Immunomodulatory effect of splenectomy in lung cancer mouse xenograft models receiving radiation therapy.

PURPOSE: This study aims to investigate the effect of splenectomy on radiation-mediated growth inhibition and immune modulation in lung cancer xenograft models. MATERIALS AND METHODS: Human non-small cell lung cancer H1299 cells and murine Lewis lung carcinoma LL/2-luc cells were injected into the right hind leg of BALB/c-nude mice and C57BL/6 mice, respectively. Splenectomy or sham operation was performed prior to tumor cell injection or before and after irradiation during tumor growth. Irradiation was delivered with 2-3 fractions of 6 Gy X-ray using a linear accelerator. Flow cytometry analysis was performed for immune cell profiling. RESULTS: Splenectomy prior to tumor injection or at early stage inhibited growth of LL/2-luc tumors but not that of H1299 tumors; however, it did not enhance the antitumor effect of radiation regardless of intervention timing. Flow cytometry analysis showed monocytic myeloid-derived suppressor cells (MDSCs) and activated CD8+ T cells increased after irradiation in the tumors of splenectomized mice, compared to those of sham-operated mice. Administration of anti-PD-1 (programmed death-1) antibodies improved the ability of splenectomy to attenuate the growth of irradiated tumors. CONCLUSION: Splenectomy has paradoxical effects on radiation-induced tumor growth inhibition, depending on tumor types and intervention timing, but it has an immune-modulating effect when combined with radiation.

Targeted Liquid Biopsy Using Irradiation to Facilitate the Release of Cell-Free DNA from a Spatially Aimed Tumor Tissue.

PURPOSE: We investigated the feasibility of using an anatomically localized, target-enriched liquid biopsy (TLB) in mouse models of lung cancer. MATERIALS AND METHODS: After irradiating xenograft mouse with human lung cancer cell lines, H1299 (NRAS proto-oncogene, GTPase [NRAS] Q61K) and HCC827 (epidermal growth factor receptor [EGFR] E746-750del), circulating (cell-free) tumor DNA (ctDNA) levels were monitored with quantitative polymerase chain reaction on human long interspersed nuclear element-1 and cell line-specific mutations. We checked dose-dependency at 6, 12, or 18 Gy to each tumor-bearing mouse leg using 6-MV photon beams. We also analyzed ctDNA of lung cancer patients by LiquidSCAN, a targeted deep sequencing to validated the clinical performances of TLB method. RESULTS: Irradiation could enhance the detection sensitivity of NRAS Q61K in the plasma sample of H1299-xenograft mouse to 4.5- fold. While cell-free DNA (cfDNA) level was not changed at 6 Gy, ctDNA level was increased upon irradiation. Using double-xenograft mouse with H1299 and HCC827, ctDNA polymerase chain reaction analysis with local irradiation in each region could specify mutation type matched to transplanted cell types, proposing an anatomically localized, TLB. Furthermore, when we performed targeted deep sequencing of cfDNA to monitor ctDNA level in 11 patients with lung cancer who underwent radiotherapy, the average ctDNA level was increased within a week after the start of radiotherapy. CONCLUSION: TLB using irradiation could temporarily amplify ctDNA release in xenograft mouse and lung cancer patients, which enables us to develop theragnostic method for cancer patients with accurate ctDNA detection.

Novel DNA-based T-Cell Activator Promotes Rapid T-Cell Activation and Expansion.

Autologous chimeric antigen receptor engineered T-cell therapies are beginning to dramatically change the outlook for patients with several hematological malignancies. Yet methods to activate and expand these cells are limited, often pose challenges to automation, and have biological limitations impacting the output of the injectable dose. This study describes the development of a novel, highly flexible, soluble DNA-based T-cell activation and expansion platform which alleviates the limitations of current technologies and provides rapid T-cell activation and expansion.

Preclinical study of influenza bivalent vaccine delivered with a two compartmental microneedle array.

Multiple vaccines can be mixed into a single combination to be a single product. However, combination vaccines have problems of complexity. In this study, microneedles were utilized in a compartmental microneedle array (CMA) to deliver two influenza vaccine strains without mixing. In this study, the CMA had two compartments, and two rectangular structures were attached to each end of the array to enable integration of the compartments with the coating equipment. The coating solution, which contained influenza vaccines for B/Yamagata (B-Y) and B/Victoria (B-V), was filled into the two reservoirs of the container. The CMA was aligned with the container for dipping the first compartment of the array into the first reservoir and the second compartment into the second reservoir. The CMA containing B-Y and B-V separately was administered to mice, and weight change and survival were compared with other groups of mice administered (a) combination vaccines with microneedles, (b) two monovalent vaccines with microneedles, (c) intramuscularly with a combination vaccine, and (d) intramuscularly with two monovalent vaccines. Plaque reduction neutralization tests were also performed to compare the CMA group with the other groups. The CMA showed a relative standard error of less than 7% between samples in dose uniformity. It also showed comparable antibody-forming efficacy compared to other groups, especially by B/Yamagata virus challenge. The CMA mice group showed better survival and weight change than mice that received intramuscular (IM) injection of the combination vaccine. In the neutralizing antibody experiment, all microneedle groups showed a higher neutralizing antibody than the IM groups. Vaccines were administered without mixing by a single administration using a CMA, and the CMA showed comparable efficacy with IM administration of the combination vaccine. Multivalent vaccines can be delivered without mixing as a single product by using a CMA.

Development and clinical study of the use of infrared radiation to accelerate the dissolution rate of a microneedle array patch (MAP).

The dissolution rate of a microneedle array patch (MAP) determines how long a MAP must remain attached to the skin (often called "wear time"). In this study, the dissolution rate of a MAP was increased, not by changing the drug formulation but by employing an infrared (IR) device that is widely used for hospital treatment and in-home therapy. A MAP with microneedles 480 µm in height was prepared using hyaluronic acid (HA). Changes in transepidermal water loss (TEWL), the surface temperature of the skin, and the dissolution rate of the MAP tips with IR irradiation were evaluated on human skin in vivo. Time for recovery from erythema that occurred after MAP attachment and IR irradiation was also evaluated. TEWL increased more than fourfold with IR irradiation. Water that evaporated as a result of IR irradiation was trapped in the skin layer by the patch, resulting in the increased dissolution rate of the MAP tips. After 10 min of IR irradiation, the height of the dissolving tips compared with their initial height increased from 41 to 56%, and the dissolved volume of the tips compared with their initial volume increased from 7 to 18%. During the 10 min of irradiation, the skin surface temperature rose from 32 to 40 °C. Erythema occurred in the early stage of treatment with IR irradiation and MAP attachment, but it abated within 2 h after removal of the MAP and cessation of IR irradiation. Through this study, it was possible to shorten the administration time of MAPs by using an IR device that could be easily accessed. This method can be applied to various types of MAPs in order to reduce the time that the MAPs must remain attached to the skin without changing the drug formulation. Graphical abstract The increase in dissolution rate of dissolving microneedle array patch (MAP) as a result of infrared radiation. a Water-soluble tips of MAP dissolved in water in skin without infrared irradiation. Dotted line indicates the initial dissolving microneedles. b Water in skin and subcutaneous layer evaporated actively with infrared irradiation and was stored under patch of MAP. Increased amount of water in skin induced faster dissolution of MAP tips.

Preparation of H1N1 microneedles by a low-temperature process without a stabilizer.

During the manufacture of H1N1 microneedles, a stabilizer is usually added to maintain the antigenicity of the vaccine. However, finding a suitable stabilizer is difficult, and the addition of a stabilizer can limit the antigen dose and the addition of an adjuvant because of the limited volume of the microneedles. In this study, the authors evaluated whether H1N1 microneedles could be fabricated without a stabilizer by keeping the production environment at a low temperature. H1N1 microneedle patches without a stabilizer were prepared in a process that involved maintaining a low temperature of 10 °C. The protective immune response to this method of drug application was investigated by comparing it with traditional intramuscular (IM) immunization and with the use of H1N1 microneedles with a stabilizer. A process-sensitive antigen, H1N1, was stabilized without the use of a stabilizer in a process that maintained a low temperature of 10 °C. The preparation process consisted of coating and drying processes. In animal experiments, mice were immunized using an array of low-temperature H1N1 microneedles without a stabilizer (LT-MN), and they showed strong antibody responses. Compared to three other application methods of traditional IM immunization, low-temperature H1N1 microneedles with a stabilizer (LT-MN-T), and room-temperature H1N1 microneedles with a stabilizer (RT-MN-T), LT-MN produced comparable results in inducing protective immunity. A plaque reduction neutralization test found that LT-MN and LT-MN-T provided greater immunity compared with IM and RT-MN-T. A process in which the temperature is maintained at 10 °C can provide successful vaccination with H1N1 microneedles without the addition of a stabilizer. This process can be applied to various temperature-sensitive biologics.

Iclaprim activity against wild-type and corresponding thymidine kinase-deficient Staphylococcus aureus in a mouse protection model.

The in vitro and in vivo antimicrobial activities of dihydrofolate reductase (DHFR) inhibitors are inhibited in the presence of free thymidine in the growth milieu and in rodent efficacy models. However, for thymidine kinase (TK) deficient mutant bacteria, the presence of free thymidine does not impact the activity of DHFR inhibitors, and these mutants were used to assess the in vivo efficacy of the DHFR inhibitor, iclaprim. The efficacies of iclaprim, trimethoprim, and vancomycin were evaluated in a systemic mouse infection model. Female CD-1 mice were infected intraperitoneally (IP) with wild-type Staphylococcus aureus ATCC 25923 (MSSA) or AW 6 (MRSA) or their corresponding isogenic TK-deficient mutant S. aureus strains AH 1246 and AH 1252. Iclaprim showed potent antibacterial activity against both the TK-deficient mutant S. aureus strains, with PD50 values of 1.8 and < 0.5 mg/kg, respectively, for strains AH 1246 and AH 1252. In contrast, poor antibacterial activity was observed against corresponding wild-type (TK competent) S. aureus strains, with PD50 values of 10.8 and 2.2 mg/kg, respectively, for strains ATCC 25923 and AW 6. This study confirms that thymidine plays an important antagonistic role when determining the efficacy of DHFR inhibitors in vivo. This is the first study to show that iclaprim is active against TK-deficient S. aureus strains in a systemic mouse infection model, and that TK-deficient mutants may be used to evaluate iclaprim's activity in rodent models in vivo.

Identification of the In Vivo Pharmacokinetics and Pharmacodynamic Driver of Iclaprim.

The neutropenic murine thigh infection model was used to define the pharmacokinetic/pharmacodynamic index linked to efficacy of iclaprim against Staphylococcus aureus ATCC 29213 and Staphylococcus pneumoniae ATCC 10813. The 24-h area under the curve (AUC)/MIC index was most closely linked to efficacy for S. aureus (R2, 0.65), while both the 24-h AUC/MIC and the percentage of time that drug concentrations remain above the MIC (%T>MIC) were strongly associated with effect (R2, 0.86 for both parameters) for S. pneumoniae.

Role of Nrf2 and protective effects of Metformin against tobacco smoke-induced cerebrovascular toxicity.

Cigarette smoking (CS) is associated with vascular endothelial dysfunction in a causative way primarily related to the TS content of reactive oxygen species (ROS), nicotine, and inflammation. TS promotes glucose intolerance and increases the risk of developing type-2 diabetes mellitus (2DM) with which it shares other pathogenic traits including the high risk of cerebrovascular and neurological disorders like stroke via ROS generation, inflammation, and blood-brain barrier (BBB) impairment. Herein we provide evidence of the role played by nuclear factor erythroid 2-related factor (Nrf2) in CS-induced cerebrobvascular/BBB impairments and how these cerebrovascular harmful effects can be circumvented by the use of metformin (MF; a widely prescribed, firstline anti-diabetic drug) treatment. Our data in fact revealed that MF activates counteractive mechanisms primarily associated with the Nrf2 pathway which drastically reduce CS toxicity at the cerebrovascular level. These include the suppression of tight junction (TJ) protein downregulation and loss of BBB integrity induced by CS, reduction of inflammation and oxidative stress, renormalization of the expression levels of the major BBB glucose transporter Glut-1 and that of the anticoagulant factor thrombomodulin. Further, we provide additional insights on the controversial interplay between Nrf2 and AMPK.

Periimplant bone regeneration in hydroxyapatite block grafts with mesenchymal stem cells and bone morphogenetic protein-2.

Hydroxyapatite (HA) blocks as an alternative material for autogenous onlay bone grafts are regarded as an insufficient substitute for osseointegration of dental implant. In this study, we evaluated the effects of dog mesenchymal stromal cells (dMSCs) with or without bone morphogenetic protein-2 (BMP) on new peri-implant bone formation after HA block graft. In four mandibular bone defects (8×8×6 mm each) in five beagle dogs, dental implants were placed with HA block loaded with autogenous dMSCs with or without BMP-2. Animals were sacrificed at eight weeks, and bone healing was evaluated among four groups consisting of 1) HA alone as a control, 2) HA+dMSCs, 3) HA+BMP-2, and 4) HA+dMSCs+BMP-2. According to histomorphometric evaluation, the MSC+BMP-2 group and the BMP-2 group showed significantly higher bone-implant-contact (BIC) length than the MSC group, while there was no significant difference in new bone formation among the groups. According to micro-CT analysis, bone volume and bone mineral density were significantly higher in the MSC+BMP-2 group compared with the control group (p<0.01 and p<0.05, respectively). BIC was significantly higher in the MSC+BMP-2 group than both the control and MSC groups (p<0.01 and p<0.05, respectively). In conclusion, our results showed that bone regeneration at peri-implant bone defects grafted with HA blocks was significantly increased by dual delivery of MSCs and BMP-2. Conversely, HA blocks with MSC or BMP-2 alone did not allow for efficient peri-implant bone regeneration.

Impact of cigarette smoke extract and hyperglycemic conditions on blood-brain barrier endothelial cells.

BACKGROUND: Diabetes and tobacco smoking are significant public health concerns which have been shown to independently impact the blood-brain barrier (BBB). Since smoking is a risk factor for diabetes and shares some of the common pathological pathways leading to metabolic abnormalities, it is hypothesized that their combination would produce additive or synergistic BBB dysfunction. Therefore, the objective of this study was to assess this hypothesis and evaluate the magnitude of these effects in vitro using hCMEC/D3 cells; a well-established human BBB endothelial cell line. METHODS: Monolayers of hCMEC/D3 cells were exposed to hyperglycemic conditions (HG; 35 mM) or 5% soluble cigarette smoke extracts (CSE, model of mainstream smoke exposure) for 12-24 h. Cells were then harvested for subsequent biochemical analyses. Transendothelial electrical resistance (TEER) and paracellular permeability to florescent dextrans were used to assess monolayer integrity. Analysis of released factors and cytokines was carried out by ELISA. Western blot (WB) analysis/immunofluorescence of relevant molecular targets was carried out. P-gp efflux activity was measured using rhodamine 123. RESULTS: Immunofluorescence and WB data showed a significant ZO-1 down-regulation by HG and/or CSE over 24 h exposure. CSE in presence of HG produced a synergistic increase in release of vascular endothelial growth factor that was accompanied by decreased TEER and augmented permeability to labeled dextrans in a size-dependent manner. Moreover, CSE increased the expression of GLUT-1 and SGLT-1 in isolated membrane fractions of hCMEC/D3 cells. The effect was amplified by HG. Both, HG and CSE elicited the membrane upregulation of P-glycoprotein (P-gp) expression which however, was not paralleled by a comparable efflux activity. Interestingly, concomitant exposure to HG and CSE evoked a marked upregulation of PECAM-1 and other pro-inflammatory markers including IL-6 and -8, when compared to each condition alone. Moreover, exposure to all tested conditions amplified (to a different degree) cellular oxidative stress response denoted by increased Nrf2 nuclear translocation. CONCLUSION: Overall, our results have clearly shown an additive pattern in the release of angiogenic and inflammatory factors following concomitant exposure to HG and CSE. This suggests the involvement of common key modulators in BBB impairment by both CS and HG possibly through the activation of oxidative stress responses.

Incidental diagnosis of vaginal schwannoma in a patient with thigh pain.

Schwannoma commonly arises from Schwann cells of the neural sheath, and is rare in the groin region. Here, we describe a vaginal schwannoma incidentally detected by magnetic resonance imaging (MRI) in a patient with thigh pain. A 43-year-old woman presented with thigh pain with burning and tingling sensations in the medial aspect of her left thigh. MRI revealed a mass lesion of heterogeneous intensity 5.2 × 5.7 cm in the left vaginal wall. The mass was resected and histology revealed schwannoma.

Squamous cell carcinoma arising in an ovarian mature cystic teratoma complicating pregnancy.

Mature cystic teratomas of the ovary (MCT) are usually observed in women of reproductive age with the most dreadful complication being malignant transformation which occurs in approximately 1% to 3% of MCTs. In this case report, we present a patient with squamous cell carcinoma which developed from a MCT during pregnancy. The patient was treated conservatively without adjuvant chemotherapy and was followed without evidence of disease for more than 60 months using conventional tools as well as positron emission tomography-computed tomography following the initial surgery. We report this case along with the review of literature.

Liver recurrence in early endometrial cancer with focal myometrial invasion.

Endometrial cancer is the most common malignancy of the female genital tract. The cancer spreads by direct extension, transtubal dissemination, lymphatic dissemination, and/or by hematogenous spread, usually results in lung metastasis, but may less commonly involve liver, brain, and bone. Here, we describe a patient with stage IA endometrial cancer who developed liver recurrence 17 months after surgery.

Case of uterine leiomyoma showing fludeoxyglucose uptake on F-18 fludeoxyglucose positron emission tomography/computed tomography.

In order not to over treat uterine leiomyoma and to avoid overlooking uterine leiomyosarcoma, a highly reliable diagnostic method has been thought. Occasionally, it is difficult to discriminate uterine leiomyoma from uterine leiomyosarcoma. Recently positron emission tomography/computed tomography (PET/CT) has been proved useful in assessing pelvic malignancies. We experienced a case of uterine leiomyoma showing increased F-18 fludeoxyglucose uptake on PET/CT in a postmenopausal woman. However, histological analysis demonstrated benign leiomyoma by the hysterectomy. Immunohistochemical analysis of glucose transporter-1 showed negative in leiomyoma. Our case indicates that uterine leiomyoma in a postmenopausal woman may show false positive result of PET/CT.

MGMT -535G>T polymorphism is associated with prognosis for patients with metastatic colorectal cancer treated with oxaliplatin-based chemotherapy.

PURPOSE: The present study analyzed the polymorphisms of DNA repair genes and their impact on the response to chemotherapy and survival of patients with colorectal cancer. PATIENTS AND METHODS: A total of 94 patients with recurrent or metastatic colorectal cancer treated with oxaliplatin-based combination chemotherapy were enrolled in the present study. The single nucleotide polymorphisms of 16 DNA repair genes were determined using a PCR-RFLP assay. RESULTS: During the median follow-up duration of 15.9 (2.1-53.0) months, 67 (71.3%) progressions and 29 (30.9%) deaths were observed. Among the 60 patients assessable for response, response to the oxaliplatin-based regimens was found in 27 (45%) patients (9 CR and 18 PR). In a logistic regression analysis adjusted to age, sex, primary site, disease status, and regimen, the POLR2C rs4937 and MSH2 rs3732183 polymorphisms were statistically associated with the response to the oxaliplatin-based chemotherapy. A multivariate survival analysis showed that the TT genotype of the MGMT (rs1625649) -535G>T polymorphism was found to correlate with a worse progression-free survival (PFS) than the combined GG + GT genotypes (HR = 3.137; 95% CI = 1.423-6.914; P = 0.005), which was also observed among the 60 evaluable patients (HR = 2.653; 95% CI = 1.101-6.392; P = 0.030) For the clinical parameters, curative resection was the most significant prognostic factor in a Cox model for PFS and overall survival (HR = 0.229 and 0.205; P < 0.001 and 0.001, respectively). CONCLUSION: The MGMT -535G>T polymorphism (rs1625649) was found to be correlated with PFS in patients with advanced colorectal cancer treated with oxaliplatin-based chemotherapy.

Hypermethylation of E-cadherin in endometrial carcinoma.

OBJECTIVE: Hypermethylation of CpG island is a common mechanism for the inactivation of tumor suppressor genes. Hypermethylation of the E-cadherin promoter region has been rarely studied in endometrial carcinoma of Korean women. The purpose of this study is to investigate methylation status of E-cadherin promoter region in endometrial carcinomas and endometrial hyperplasias, and analyze the correlation with clinicopathologic variables in endometrial carcinomas. METHODS: We examined the methylation status of the E-cadherin promoter region using methylation specific polymerase chain reaction and immunohistochemical expression (IHC) of E-cadherin in 30 endometrioid endometrial carcinomas and 20 endometrial hyperplasias, and correlated these results with various clinicopathological factors of endometrial carcinomas. RESULTS: Decreased expression of E-cadherin was detected in 13 of 30 (43.3%) endometrial carcinomas and in 1 of 20 (5%) endometrial hyperplasias (p=0.009). Promoter hypermethylation was detected in 12 of 30 (40%) endometrial carcinomas and 2 of 20 (10%) endometrial hyperplasias (p=0.015). Methylation status did not have a significant influence on the tumor grade and lymph node metastasis. However, the hypermethylation rate was significantly higher in stage above Ic (p=0.025). Decreased expression of E-cadherin was associated with tumor grade, tumor stage, and lymph node metastasis in endometrial carcinomas (p=0.01, p=0.02, p=0.03). There was no correlation between DNA hypermethylation and decreased expression of E-cadherin in endometrial carcinomas (p>0.05). CONCLUSION: These results indicate that hypermethylation of E-cadherin promoter region is a frequent event in endometrial carcinoma, which may play an important role in the progression of carcinogenesis. Also, the promoter methylation of E-cadherin in endometrial carcinoma was found to be significantly associated with higher stage above Ic.

An ovarian mucinous cystadenocarcinoma arising from mature cystic teratoma with para-aortic lymph node metastasis: a case report.

Malignant transformation of a mature cystic teratoma (MCT) is an uncommon complication. The most common form of malignant transformation of a MCT is squamous cell carcinoma, representing 75% of malignant transformations. The frequency of malignant transformation of MCT to adenocarcinoma is just 6.8%. To the best of our knowledge, no case of para-aortic lymph node metastasis in mucinous adenocarcinoma arising from MCT has been reported before. The prognosis of malignant transformation of the MCT is very poor. Here, we report an unusual case of a 41-year-old woman with mucinous adenocarcinoma arising from MCT with para-aortic lymph node metastasis.

Expression of Cyclooxygenase-2 and Bcl-2 in human gastric adenomas.

BACKGROUND: Cyclooxygenase (COX)-2 is the rate-limiting enzyme in prostaglandin synthesis. An increased expression has been implicated in the development and progression of human gastric cancers and colorectal adenomas and cancers. This study aimed to determine the involvement and association of COX-2 and Bcl-2 in precancerous gastric adenomas. METHODS: Seventy-nine gastric polyps were obtained by endoscopic mucosal resection or polypectomy from January, 2000 to July, 2003. Immunohistochemical expression of COX-2 and Bcl-2 was observed, and their relationships with various clinicopathological factors were analyzed. RESULTS: Histologically, 13 hyperplastic polyps and 66 tubular adenomas, of which 17 showed high-grade dysplasia, were observed. Increased COX-2 expression was observed in low-grade and high-grade tubular adenomas compared to hyperplastic polyps (p=0.004 and p=0.001, respectively). COX-2 expression was significantly higher in larger (>1 cm) compared with smaller (<1 cm) tubular adenomas (o=0.034), but no relation was observed in hyperplastic polyps. While Bcl-2 expression differed significantly according to histology, increased Bcl-2 expression was observed especially in COX-2 positive low-grade tubular adenomas. CONCLUSION: COX-2 expression increased in a size-dependent manner in tubular adenomas, suggesting a role in polyp growth. The increased expression of Bcl-2 in tubular adenomas, especially in COX-2 positive tubular adenomas, suggests that COX-2 action may be related to Bcl-2 expression.