior superficial, and posterior deep (PD) regions of the supraspinatus muscle. To measure the stiffness of the supraspinatus musculotendinous unit during surgery, the supraspinatus tendon was axially stretched until the anatomic insertion site was reached, and force per deformation was recorded. The correlation between stiffness of the supraspinatus and SWE value in each region of the supraspinatus muscle or Goutallier stage was determined. In addition, patients were divided into 2 groups: (1) In the complete footprint coverage group, greater than 50% of the footprint was covered during the stiffness measurement, and (2) in the incomplete footprint coverage group, less than 50% of the footprint was covered during the stiffness measurement. Differences in SWE value and Goutallier stage were measured between the 2 groups. RESULTS: The best correlation of stiffness with the SWE modulus of the PD muscle of the supraspinatus was R = 0.69, and the correlation of stiffness with the Goutallier stage on MRI was R = 0.48. The SWE value of the PD region was greater in the incomplete footprint coverage group than in the complete footprint coverage group, although the Goutallier stage was not significantly different. CONCLUSIONS: The highest correlation with stiffness of the supraspinatus musculotendinous unit was with the SWE modulus of the PD muscle, as compared with SWE evaluation of the other regions or the Goutallier stage on MRI. Ultrasound SWE can predict the stiffness of the supraspinatus musculotendinous unit best. CLINICAL RELEVANCE: Rotator cuff retraction adds difficulty to arthroscopic rotator cuff repair. Ultrasound SWE may be used for presurgical planning.

A new technique of surgical planning for anterior cruciate ligament reconstruction: Feasibility Assessment of Shear Wave Elastography to Tendon of Semitendinosus Muscle.

There is currently no method to quantify the quality of the harvested graft before anterior cruciate ligament (ACL) reconstruction. The purpose of this study was to explore a method for measuring stiffness of the normal tendon of the semitendinosus muscle using shear wave elastography (SWE). Our investigation comprised two steps. First, we determined orientation of the semitendinosus tendon fibers in 20 lower legs of embalmed cadavers. Second, we investigated the feasibility of quantifying stiffness of the normal semitendinosus tendon by SWE in 24 subjects (48 legs: 24 male and 24 female subjects) in vivo. Measured values were compared between male and female subjects. The point at the intersection of the semitendinosus tendon with the axial section through the center of the patella mostly was the middle of the semitendinosus tendon in the cadavers. The SWE modulus in all of the subjects could be measured on this point in vivo. The SWE modulus of males and females were 474.0 ± 71.9 kPa and 396.9 ± 104.5 kPa, respectively. The SWE modulus of males was significantly larger than that of females (P < 0.01). This study demonstrates that stiffness of the tendon of the semitendinosus muscle can be measured by SWE. It also shows that tendon of the semitendinosus muscle in males is stiffer than in females. Clin. Anat. 31:404-408, 2018. © 2017 Wiley Periodicals, Inc.

Influence of cleaning methods on resin bonding to saliva-contaminated zirconia.

OBJECTIVE: The aim of this study was to investigate the influence of different cleaning methods on the shear bond strengths of 2 resin cements to saliva-contaminated zirconia. METHODS: After saliva contamination, alumina-blasted zirconia specimens were cleaned with 1 of 5 methods of water-rinsing (SA), K-etchant GEL phosphoric acid (PA), Ivoclean (IC), AD Gel (ADG), or additional alumina-blasting (AB). Alumina-blasted zirconia without saliva contamination was used as control group (Cont). Composite cylinders were bonded to the zirconia with 1 of 2 dual-cured resin cements. The bond strengths were measured by shear testing after 24 hours (TC0) and after thermal cycling at 4°C-60°C (TC10 000) and specimen surfaces were evaluated using X-ray photoelectron spectroscopy (XPS). Data were statistically analyzed using 3-way analysis of variance and Tukey test (α = 0.05). RESULTS: There were no significant differences in the bond strengths of 2 resin cements between the Cont ADG, and AB groups before and after TCs (P > .05). SA, PA, and IC groups did not exhibit durable resin bonding to zirconia. XPS showed that carbon and nitrogen increased in the SA group in comparison to the Cont group. The concentration of carbon in other 4 groups returned to the concentration range of the Cont group; however, nitrogen was not detected in the only AB group. CONCLUSIONS: Saliva contamination significantly reduced the bond strength of 2 resin cements to zirconia. Additional AB or cleaning with ADG resulted in effective cleaning of saliva contamination and preserved resin cement bond strength to zirconia. CLINICAL SIGNIFICANCE: Saliva contamination occurs during clinical procedures for adjustment of zirconia ceramic restorations in the oral environment. AD Gel application is effective for removing saliva contaminants on the alumina-blasted zirconia surface beforehand by the dental laboratory instead of additional AB since AD Gel application and AB had a similar effect on the removal of organic components of saliva.

A keratan sulfate disaccharide prevents inflammation and the progression of emphysema in murine models.

Emphysema is a typical component of chronic obstructive pulmonary disease (COPD), a progressive and inflammatory airway disease. However, no effective treatment currently exists. Here, we show that keratan sulfate (KS), one of the major glycosaminoglycans produced in the small airway, decreased in lungs of cigarette smoke-exposed mice. To confirm the protective effect of KS in the small airway, a disaccharide repeating unit of KS designated L4 ([SO3--6]Galß1-4[SO3--6]GlcNAc) was administered to two murine models: elastase-induced-emphysema and LPS-induced exacerbation of a cigarette smoke-induced emphysema. Histological and microcomputed tomography analyses revealed that, in the mouse elastase-induced emphysema model, administration of L4 attenuated alveolar destruction. Treatment with L4 significantly reduced neutrophil influx, as well as the levels of inflammatory cytokines, tissue-degrading enzymes (matrix metalloproteinases), and myeloperoxidase in bronchoalveolar lavage fluid, suggesting that L4 suppressed inflammation in the lung. L4 consistently blocked the chemotactic migration of neutrophils in vitro. Moreover, in the case of the exacerbation model, L4 inhibited inflammatory cell accumulation to the same extent as that of dexamethasone. Taken together, L4 represents one of the potential glycan-based drugs for the treatment of COPD through its inhibitory action against inflammation.

Application of shear wave elastography for the gastrocnemius medial head to tennis leg.

Muscle strain of the gastrocnemius medial head mainly occurs at the musculotendinous junction (MTJ), and stiffness is a risk factor. Shear wave elastography (SWE) measures elasticity by determining the propagation velocity. The aim of this study was to measure the elasticity of the normal muscle and aponeurosis in the MTJ of the gastrocnemius medial head using SWE, thus obtaining information relevant to muscle strain at this point. Forty-one volunteers (82 legs) were recruited and the gastrocnemius medial heads were examined at four points: three on the aponeurosis, namely at the center of the MTJ (Central), 10 mm proximal to it (Proximal) and 10 mm distal to it (Distal); and at one on the muscle belly (Muscle). The measurements were compared among the points, between males and females, and between younger and middle-aged subjects. Correlations between the elastic modulus and age were also examined. The elastic moduli at Proximal, Central, Distal, and Muscle were 2.82 ± 0.53 m/s, 3.43 ± 0.83 m/s, 4.83 ± 1.56 m/s, and 2.25 ± 0.43 m/s, respectively. These values differed significantly among the points of the aponeurosis, Distal having the highest modulus followed by Central. The elastic moduli were significantly greater in males than females at Distal and Muscle and in younger subjects than middle-aged subjects at Muscle. No significant correlations between elastic modulus and age were observed for any point. SWE could be a feasible method for quantifying the elasticity of muscle and aponeurosis in the MTJ of the gastrocnemius medial head. Clin. Anat. 30:114-119, 2017. © 2016 Wiley Periodicals, Inc.

Microhardness of dual-polymerizing resin cements and foundation composite resins for luting fiber-reinforced posts.

STATEMENT OF PROBLEM: The optimal luting material for fiber-reinforced posts to ensure the longevity of foundation restorations remains undetermined. PURPOSE: The purpose of this study was to evaluate the suitability of 3 dual-polymerizing resin cements and 2 dual-polymerizing foundation composite resins for luting fiber-reinforced posts by assessing their Knoop hardness number. MATERIAL AND METHODS: Five specimens of dual-polymerizing resin cements (SA Cement Automix, G-Cem LincAce, and Panavia F2.0) and 5 specimens of dual-polymerizing foundation composite resins (Clearfil DC Core Plus and Unifil Core EM) were polymerized from the top by irradiation for 40 seconds. Knoop hardness numbers were measured at depths of 0.5, 2.0, 4.0, 6.0, 8.0, and 10.0 mm at 0.5 hours and 7 days after irradiation. Data were statistically analyzed by repeated measures ANOVA, 1-way ANOVA, and the Tukey compromise post hoc test (α=.05). RESULTS: At both times after irradiation, the 5 resins materials showed the highest Knoop hardness numbers at the 0.5-mm depth. At 7 days after irradiation, the Knoop hardness numbers of the resin materials did not differ significantly between the 8.0-mm and 10.0-mm depths (P>.05). For all materials, the Knoop hardness numbers at 7 days after irradiation were significantly higher than those at 0.5 hours after irradiation at all depths (P<.05). At 7 days after irradiation, the Knoop hardness numbers of the 5 resin materials were found to decrease in the following order: DC Core Plus, Unifil Core EM, Panavia F2.0, SA Cement Automix, and G-Cem LincAce (P<.05). CONCLUSIONS: The Knoop hardness number depends on the depth of the cavity, the length of time after irradiation, and the material brand. Although the Knoop hardness numbers of the 2 dual-polymerizing foundation composite resins were higher than those of the 3 dual-polymerizing resin cements, notable differences were seen among the 5 materials at all depths and at both times after irradiation.

Conservative Treatment Using Platelet-Rich Plasma for Acute Anterior Cruciate Ligament Injuries in Highly Active Patients: A Retrospective Survey.

Background The recommended treatment for anterior cruciate ligament (ACL) injuries in active, sports-oriented patients is reconstructive surgery in order to regain stability and prevent secondary meniscal injuries. However, ACL reconstruction requires a long recovery period and poses issues such as decreased muscle strength due to tendon harvesting and postoperative osteoarthritis (OA) progression, thereby raising significant expectations for the advancement of conservative treatments. Recent studies have shown that platelet-rich plasma (PRP) therapy, which utilizes the tissue repair-promoting property of platelets, is effective for ACL injuries. Methods We administered PRP therapy within six weeks after ACL injury in patients who expressed the desire for an early return to sports through conservative care. After the treatment, patients wore a simple brace that limited deep flexion but placed no restrictions on weight bearing. Four months was the standard goal established for returning to pre-injury condition, and, depending on the target level, timing, and knee condition, we adjusted the additional PRP treatments and rehabilitation approach, gradually authorizing the patients' return to sport. We assessed the ligament repair status by magnetic resonance imaging (MRI) just before the full return to sports. A retrospective survey was conducted to evaluate the status of ligament repair and the condition of return-to-sport in patients with ACL injuries who underwent conservative treatment using PRP. Results The average patient age was 32.7 years and the average treatment was 2.8 PRP sessions. MRI evaluations confirmed that ligament continuity was regained in all cases. All the patients returned to their pre-injury level (Tegner Activity Scale 7.0) in an average of 139.5 days, but there was one instance of re-rupture following the return to sports. Conclusion All patients with ACL injury who underwent PRP therapy regained ligament continuity and returned to sport successfully with only one case of re-rupture.

Circulating tumor-associated antigen-specific IFNγ+4-1BB+ CD8+ T cells as peripheral biomarkers of treatment outcomes in patients with pancreatic cancer.

CD8+ T cells affect the outcomes of pancreatic ductal adenocarcinoma (PDAC). Using tissue samples at pre-treatment to monitor the immune response is challenging, while blood samples are beneficial in overcoming this limitation. In this study, we measured peripheral antigen-specific CD8+ T cell responses against four different tumor-associated antigens (TAAs) in PDAC using flow cytometry and investigated their relationships with clinical features. We analyzed the optimal timing within the treatment course for effective immune checkpoint inhibition in vitro. We demonstrated that the frequency of TAA-specific IFNγ+4-1BB+ CD8+ T cells was correlated with a fold reduction in CA19-9 before and after neoadjuvant therapy. Moreover, patients with TAA-specific IFNγ+4-1BB+ CD8+ T cells after surgery exhibited a significantly improved disease-free survival. Anti-PD-1 treatment in vitro increased the frequency of TAA-specific IFNγ+4-1BB+ CD8+ T cells before neoadjuvant therapy in patients, suggesting the importance of the timing of anti-PD-1 inhibition during the treatment regimen. Our results indicate that peripheral immunophenotyping, combined with highly sensitive identification of TAA-specific responses in vitro as well as detailed CD8+ T cell subset profiling via ex vivo analysis, may serve as peripheral biomarkers to predict treatment outcomes and therapeutic efficacy of immunotherapy plus neoadjuvant chemotherapy.

A single dose of lipopolysaccharide into mice with emphysema mimics human chronic obstructive pulmonary disease exacerbation as assessed by micro-computed tomography.

Chronic obstructive pulmonary disease (COPD), manifested as emphysema and chronic airway obstruction, can be exacerbated by bacterial and viral infections. Although the frequency of exacerbations increases as the disease progresses, the mechanisms underlying this phenomenon are largely unknown, and there is a need for a simple in vivo exacerbation model. In this study, we compared four groups of mice treated with PBS alone, elastase alone, LPS alone, and elastase plus LPS. A single intratracheal administration of LPS to mice with elastase-induced emphysema provoked infiltration of inflammatory cells, especially CD8(+) T cells, into alveolar spaces and increased matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, and perforin production in bronchoalveolar lavage fluid at the acute inflammatory phase compared with the other groups. We also measured the percentage of low-attenuation area (LAA%) in the above mice using micro-computed X-ray tomography. The LAA% was the most sensitive parameter for quantitative assessments of emphysema among all the parameters evaluated. Using the parameter of LAA%, we found significantly more severe alveolar destruction in the group treated with elastase plus LPS compared with the other groups during long-term longitudinal observations. We built three-dimensional images of the emphysema and confirmed that the lungs of elastase plus LPS-treated mice contained larger emphysematous areas than mice treated with elastase alone. Although human exacerbation of COPD is clinically and pathologically complicated, this simple mouse model mimics human cases to some extent and will be useful for elucidating its mechanism and developing therapeutic strategies.

Sensitivity of heterozygous α1,6-fucosyltransferase knock-out mice to cigarette smoke-induced emphysema: implication of aberrant transforming growth factor-ß signaling and matrix metalloproteinase gene expression.

We previously demonstrated that a deficiency in core fucosylation caused by the genetic disruption of α1,6-fucosyltransferase (Fut8) leads to lethal abnormalities and the development of emphysematous lesions in the lung by attenuation of TGF-ß1 receptor signaling. Herein, we investigated the physiological relevance of core fucosylation in the pathogenesis of emphysema using viable heterozygous knock-out mice (Fut8(+/-)) that were exposed to cigarette smoke (CS). The Fut8(+/-) mice exhibited a marked decrease in FUT8 activity, and matrix metalloproteinase (MMP)-9 activities were elevated in the lung at an early stage of exposure. Emphysema developed after a 3-month CS exposure, accompanied by the recruitment of large numbers of macrophages to the lung. CS exposure substantially and persistently elevated the expression level of Smad7, resulting in a significant reduction of Smad2 phosphorylation (which controls MMP-9 expression) in Fut8(+/-) mice and Fut8-deficient embryonic fibroblast cells. These in vivo and in vitro studies show that impaired core fucosylation enhances the susceptibility to CS and constitutes at least part of the disease process of emphysema, in which TGF-ß-Smad signaling is impaired and the MMP-mediated destruction of lung parenchyma is up-regulated.

Flagellin/Toll-like receptor 5 response was specifically attenuated by keratan sulfate disaccharide via decreased EGFR phosphorylation in normal human bronchial epithelial cells.

Bacterial or viral infection of the airway plays a critical role in the pathogenesis and exacerbation of chronic obstructive pulmonary disease (COPD) which is expected to be the 3rd leading cause of death by 2020. The induction of inflammatory responses in immune cells as well as airway epithelial cells is observed in the disease process. There is thus a pressing need for the development of new therapeutics. Keratan sulfate (KS) is the major glycosaminoglycans (GAGs) of airway secretions, and is synthesized by epithelial cells on the airway surface. Here we report that a KS disaccharide, [SO3(-)-6]Galß1-4[SO3(-)-6]GlcNAc, designated as L4, suppressed the production of Interleukin-8 (IL-8) stimulated by flagellin, a Toll-like receptor (TLR) 5 agonist, in normal human bronchial epithelial (NHBE) cells. Such suppressions were not observed by other L4 analogues, N-acetyllactosamine or chondroitin-6-sulfate disaccharide. Moreover, treatment of NHBE cells with L4 inhibited the flagellin-stimulated phosphorylation of epidermal growth factor receptor (EGFR), the down stream signaling pathway of TLRs in NHBE cells. These results suggest that L4 specifically blocks the interaction of flagellin with TLR5 and subsequently suppresses IL-8 production in NHBE cells. Taken together, L4 represents a potential molecule for prevention and treatment of airway inflammatory responses to bacteria infections, which play a critical role in exacerbation of COPD.

Integration of the mouse sperm fertilization-related protein equatorin into the acrosome during spermatogenesis as revealed by super-resolution and immunoelectron microscopy.

Spermatids must precisely integrate specific molecules into structurally supported domains that develop during spermatogenesis. Once established, the architecture of the acrosome contributes to the acrosome reaction, which occurs prior to gamete interaction in mammals. The present study aims to clarify the morphology associated with the integration of the mouse fertilization-related acrosomal protein equatorin (mEQT) into the developing acrosome. EQT mRNA was first detected by in situ hybridization in round spermatids but disappeared in early elongating spermatids. The molecular size of mEQT was approximately 65 kDa in the testis. Developmentally, EQT protein was first detected on the nascent acrosomal membrane in round spermatids at approximately step 3, was actively integrated into the acrosomal membranes of round spermatids in the following step and then participated in acrosome remodeling in elongating spermatids. This process was clearly visualized by high-resolution fluorescence microscopy and super-resolution stimulated emission depletion nanoscopy by using newly generated C-terminally green-fluorescent-protein-tagged mEQT transgenic mice. Immunogold electron microscopy revealed that mEQT was anchored to the acrosomal membrane, with the epitope region observed as lying 5-70 nm away from the membrane and was associated with the electron-dense acrosomal matrix. This new information about the process of mEQT integration into the acrosome during spermatogenesis should provide a better understanding of the mechanisms underlying not only acrosome biogenesis but also fertilization and male infertility.