Dickkopf-1 Expression as a Marker for Predicting Clinical Outcome in Esophageal Squamous Cell Carcinoma

Background and Objectives  Dickkopf-1 (DKK1) is the inhibitor of the canonical Wnt signaling pathway, however it is highly transactivated in various cancers, suggesting the presence of unknown mechanism. Its implication in human esophageal squamous cell carcinoma (ESCC) has not been sufficiently investigated. Patients and Methods  We evaluated DKK1 protein expression in resected specimens from 170 patients with ESCC by immunohistochemistry. Tumors were categorized as positive or negative for DKK1. The relationships between DKK1 expression in ESCC and various clinicopathological parameters and prognosis (disease-free survival; DFS) were analyzed separately. Results  Immunohistochemically, 72 (42.4%) tumors were DKK1 positive while no significant staining was observed in the normal squamous epithelium except for few basal cells. There was no significant relationship between DKK1 expression in ESCC and any of the clinicopathological parameters tested in this study. Patients with DKK1-positive tumors had poorer DFS than those with negative ESCC (5-year DFS; 31.5% versus 53.6%, P = 0.0062). Univariate analysis showed a significant relationship between pT [hazard ratio (HR) = 2.944, 95% confidence interval (CI) = 1.713–5.059, P < 0.0001], number of pN (HR = 2.836, 95% CI = 1.866–4.309, P < 0.0001), lymphatic invasion (HR = 2.892, 95% CI = 1.336–6.262, P = 0.0070), and DKK1 expression (HR = 1.763, 95% CI = 1.167–2.663, P = 0.0071) and DFS. Multivariate analysis including the above four parameters identified pT (HR = 2.053, 95% CI = 1.157–3.645, P = 0.0140), pN number (HR = 2.107, 95% CI = 1.362–3.260, P = 0.0008), and DKK1 expression (HR = 1.813, 95% CI = 1.195–2.751, P = 0.0052) as independent and significant prognostic factors for DFS. Conclusion  Our data suggest the usefulness of DKK1 as a novel predictor of poor prognosis of patients with ESCC after curative resection and also as a therapeutic target for future tailored therapies against ESCC.     

Vascular Endothelial Growth Factor C Promotes Lymph Node Metastasis in a Rectal Cancer Orthotopic Model

Purpose Vascular endothelial growth factor C (VEGF-C), a novel member of the vascular endothelial growth factor family, is a relatively specific lymphangiogenic growth factor. It has been suggested that increased expression of VEGF-C in primary tumors is correlated with lymph node metastasis. We conducted this study to determine whether VEGF-C directly affects lymphangiogenesis and lymph node metastasis in colorectal cancer.Methods For an accurate analysis and clear visualization of metastases, the rectal cancer cell line, DLD1, was engineered to stably express green fluorescent protein (GFP) (DLD1/GFP). We implanted DLD1/GFP cells overexpressing VEGF-C orthotopically into the rectal walls of nude mice.Results Lymph node metastasis was confirmed in all (100%) of the mice bearing DLD1/GFP-VEGF-C tumors, but in only 25% of the mice bearing control tumors. There were more lymph node metastases per mouse in the mice bearing DLD1/GFP-VEGF-C tumors than in the mice bearing control tumors. There were no differences in cell growth and motility in vitro or in the resulting tumor volume from the implanted cells between the two groups. Immunohistochemical staining revealed that VEGF-C induced the growth of lymphatic vessels, which were enlarged in the tumor periphery and contained tumor cell emboli.Conclusion These results suggest that VEGF-C-induced lymphangiogenesis mediates tumor spread and the formation of lymph node metastasis.     

Glutamine stimulates amino acid transport during ischemia-reperfusion in human intestinal epithelial cells

BACKGROUND: The potential mechanism of intestinal ischemia-reperfusion (I/R) injury includes oxygen-derived toxic free radicals. We tested the hypothesis that glutamine increases intracellular glutathione, a protective substrate against oxidative stress, by stimulating membrane amino acid transport during I/R using human intestinal epithelial cell line Caco-2. METHODS: Ischemic conditions were obtained by combining both hypoxic (1%O2-5%CO2-94% N2) and nutrient-deprived (Phosphate-Buffered Saline; PBS) conditions. After 2 h of ischemia, re-oxygenation (5%CO2-95% air) was initiated and the culture medium was changed to PBS, PBS supplemented with amino acids (A.A.), and PBS supplemented with 2 mm glutamine plus amino acids (Gln) (reperfusion). After 4 h of reperfusion, the transport of 3H-glutamine, 3H-glutamate, and 3H-leucine was assayed and intracellular glutathione was measured. 3H-thymidine incorporation was measured for the determination of DNA synthesis. Data (mean +/- SD) were analyzed by ANOVA. RESULTS: Ischemia decreased Na+-dependent glutamine, Na+-dependent glutamate, and Na+-independent leucine transport compared with control (P < 0.01). After reperfusion, glutamine and glutamate transport in the PBS and A.A. groups decreased significantly compared with control (P < 0.01), whereas glutamine supplementation increased glutamine transport to the levels in control (P < 0.01) and partially increased glutamate transport (P < 0.01). Leucine transport significantly increased in the A.A. and Gln groups compared with the PBS group. Glutamine significantly increased intracellular glutathione and DNA synthesis compared with the PBS and A.A. groups (P < 0.01). CONCLUSIONS: This study demonstrated that glutamine up-regulates amino acid transport during I/R in human intestinal epithelial cells, possibly resulting in increased intracellular glutathione and DNA synthesis.     

Hydroxyapatite-coating of pedicle screws improves resistance against pull-out force in the osteoporotic canine lumbar spine model: a pilot study.

BACKGROUND CONTEXT: In patients with spinal osteoporosis, the early achievement and maintenance of a biological bond between the pedicle screw and bone is important to avoid screw loosening complications. There are few reports of in vivo investigations involving biomechanical and histological evaluations in the osteoporotic spine. PURPOSE: To evaluate the effect of hydroxyapatite (HA)-coating on the pedicle screw in the osteoporotic lumbar spine and to investigate the relationship between resistance against the screw pull-out force and bone mineral density (BMD) of the vertebral body. STUDY DESIGN/SETTING: Mechanical and pathological investigations in the lumbar spine. METHODS: Two 24-month-old female beagle dogs were fed a calcium-free dog chow for 6 months after ovariectomy (OVX). BMD (in g/cm2) was measured by dual energy X-ray absorptiometry at pre-OVX and 6 months after OVX. Pedicle screws were placed from L1 to L6 at 6 months after OVX. Twenty-four pure titanium cortical screws (Synthes, #401-114) were used as pedicle screws (Ti-PS). Of these, 12 screws had HA-coating (HA-PS). The HA-PS screws were inserted into the right pedicles and the Ti-PS were inserted into the left pedicles. Ten days after this procedure, the lumbar spines were removed en bloc for screw pull-out testing and histological evaluation. RESULTS: The mean BMD value of the lumbar vertebrae 6 months after the OVX was 0.549+/-0.087 g/cm2, which was significantly less than the pre-OVX mean BMD of 0.603+/-0.092 g/cm2 (p < 0.001). The mean resistance against the pull-out force for the HA-PS was significantly greater at 165.6+/-26.5N than in the Ti-PS (103.1+/-30.2N, p < .001). The histological sections in the HA-PS clearly revealed new bone bonding with the apatite coating but only fibrous tissue bonding in the Ti-PS. CONCLUSIONS: The results of this study showed that the resistance to the pull-out force of HA-PS is 1.6 times that of Ti-PS. Furthermore, HA-PS has superior biological bonding to the surrounding bone, as early as 10 days after surgery in this osteoporotic spine model. Thus, in patients with osteoporosis, coating of the pedicle screw with HA may provide better stability and bonding between the pedicle screw and bone in the early postoperative period.     

Bilateral testicular tumors recurring 31 years after the initial treatment: a case report and the literature review

We herein report a case of bilateral testicular germ cell tumor recurring 31 years after right high inguinal orchiectomy. In 2003, a 62-year-old man presented with a mass in the left testis. Ultrasonography demonstrated three hypoechoic mass and microlithiasis of the left testis. Abdominal and breast computed tomography revealed no lymph adenopathy and any metastasis. The preoperative diagnosis was stage I testicular tumor and subsequently left high orchiectomy was performed. Histological examination revealed typical seminoma. At present, the patient is free from recurrence after the surgery. To our knowledge, 166 cases were reported in Japan. Approximately fifty percent of metachronal bilateral testicular tumors previously reported have been recurred after five years and more from the initial surgery. In the testicular cancer, long-term follow-up and self examination of the scrotum are of great importance. We review the metachronal bilateral testicular germ cell tumors previously reported in Japan.     

Dual role of vascular endothelial growth factor in hepatic ischemia-reperfusion injury

BACKGROUND: Vascular endothelial growth factor (VEGF), a major angiogenic factor, mediates a variety of disease conditions through promotion of angiogenesis. It also plays a critical role as a potent proinflammatory cytokine in a variety of physiologic and pathologic immune responses. In the present study, we evaluated the expression of VEGF in hepatic warm ischemia-reperfusion (I/R) injury and examined the effect of recombinant human (rh)VEGF administration in an established murine model. METHOD: The expression of VEGF in the liver was assessed by quantitative real-time polymerase chain reaction and immunohistochemistry during I/R injury using 70% partial hepatic ischemia model. The effect of rhVEGF administration on I/R injury was evaluated by measuring liver function and histology. In addition, local inducible nitric oxide synthase (iNOS) and endothelial NO synthase expressions were examined to address the underlying mechanisms. RESULTS: The local expression of VEGF was significantly up-regulated at 2 hours after reperfusion after 60 minutes of ischemia compared with that in the naive liver. VEGF was expressed predominantly in CD11b+ cells infiltrating into the ischemic liver. The administration of rhVEGF had a significant protective effect on ischemic injury in the liver. This effect was associated with the up-regulation of iNOS expression in the rhVEGF-treated liver. CONCLUSION: We demonstrate a dual role of VEGF in hepatic warm I/R injury. Although endogenous VEGF is expressed and functional to initiate hepatic I/R injury, exogenous rhVEGF has a beneficial effect on the ischemic liver. These data may provide new insights into the role of VEGF as well as pathophysiology of hepatic I/R injury.     

Function of the vascular endothelial growth factor receptors Flt-1 and Flk-1/KDR in the alloimmune response in vivo

BACKGROUND: We have recently reported that vascular endothelial growth factor (VEGF) functions as a proinflammatory cytokine to regulate the trafficking of leukocytes into allografts in the early posttransplant period. VEGF binds two major VEGF receptors: VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR). Here, we wished to investigate the expression and function of VEGF receptors in the process of acute allograft rejection in vivo. METHODS: We performed fully MHC-mismatched C57BL/6 (H-2b) into BALB/c (H-2d) vascularized heterotopic murine cardiac transplants and we examined the expression of VEGF and VEGF receptors by immunohistochemistry during acute allograft rejection. Next, we treated mice with specific neutralizing monoclonal antibodies against murine VEGFR-1 and VEGFR-2 and examined their effect on the development of acute allograft rejection by histology and by analysis of graft survival. The intragraft expression of cytokines and chemokines were also evaluated by quantitative real-time PCR analysis. RESULTS: The expression of VEGF, VEGFR-1 and VEGFR-2 were significantly up-regulated during allograft rejection as compared to isografts. Administration of either anti-VEGFR-1 or anti-VEGFR-2 alone failed to inhibit allograft rejection. However, coadministration of both antibodies together inhibited leukocyte infiltration of allografts and prolonged allograft survival. Furthermore, the effect of VEGFR blockade was associated with the downregulation of intragraft cytokine and chemokine expression. CONCLUSIONS: Our data suggest that VEGF-VEGFR interactions function in the alloimmune response in vivo. Targeting VEGFRs may represent a novel therapy to protect allografts following clinical transplantation.     

Improvement of the sentinel lymph node detection rate of cervical sentinel lymph node biopsy using real‐time fluorescence navigation with indocyanine green in head and neck skin cancer

The standard technique using lymphoscintigraphy, blue dye and a gamma probe has established a reliable method for sentinel node biopsy for skin cancer. However, the detection rate of cervical sentinel lymph nodes (SLN) is generally lower than that of inguinal or axillary SLN because of the complexity of lymphatic drainage in the head and neck region and the “shine‐through” phenomenon. Recently, indocyanine green fluorescence imaging has been reported as a new method to detect SLN. We hypothesized that fluorescence navigation with indocyanine green in combination with the standard technique would improve the detection rate of cervical sentinel nodes. We performed cervical sentinel node biopsies using the standard technique in 20 basins of 18 patients (group A) and using fluorescence navigation in combination with the standard technique in 12 basins of 16 patients (group B). The mean number of sentinel nodes was two per basin (range, 1–4) in group A and three per basin (range, 1–5) in group B. The detection rate of sentinel nodes was 83% (29/35) in group A and 95% (36/38) in group B. The false‐negative rate was 6% (1/18 patients) in group A and 0% in group B. Fluorescence navigation with indocyanine green may improve the cervical sentinel node detection rate. However, greater collection of data regarding the usefulness of cervical sentinel node biopsy using indocyanine green is necessary.     

An inverse correlation of VZV skin-test reaction,but not antibody,with severity of herpes zoster skin symptoms and zoster-associated pain

BackgroundCell-mediated immunity (CMI) has been considered to be related to the development of herpes zoster (HZ). However, there have been no large-scale prospective studies on the relationship between VZV-specific CMI and severity of HZ.ObjectiveWe carried out a large-scale prospective cohort study to clarify the relationship between immunological factors for varicella-zoster virus (VZV) and the clinical severity of HZ.MethodsWe carried out a cohort study on VZV immunity in a population living on an island cluster, Shozu County in Japan, and examined the people who developed HZ during a median follow-up period of 2 years, with a focus on the relationship between cell-mediated and humoral immunity and the severity of skin lesions and zoster-associated pain. A total of 12,522 people over the age of 50 were enrolled in this study, and 258 registrants were diagnosed as HZ. CMI was measured by VZV skin test, and humoral immunity was assessed with serological tests (neutralization test, immunoadherence hemagglutination test, and gpELISA test) for VZV-specific antibodies.ResultsCMI to VZV assessed by VZV skin test showed a significant inverse relationship to the severity of HZ skin lesions, and also to the severity of acute and subacute pain. Furthermore, weak response to the VZV skin test was associated with a high risk of post-herpetic neuralgia. In contrast, VZV-specific antibody titer was not associated with the severity of skin lesions and zoster-associated pain.ConclusionVZV-specific CMI, but not humoral immunity, may play a key role in controlling the severity of HZ skin lesions and zoster-associated pain.