SCD1 inhibition enhances the effector functions of CD8+ T cells via ACAT1-dependent reduction of esterified cholesterol.

We previously reported that the inhibition of stearoyl-CoA desaturase 1 (SCD1) enhances the antitumor function of CD8+ T cells indirectly via restoring production of DC recruiting chemokines by cancer cells and subsequent induction of antitumor CD8+ T cells. In this study, we investigated the molecular mechanism of direct enhancing effects of SCD1 inhibitors on CD8+ T cells. In vitro treatment of CD8+ T cells with SCD1 inhibitors enhanced IFN-γ production and cytotoxic activity of T cells along with decreased oleic acid and esterified cholesterol, which is generated by cholesterol esterase, acetyl-CoA acetyltransferase 1 (ACAT1), in CD8+ T cells. The addition of oleic acid or cholesteryl oleate reversed the enhanced functions of CD8+ T cells treated with SCD1 inhibitors. Systemic administration of SCD1 inhibitor to MCA205 tumor-bearing mice enhanced IFN-γ production of tumor-infiltrating CD8+ T cells, in which oleic acid and esterified cholesterol, but not cholesterol, were decreased. These results indicated that SCD1 suppressed effector functions of CD8+ T cells through the increased esterified cholesterol in an ACAT1-dependent manner, and SCD1 inhibition enhanced T cell activity directly through decreased esterified cholesterol. Finally, SCD1 inhibitors or ACAT1 inhibitors synergistically enhanced the antitumor effects of anti-PD-1 antibody therapy or CAR-T cell therapy in mouse tumor models. Therefore, the SCD1-ACAT1 axis is regulating effector functions of CD8+ T cells, and SCD1 inhibitors, and ACAT1 inhibitors are attractive drugs for cancer immunotherapy.

Macrophage depletion using clodronate liposomes reveals latent dysfunction of the hematopoietic microenvironment associated with persistently imbalanced M1/M2 macrophage polarization in a mouse model of hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH), a hyperinflammatory syndrome, is caused by the incessant activation of lymphocytes and macrophages, resulting in damage to organs, including hematopoietic organs. Recently, we demonstrated that repeated lipopolysaccharide (LPS) treatment induces HLH-like features in senescence-accelerated (SAMP1/TA-1) mice but not in senescence-resistant control (SAMR1) mice. Hematopoietic failure in LPS-treated SAMP1/TA-1 mice was attributed to hematopoietic microenvironment dysfunction, concomitant with severely imbalanced M1 and M2 macrophage polarization. Macrophages are a major component of the bone marrow (BM) hematopoietic microenvironment. Clodronate liposomes are useful tools for in vivo macrophage depletion. In this study, we depleted macrophages using clodronate liposomes to determine their role in the hematopoietic microenvironment in SAMP1/TA-1 and SAMR1 mice. Under clodronate liposome treatment, the response between SAMR1 and SAMP1/TA-1 mice differed as follows: (1) increase in the number of activated M1 and M2 macrophages derived from newly generated macrophages and M2-dominant and imbalanced M1 and M2 macrophage polarization in the BM and spleen; (2) severe anemia and thrombocytopenia; (3) high mortality rate; (4) decrease in erythroid progenitors and B cell progenitors in the BM; and (5) decrease in the mRNA expression of erythroid-positive regulators such as erythropoietin and increase in that of erythroid- and B lymphoid-negative regulators such as interferon-γ in the BM. Depletion of residual macrophages in SAMP1/TA-1 mice impaired hematopoietic homeostasis, particularly erythropoiesis and B lymphopoiesis, owing to functional impairment of the hematopoietic microenvironment accompanied by persistently imbalanced M1/M2 polarization. Thus, macrophages play a vital role in regulating the hematopoietic microenvironment to maintain homeostasis.

Anorectal Transplantation: The First Long-term Success in a Canine Model.

OBJECTIVE: Anorectal transplantation is a challenging procedure but a promising option for patients with weakened or completely absent anorectal function. SUMMARY BACKGROUND DATA: We constructed a canine model of anorectal transplantation, evaluated the long-term outcomes, and controlled rejection and infection in allotransplantation. METHODS: In the pudendal nerve function study, 6 dogs were randomly divided into 2 groups, transection and anastomosis, and were compared with a control using anorectal manometry, electromyography, and histological examination. In the anorectal transplantation model, 4 dogs were assigned to 4 groups: autotransplant, allotransplant with immunosuppression, allotransplant without immunosuppression, and normal control. Long-term function was evaluated by defecography, videography, and histological examination. RESULTS: In the pudendal nerve function study, anorectal manometry indicated that the anastomosis group recovered partial function 6 months postoperatively. Microscopically, the pudendal nerve and the sphincter muscle regenerated in the anastomosis group. Anorectal transplantation was technically successful with a 3-stage operation: colostomy preparation, anorectal transplantation, and stoma closure. The dog who underwent allotransplantation and immunosuppression had 2 episodes of mild rejection, which were reversed with methylprednisolone and tacrolimus. The dog who underwent allotransplantation without immunosuppression had a severe acute rejection that resulted in graft necrosis. Successful dogs had full defecation control at the end of the study. CONCLUSIONS: We describe the critical role of the pudendal nerve in anorectal function and the first long-term success with anorectal transplantation in a canine model. This report is a proof-of-concept study for anorectal transplantation as a treatment for patients with an ostomy because of anorectal dysfunction.

GIT1 is an untolerized autoantigen involved in immunologic disturbance of spermatogenesis.

Male haploid cells, spermatids and spermatozoa, that appear after the establishment of immune tolerance express novel cell surface and intracellular proteins that can be recognized as foreign antigens by the self-immune system. However, these germ cells do not normally evoke a pathological immune response. The immune-privileged micro-circumstance in testis involving the blood-testis-barrier formed by Sertoli cells protects these germ cells from autoimmune attack. We recently found that immunization with heat shock protein family A member 4-like (HSPA4L), one of the new differentiation antigens of haploid cells, induced experimental autoimmune orchitis (EAO) in A/J male mice. In this study, we focused on G protein-coupled receptor kinase interacting protein-1 (GIT1), another haploid cell-specific differentiation antigen, to investigate whether GIT1 is a target autoantigen for EAO induction. GIT1 emulsified with complete Freund's adjuvant was injected subcutaneously into the mice inguinal region once on day 0 and again on day 14, and the optimum condition of EAO induction was determined. Mice immunized with 200 µg GIT1 showed significantly higher incidence of EAO than that of immunization with other concentrations. In particular, significant lymphocytic inflammation and extensive aspermatogenesis were observed in these mice at 120 days after the first immunization. These findings indicate that GIT1 is also a target antigen that induces EAO, like HSPA4L.

Imbalanced M1 and M2 Macrophage Polarization in Bone Marrow Provokes Impairment of the Hematopoietic Microenvironment in a Mouse Model of Hemophagocytic Lymphohistiocytosis.

Lipopolysaccharide (LPS) treatment induced hemophagocytic lymphohistiocytosis in senescence-accelerated mice (SAMP1/TA-1), but not in senescence-resistant control mice (SAMR1). SAMP1/TA-1 treated with LPS exhibited functional impairment of the hematopoietic microenvironment, which disrupted the dynamics of hematopoiesis. Macrophages are a major component of the bone marrow (BM) hematopoietic microenvironment, which regulates hematopoiesis. Qualitative and quantitative changes in activated macrophages in LPS-treated SAMP1/TA-1 are thought to contribute to the functional deterioration of the hematopoietic microenvironment. Thus, we examined the polarization of pro-inflammatory (M1) and anti-inflammatory (M2) macrophages, and the dynamics of macrophage production in the BM of SAMP1/TA-1 and SAMR1 after LPS treatment. After LPS treatment, the proportions of M1 and M2 macrophages and the numbers of macrophage progenitor (CFU-M) cells increased in both SAMP1/TA-1 and SAMR1. However, compared to the SAMR1, the increase in the M1 macrophage proportion was prolonged, and the increase in the M2 macrophage proportion was delayed. The increase in the number of CFU-M cells was prolonged in SAMP1/TA-1 after LPS treatment. In addition, the levels of transcripts encoding an M1 macrophage-inducing cytokine (interferon-γ) and macrophage colony-stimulating factor were markedly increased, and the increases in the levels of transcripts encoding M2 macrophage-inducing cytokines (interleukin (IL)-4, IL-10, and IL-13) were delayed in SAMP1/TA-1 when compared to SAMR1. Our results suggest that LPS treatment led to the severely imbalanced polarization of activated M1/M2 macrophages accompanied by a prolonged increase in macrophage production in the BM of SAMP1/TA-1, which led to the impairment of the hematopoietic microenvironment, and disrupted the dynamics of hematopoiesis.

Improvement in Outcomes After Cardiac Arrest and Resuscitation by Inhibition of S-Nitrosoglutathione Reductase.

BACKGROUND: The biological effects of nitric oxide are mediated via protein S-nitrosylation. Levels of S-nitrosylated protein are controlled in part by the denitrosylase, S-nitrosoglutathione reductase (GSNOR). The objective of this study was to examine whether GSNOR inhibition improves outcomes after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). METHODS: Adult wild-type C57BL/6 and GSNOR-deleted (GSNOR-/-) mice were subjected to potassium chloride-induced CA and subsequently resuscitated. Fifteen minutes after a return of spontaneous circulation, wild-type mice were randomized to receive the GSNOR inhibitor, SPL-334.1, or normal saline as placebo. Mortality, neurological outcome, GSNOR activity, and levels of S-nitrosylated proteins were evaluated. Plasma GSNOR activity was measured in plasma samples obtained from post-CA patients, preoperative cardiac surgery patients, and healthy volunteers. RESULTS: GSNOR activity was increased in plasma and multiple organs of mice, including brain in particular. Levels of protein S-nitrosylation were decreased in the brain 6 hours after CA/CPR. Administration of SPL-334.1 attenuated the increase in GSNOR activity in brain, heart, liver, spleen, and plasma, and restored S-nitrosylated protein levels in the brain. Inhibition of GSNOR attenuated ischemic brain injury and improved survival in wild-type mice after CA/CPR (81.8% in SPL-334.1 versus 36.4% in placebo; log rank P=0.031). Similarly, GSNOR deletion prevented the reduction in the number of S-nitrosylated proteins in the brain, mitigated brain injury, and improved neurological recovery and survival after CA/CPR. Both GSNOR inhibition and deletion attenuated CA/CPR-induced disruption of blood brain barrier. Post-CA patients had higher plasma GSNOR activity than did preoperative cardiac surgery patients or healthy volunteers ( P<0.0001). Plasma GSNOR activity was positively correlated with initial lactate levels in postarrest patients (Spearman correlation coefficient=0.48; P=0.045). CONCLUSIONS: CA and CPR activated GSNOR and reduced the number of S-nitrosylated proteins in the brain. Pharmacological inhibition or genetic deletion of GSNOR prevented ischemic brain injury and improved survival rates by restoring S-nitrosylated protein levels in the brain after CA/CPR in mice. Our observations suggest that GSNOR is a novel biomarker of postarrest brain injury as well as a molecular target to improve outcomes after CA.

Cardioprotection via Metabolism for Rat Heart Preservation Using the High-Pressure Gaseous Mixture of Carbon Monoxide and Oxygen.

The high-pressure gas (HPG) method with carbon monoxide (CO) and oxygen (O2) mixture maintains the preserved rat heart function. The metabolites of rat hearts preserved using the HPG method (HPG group) and cold storage (CS) method (CS group) by immersion in a stock solution for 24 h were assessed to confirm CO and O2 effects. Lactic acid was significantly lower and citric acid was significantly higher in the HPG group than in the CS group. Moreover, adenosine triphosphate (ATP) levels as well as some pentose phosphate pathway (PPP) metabolites and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were significantly higher in the HPG group than in the CS group. Additionally, reduced glutathione (GSH), which protects cells from oxidative stress, was also significantly higher in the HPG group than in the CS group. These results indicated that each gas, CO and O2, induced the shift from anaerobic to aerobic metabolism, maintaining the energy of ischemic preserved organs, shifting the glucose utilization from glycolysis toward PPP, and reducing oxidative stress. Both CO and O2 in the HPG method have important effects on the ATP supply and decrease oxidative stress for preventing ischemic injury. The HPG method may be useful for clinical application.

The effectiveness of traditional Japanese medicine Goshajinkigan in irradiation-induced aspermatogenesis in mice.

BACKGROUND: Infertility and gonadal dysfunction are well known side-effects by cancer treatment in males. In particularly, chemotherapy and radiotherapy induced testicular damage, resulting in prolonged azoospermia. However, information regarding therapeutics to treat spermatogenesis disturbance after cancer treatment is scarce. Recently, we demonstrated that Goshajinkigan, a traditional Japanese medicine, can completely rescue severe busulfan-induced aspermatogenesis in mice. In this study, we aimed to detect the effects of Goshajinkigan on aspermatogenesis after irradiation. METHODS: This is animal research about the effects of traditional Japanese medicine on infertility after cancer treatment. C57BL/6 J male mice received total body irradiation (TBI: a single dose of 6Gy) at 4 weeks of age and after 60 days were reared a Goshajinkigan (TJ107)-containing or TJ107-free control diet from day 60 to day 120. Then, two untreated females were mated with a single male from each experimental group. On day 60, 120 and 150, respectively, the sets of testes and epididymis of the mice in each group after deep anesthetization were removed for histological and cytological examinations. RESULTS: Histological and histopathological data showed that 6Gy TBI treatment decreased the fertility rate (4/10) in the control diet group; in contrast, in the TJ107-diet group, the fertility rate was 10/10 (p < 0.05 vs. 6Gy group). Supplementation with TJ107 was found to rescue the disrupted inter-Sertoli tight junctions via the normalization of claudin11, occludin, and ZO-1 expression and reduce serum anti-germ cell autoantibodies. CONCLUSIONS: These findings show the therapeutic effect on TBI-induced aspermatogenesis and the recovering disrupted gonadal functions by supplementation with TJ107.

Transverse ligament of the elbow joint: an anatomic study of cadavers.

BACKGROUND: The medial collateral ligament of the elbow joint consists of the anterior oblique ligament (AOL), posterior oblique ligament (POL), and transverse ligament (TL). This study aimed to clarify the structure of the TL, with a focus on the continuity between the TL and AOL. METHODS: A total of 42 cadavers (18 males, 24 females) were dissected at Aichi Medical University between 2016 and 2018. Cases of elbow deformity or atrophy were excluded, and 60 elbows (15 males, 15 females) were dissected to assess the fibers of both the TL and AOL using a stereomicroscope. RESULTS: The TL could be detected in all elbows and always continued to the AOL. The TL was classified into 2 types. The TLs continuing to the distal half of the AOL (type I) were observed in 44 elbows (73.3%), whereas the TLs continuing to the entire AOL (type II) were found in 16 elbows (26.7%). Type II TLs were significantly more frequently observed in the elbows of females than in those of males (P = .041). Stereomicroscopic observation revealed that the TL fibers entered perpendicularly to the distal half of the AOL in both types. CONCLUSIONS: The TL frequently continues to the distal half of the AOL, but rarely continues to the entire AOL. The TLs continuing to the entire AOL were more frequently detected in the elbows of females than in those of males. The TL possibly contributes to medial elbow stability via its continuity to the AOL.

High-Pressure Carbon Monoxide and Oxygen Mixture is Effective for Lung Preservation.

(1) Background: Heme oxygenase-1 (HO-1) degrades heme and generates carbon monoxide (CO), producing various anti-inflammatory, anti-oxidative, and anti-apoptotic effects. This study aimed to confirm the effects of CO on the ischemia-reperfusion injury (IRI) of donor lungs using a high-pressure gas (HPG) preservation method. (2) Methods: Donor rat and canine lungs were preserved in a chamber filled with CO (1.5 atm) and oxygen (O2; 2 atm) and were ventilated with either CO and O2 mixture (CO/O2 group) or air (air group) immediately before storage. Rat lungs were subjected to heterotopic cervical transplantation and evaluated after reperfusion, whereas canine lungs were subjected to allogeneic transplantation and evaluated. (3) Results: Alveolar hemorrhage in the CO/O2 group was significantly milder than that in the air group. mRNA expression levels of HO-1 remained unchanged in both the groups; however, inflammatory mediator levels were significantly lower in the CO/O2 group than in the air group. The oxygenation of graft lungs was comparable between the two groups, but lactic acid level tended to be higher in the air group. (4) Conclusions: The HO-1/CO system in the HPG preservation method is effective in suppressing IRI and preserving donor lungs.

S-Nitrosylation of Calcium-Handling Proteins in Cardiac Adrenergic Signaling and Hypertrophy.

RATIONALE: The regulation of calcium (Ca(2+)) homeostasis by ß-adrenergic receptor (ßAR) activation provides the essential underpinnings of sympathetic regulation of myocardial function, as well as a basis for understanding molecular events that result in hypertrophic signaling and heart failure. Sympathetic stimulation of the ßAR not only induces protein phosphorylation but also activates nitric oxide-dependent signaling, which modulates cardiac contractility. Nonetheless, the role of nitric oxide in ßAR-dependent regulation of Ca(2+) handling has not yet been explicated fully. OBJECTIVE: To elucidate the role of protein S-nitrosylation, a major transducer of nitric oxide bioactivity, on ßAR-dependent alterations in cardiomyocyte Ca(2+) handling and hypertrophy. METHODS AND RESULTS: Using transgenic mice to titrate the levels of protein S-nitrosylation, we uncovered major roles for protein S-nitrosylation, in general, and for phospholamban and cardiac troponin C S-nitrosylation, in particular, in ßAR-dependent regulation of Ca(2+) homeostasis. Notably, S-nitrosylation of phospholamban consequent upon ßAR stimulation is necessary for the inhibitory pentamerization of phospholamban, which activates sarcoplasmic reticulum Ca(2+)-ATPase and increases cytosolic Ca(2+) transients. Coincident S-nitrosylation of cardiac troponin C decreases myocardial sensitivity to Ca(2+). During chronic adrenergic stimulation, global reductions in cellular S-nitrosylation mitigate hypertrophic signaling resulting from Ca(2+) overload. CONCLUSIONS: S-Nitrosylation operates in concert with phosphorylation to regulate many cardiac Ca(2+)-handling proteins, including phospholamban and cardiac troponin C, thereby playing an essential and previously unrecognized role in cardiac Ca(2+) homeostasis. Manipulation of the S-nitrosylation level may prove therapeutic in heart failure.

Morphological study of the neurovascular bundle to elucidate nerve damage in pelvic surgery.

PURPOSE: Postoperative sexual and urinary dysfunction may occur after rectal cancer surgery involving the pelvis, but this problem cannot be solved. The aim of this study was to examine the nerve morphology of the neurovascular bundle in cadavers to determine possible causes of nerve damage during surgery. METHODS: Twenty-two formalin-fixed cadavers were used in the study. The cadavers were donated to the Tokyo Medical University. The study comprised histological evaluation of paraffin-embedded bilateral neurovascular bundle specimens from the cadavers. Four slides of 3-cm thick were made every 1 cm in a plane perpendicular to the rectum towards the pelvic floor from the peritoneal reflection in bilateral neurovascular bundles in 22 cadavers. The number of nerves, the mean nerve area, and the mean nerve diameter were measured in each slide. RESULTS: The results were categorized into cases with high (group H) and low (group L) positions of the pelvis 1 cm above and 2 cm below the peritoneal reflection, respectively. There was no significant difference in the number of nerves between these groups. The nerve area and nerve diameter were significantly smaller in group L, and these characteristics were more marked in males. CONCLUSIONS: Our results show that the nerves of the neurovascular bundle became smaller in the deep pelvis. This may cause these nerves to be more susceptible to injury, resulting in nerve damage in the deep pelvis that leads to postoperative dysfunction. Particularly, this type of nerve damage may be a cause of postoperative sexual dysfunction in males.

Mitochondria-targeted hydrogen sulfide donor AP39 improves neurological outcomes after cardiac arrest in mice.

AIMS: Mitochondria-targeted hydrogen sulfide donor AP39, [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl)phenoxy)decyl) triphenylphosphonium bromide], exhibits cytoprotective effects against oxidative stress in vitro. We examined whether or not AP39 improves the neurological function and long term survival in mice subjected to cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). METHODS: Adult C57BL/6 male mice were subjected to 8 min of CA and subsequent CPR. We examined the effects of AP39 (10, 100, 1000 nmol kg(-1)) or vehicle administered intravenously at 2 min before CPR (Experiment 1). Systemic oxidative stress levels, mitochondrial permeability transition, and histological brain injury were assessed. We also examined the effects of AP39 (10, 1000 nmol kg(-1)) or vehicle administered intravenously at 1 min after return of spontaneous circulation (ROSC) (Experiment 2). ROSC was defined as the return of sinus rhythm with a mean arterial pressure >40 mm Hg lasting at least 10 seconds. RESULTS: Vehicle treated mice subjected to CA/CPR had poor neurological function and 10-day survival rate (Experiment 1; 15%, Experiment 2; 23%). Administration of AP39 (100 and 1000 nmol kg(-1)) 2 min before CPR significantly improved the neurological function and 10-day survival rate (54% and 62%, respectively) after CA/CPR. Administration of AP39 before CPR attenuated mitochondrial permeability transition pore opening, reactive oxygen species generation, and neuronal degeneration after CA/CPR. Administration of AP39 1 min after ROSC at 10 nmol kg(-1), but not at 1000 nmol kg(-1), significantly improved the neurological function and 10-day survival rate (69%) after CA/CPR. CONCLUSION: The current results suggest that administration of mitochondria-targeted sulfide donor AP39 at the time of CPR or after ROSC improves the neurological function and long term survival rates after CA/CPR by maintaining mitochondrial integrity and reducing oxidative stress.

Neonatal estrogen treatment with ß-estradiol 17-cypionate induces in post-pubertal mice inflammation in the ductuli efferentes, epididymis, and vas deferens, but not in the testis, provoking obstructive azoospermia.

Neonatal estrogen treatment (NET) induces morphological changes in male reproductive organs. NET with ß-estradiol 17-cypionate is reported to induce inflammation with stromal-epithelial abnormalities in the prostate and seminal vesicles in post-pubertal mice. The aim of this study was to investigate the histopathology of the testis, ductuli efferentes, epididymis, and vas deferens in mice after NET with ß-estradiol 17-cypionate. No morphological changes in these organs were observed until 4 weeks after NET. However, some inflammatory cells were found in epididymis and vas deferens 6 weeks after NET. Eight weeks after NET, inflammatory cells spread to the ductuli efferentes and inflammation was severe from 6 to 12 weeks after NET. Inflammatory cells were never seen in the whole testis, but cystic dilatation of the rete testes with spermatogenic disturbance was found around the mediastinum testis. Many inflammatory cells emigrated into the lumen of the epididymis, resulting in complete absence of spermatozoa in the vas deferens. Most of the inflammatory cells penetrating into the epithelial layers of epididymal ducts were neutrophils. These results indicate that in post-pubertal mice, NET with ß-estradiol 17-cypionate induces inflammation in the ductuli efferentes, epididymis, and vas deferens, but not in the testis, provoking obstructive azoospermia.

Evaluation of the Angle Between the Long Axis of the Femoral Anterior Cruciate Ligament Footprint and Bony Morphology of the Knee: A Cadaveric Descriptive Study.

Purpose: There have been numerous studies of the anterior cruciate ligament (ACL) anatomy, but few have focused on the long axis angle of the femoral ACL footprint. This study investigated the angle between the long axis of the femoral ACL footprint and the bony morphology of the knee. Methods: This study is a cadaveric descriptive study. Thirty non-paired formalin-fixed knees of Japanese cadavers were used. Anteromedial (AM) and posterolateral (PL) bundles were identified according to the tension pattern differences during the complete range of motion of the knee. In the ACL femoral footprint, there is a fold between the mid-substance insertion site and fan-like extension fibers. After identifying AM and PL bundles of mid-substance fibers, the mid-substance and fan-like extension fibers were divided into those bundles and stained. We defined the line passing through the center of the AM and PL bundles as the long axis of the ACL. The center points of each of the four areas and the angle between the long axis of the ACL and the bony morphology of the knee were calculated using Image J software. Results: The mean angle between the axis of the femoral shaft and the long axis of the ACL mid-substance insertion was 28.8 ± 12.2 degrees. The mean angle between the Blumensaat line and the long axis of the mid-substance was 54.2 ± 13.5 degrees. Conclusion: The mean angle between the axis of the femoral shaft and the long axis of the femoral ACL footprint was approximately 29 degrees. There is a wide variation in the long axis of the femoral ACL footprint. To achieve better clinical results through a more anatomically accurate reconstruction, it can be beneficial to replicate the ACL femoral footprint along its native long axis.

FADS2 confers SCD1 inhibition resistance to cancer cells by modulating the ER stress response.

Stearoyl-CoA desaturase 1 (SCD1) is an attractive target for cancer therapy. However, the clinical efficacy of SCD1 inhibitor monotherapy is limited. There is thus a need to elucidate the mechanisms of resistance to SCD1 inhibition and develop new therapeutic strategies for combination therapy. In this study, we investigated the molecular mechanisms by which cancer cells acquire resistance to endoplasmic reticulum (ER) stress-dependent cancer cell death induced by SCD1 inhibition. SCD1 inhibitor-sensitive and -resistant cancer cells were treated with SCD1 inhibitors in vitro, and SCD1 inhibitor-sensitive cancer cells accumulated palmitic acid and underwent ER stress response-induced cell death. Conversely, SCD1-resistant cancer cells did not undergo ER stress response-induced cell death because fatty acid desaturase 2 (FADS2) eliminated the accumulation of palmitic acid. Furthermore, genetic depletion using siRNA showed that FADS2 is a key determinant of sensitivity/resistance of cancer cells to SCD1 inhibitor. A549 cells, an SCD1 inhibitor-resistant cancer cell line, underwent ER stress-dependent cancer cell death upon dual inhibition of SCD1 and FADS2. Thus, combination therapy with SCD1 inhibition and FADS2 inhibition is potentially a new cancer therapeutic strategy targeting fatty acid metabolism.

Conditionally replicative adenoviral vectors for imaging the effect of chemotherapy on pancreatic cancer cells.

Pancreatic cancer has a poor prognosis after complete macroscopic resection combined with chemotherapy. Even after neoadjuvant chemotherapy, R0 resection is often not possible. Moreover, current imaging techniques cannot reliably distinguish viable cancer cells from scar tissue at the resectional margin. We investigated the use of a conditionally replicative adenovirus (CRAd), Ad5/3Cox2CRAd-ΔE3ADP-Luc, for imaging the effects of chemotherapy. The CRAd infectivity of pancreatic cancer cells was enhanced by a chimeric Ad5/3 fiber, E1A expression was under the control of the Cox2 promoter, and the luciferase gene was inserted adjacent to the adenovirus death protein (ADP) gene. Subcutaneous xenografts of the pancreatic cancer cell line MiaPaCa-2 were established in 24 BALB/c nu/nu mice. When xenografts reached a diameter of 4-6 mm (day 1), the mice were injected i.p. with either PBS (group A; n = 12) or 1000 mg/kg gemcitabine (group B; n = 12), weekly. On days 19, 26, 33, and 40, CRAd were injected intratumorally into three mice in groups A and B. Bioluminescence was imaged 72 h after CRAd injection, and gross tumor volumes were measured then tumors were removed for ex vivo histopathology using H&E and Ki-67 staining. Correlations between gross tumor volume, pathological evaluation of the percentage of viable tumor area, and CRAd bioluminescence were analyzed. Bioluminescence correlated closely with the percentage of viable tumor area (R = 0.96), but not with gross tumor volume (R = 0.31). Therefore, CRAds might be reliable imaging tools for monitoring chemotherapy in pancreatic cancer, and could improve our ability to distinguish viable tumor cells from scar tissue.

The effects of adjuvants on autoimmune responses against testicular antigens in mice.

Experimental autoimmune orchitis (EAO) is a model of immunologic male infertility and pathologically characterized by lymphocytic inflammation, which causes breakdown of the testicular immune privilege with spermatogenic disturbance. Generally, murine EAO is induced by immunization with testicular homogenate (TH) from the testes of donor mice + complete Freund's adjuvant (CFA) + Bordetella pertussigens (BP), and it has been considered that treatment with these two adjuvants is required to enhance the immune response against testicular antigens. However, there remains a possibility that CFA and BP may affect autoimmune responses against the testicular antigens without TH. In the present study, we examined this possibility using real-time RT-PCR, Western blotting and immunohistochemical staining. The results demonstrated that immunization with TH in combination with CFA and BP evoked more severe EAO than that with only TH. Real-time RT-PCR analyses revealed that Fas mRNA expression in TH+CFA+BP-induced EAO was significantly higher than that in TH-induced EAO. Interestingly, IL-6 mRNA expression dramatically increased in TH+CFA+BP-induced EAO; however, no apparent change in IL-6 mRNA expression occurred in TH-induced EAO. It was also noted that treatment with CFA and BP alone augmented autoimmune reactions against some testicular autoantigens. These results indicates that these adjuvants are helpful in evoking severe EAO, and treatment with the adjuvants alone can evoke autoimmune reactions against some testicular autoantigens despite the use of no TH.

Effects on the local immunity in the testis by exposure to di-(2-ethylhexyl) phthalate (DEHP) in mice.

Exposure to di-(2-ethylhexyl) phthalate (DEHP) has been reported to induce spermatogenic disturbance through oxidant stress and affect the immune system as an adjuvant. However, the effect of DEHP on the testicular immune microenvironment has not yet been investigated. In the present study, we examined the testicular immune microenvironment after exposure to doses of DEHP, previously identified as no-observed-adverse-effect levels. Adult male mice were administered food containing 0%, 0.01% or 0.1% DEHP and then testes were analyzed. The results showed that a slight but significant spermatogenic disturbance appeared in the 0.1% DEHP group but not in the 0.01% DEHP group at 8 weeks. It was also demonstrated that lymphocytes and F4/80- and MHC class II- positive cells were significantly increased with the elevation of IL-10 and IFN-γ mRNA expressions in the testes of not only the 0.1% DEHP group but also the 0.01% DEHP group at 8 weeks. Histochemical analyses involving horseradish peroxidase (HRP) as a tracer showed that a little blood-borne HRP had infiltrated into the lumen of a few seminiferous tubules beyond the blood-testis-barrier in both the 0.1% and 0.01% DEHP groups at 8 weeks. This indicates that a dose of DEHP that has little effects on spermatogenesis can change the testicular immune microenvironment with functional damage of the blood-testis barrier.

Cadmium exposure increases susceptibility to testicular autoimmunity in mice.

Cadmium, one of various environmental toxicants, is known to suppress systemic immunity and to injure the testicular capillary endothelia with resultant necrosis of testicular tissues in mice and rats treated with high doses. Recently, it also became evident that cadmium can affect the integrity of the blood-testis barrier (BTB), the endocrine function of Leydig cells, apoptosis of germ cells and systemic immunity, even on treatment with a low dose that does not induce spermatogenic disturbance. Experimental autoimmune orchitis (EAO), i.e., an organ-specific autoimmunity of the testis, can be induced by repeated immunization with testicular antigens, and its pathology is characterized by lymphocytic inflammation and spermatogenic disturbance. In the present study, we investigated the morphological and functional changes of testes in mice treated with a low dose of cadmium chloride (CdCl2 ) and also examined its toxicity as to susceptibility to EAO. The results showed that exposure to 3 mg CdCl2 kg(-1) body weight did not affect the spermatogenic state. However, the BTB at the tubuli recti and the rete testis, but not the seminiferous tubules, was slightly weakened, and intra-testicular mRNA expression of interleukin (IL)-6, tumor necrosis factor-α and IL-1ß was significantly increased by the CdCl2 treatment. Furthermore, immunization with testicular antigens after the CdCl2 exposure significantly augmented the EAO severity. Therefore, exposure to a low dose of CdCl2 induces no significant disturbance of spermatogenesis, however, it does change the immunological microcircumstances in the testis, resulting in increased susceptibility to testicular autoimmunity.