Establishment of animal models to analyze the kinetics and distribution of human tumor antigen-specific CD8 T cells

Many patients develop tumor antigen-specific T cell responses detectable in peripheral blood mononuclear cells (PBMCs) following cancer vaccine. However, measurable tumor regression is observed in a limited number of patients receiving cancer vaccines. There is a need to re-evaluate systemically the immune responses induced by cancer vaccines. Here, we established animal models targeting two human cancer/testis antigens, NY-ESO-1 and MAGE-A4. Cytotoxic T lymphocyte (CTL) epitopes of these antigens were investigated by immunizing BALB/c mice with plasmids encoding the entire sequences of NY-ESO-1 or MAGE-A4. CD8⁺ T cells specific for NY-ESO-1 or MAGE-A4 were able to be detected by ELISPOT assays using antigen presenting cells pulsed with overlapping peptides covering the whole protein, indicating the high immunogenicity of these antigens in mice. Truncation of these peptides revealed that NY-ESO-1-specific CD8⁺ T cells recognized D^d-restricted 8mer peptides, NY-ESO-1_81-88. MAGE-A4-specific CD8⁺ T cells recognized D^d-restricted 9mer peptides, MAGE-A4_265-273. MHC/peptide tetramers allowed us to analyze the kinetics and distribution of the antigen-specific immune responses, and we found that stronger antigen-specific CD8⁺ T cell responses were required for more effective anti-tumor activity. Taken together, these animal models are valuable for evaluation of immune responses and optimization of the efficacy of cancer vaccines.     

Sevoflurane metabolite production in a small cohort of coronary artery bypass graft surgery patients

OBJECTIVE: To determine hepatic and renal effects of hexafluoroisopropanol in patients undergoing coronary artery bypass graft surgery under sevoflurane anesthesia. DESIGN: Prospective, clinical comparison. SETTING: University hospital. PARTICIPANTS: Adult patients scheduled for coronary artery bypass graft surgery (n = 56) were divided into 3 groups according to renal function: group 1, patients with normal renal function (plasma creatinine <1.7 mg/dL), subdivided into 2 groups (group 1a and group 1b), and group 2, patients with impaired renal function (plasma creatinine > or = 1.7 mg/dL). INTERVENTIONS: Anesthesia was maintained with fentanyl, 20 microg/kg, and sevoflurane. In group 1a and group 2, sevoflurane dosage was 0.5 minimum alveolar concentration (MAC). In group 1b, it was 1.0 MAC of sevoflurane. During cardiopulmonary bypass, the same concentration of sevoflurane was given through a membrane oxygenator. MEASUREMENTS AND MAIN RESULTS: Serum hexafluoroisopropanol concentration was measured before induction of anesthesia, at the initiation of cardiopulmonary bypass, at the release of the aortic cross-clamp, at the end of cardiopulmonary bypass, at the end of surgery, and on the 1st postoperative day. Blood urea nitrogen, creatinine, 24-hour urinary output, aspartate aminotransferase, alanine aminotransferase, and total bilirubin were measured at preoperative evaluation, at the end of surgery, and on the 1st and 3rd postoperative days. The levels of hexafluoroisopropanol increased and peaked on the 1st postoperative day. Laboratory values showed no significant differences among all groups. CONCLUSION: The serum level of hexafluoroisopropanol after 0.5 MAC of sevoflurane anesthesia does not aggravate hepatic and renal functions.     

Perindopril effect on uncoupling protein and energy metabolism in failing rat hearts

Uncoupling proteins, inner mitochondrial membrane proton transporters, are important for regulating myocardial energy efficiency. We investigated the effects of the ACE inhibitor perindopril on cardiac performance, myocardial energy efficiency, and uncoupling protein expression in an aortic regurgitation rat model. Twenty male Sprague-Dawley rats, in which aortic regurgitation was produced, were divided into untreated and perindopril-treated (5 mg x kg(-1) x d(-1)) rats. The treatments were initiated 3 days after operation. Ten control rats were sham-operated. Measurements of blood pressure and echocardiography were repeated before and 100 days after operation (endpoint). Left ventricular uncoupling protein-2 expression, creatine phosphate, and adenosine triphosphate were measured at endpoint. In perindopril-treated rats, systolic and diastolic blood pressure decreased after treatment (92+/-4/65+/-2 mm Hg). At endpoint, left ventricular end-diastolic dimension in untreated (10.7+/-0.2 mm) and treated rats (9.2+/-0.2 mm) was increased, and fractional shortening was reduced in untreated rats (28+/-1%) but did not change in treated rats (36+/-2%). Uncoupling protein-2 mRNA expression increased in untreated rats (3.7-fold) and was suppressed by perindopril (1.5-fold). The creatine phosphate was reduced in untreated rats (10.6+/-0.7 micro mol/g) but not in treated rats (15.9+/-2.0 micro mol/g). In the chronic stage of aortic regurgitation, perindopril improved cardiac performance and myocardial energy efficiency, in which the suppression of uncoupling protein-2 may play an important role.     

N-cadherin signals through Rac1 determine the localization of connexin 43 in cardiac myocytes

It has been proposed that the formation of gap junction is influenced by adherens junction in cardiac myocytes. To examine whether signals through N-cadherin are involved in the distribution of connexin 43 (Cx43), the distribution of cell-cell adhesion molecules, N-cadherin and Cx43, was analyzed in aligned cardiac myocytes. To induce cell orientation running in parallel to tension direction, neonatal rat cardiac myocytes were plated for 3 hours and exposed to 20% cyclic stretch for 24 hours on silicone dishes. The aligned cells cultured for 0-5 days were immunostained with anti- N-cadherin or anti-Cx43 antibody. After cultivation for 3-5 days, following the accumulation of N-cadherin, Cx43 was localized at the longitudinal cell termini. Adenoviral gene transfer of dominant negative N-cadherin significantly attenuated the localization of Cx43 at the longitudinal cell termini, suggesting that Cx43 localization is regulated downstream of N-cadherin. In the process of Cx43 localization, Rho family proteins, RhoA and Rac1, were activated, but not Cdc42. RhoA and Rac1 activation was inhibited by the transfection of dominant negative N-cadherin, indicating that RhoA and Rac1 were activated by N-cadherin in the oriented cardiac myocytes. The inhibition of Rho family proteins by Rho GDI significantly attenuated the accumulation of Cx43, but not that of N-cadherin. Furthermore, the translocation of Cx43 to longitudinal cell termini was prevented by the inhibition of Rac1, but not RhoA. Collectively, these findings suggest that the localization of Cx43 was determined through the Rac1 pathway downstream of N-cadherin in cardiac myocytes.     

Strict glycemic control ameliorates the increase of carotid IMT in patients with type 2 diabetes

The aim of this study was to investigate the effect of strict glycemic control on the carotid artery intima-media thickness (IMT) in type 2 diabetic patients who initially had good glycemic control (HbA1c between 5.8 and 6.4 %). The subjects were 67 patients showing deterioration of the mean HbA1c over 3 years by more than 0.2% from baseline (D group) and 33 subjects showing improvement of the mean HbA1c by more than 0.2% from baseline (A group). The clinical characteristics and annual change of IMT during the observation period were compared between the two groups in a 3-year retrospective longitudinal study. The baseline characteristics and the mean values of BMI, blood pressure, and serum lipids during the study period did not differ significantly between the two groups. However, the mean HbA1c of A group was significantly lower than that of D group (5.67 +/- 0.10 vs. 6.28 +/- 0.08, mean +/- SE, p<0.001). The adjusted annual increase rate of IMT was significantly less in A group than in D group (-0.035 +/- 0.019 vs. 0.036 +/- 0.015 mm, M +/- SEM, p<0.001). These results indicate that further improvement of glycemic control from a good HbA1c value can prevent an increase of IMT in type 2 diabetic patients.     

A case of asymptomatic racemose hemangioma treated by bronchial artery embolization]

A 69-year-old man had an abnormal shadow on chest X-ray and bronchoscopic examination showed that left B4 was completely occluded by a tumor. A non-pulsatile polypoid nodule was also found in right B'. The tumor in the left B4 was diagnosed as carcinoid, but the nodule in right B' was suspected to be hemangioma and biopsy was not performed. Bronchial arteriography revealed hypervascularization with dilated vessels distributing to the lingular lobe and convoluted and a dilated bronchial artery extending to the right upper lobe. From these findings, we diagnosed racemose hemangioma of the bronchial artery of the right upper lobe. After bronchial artery embolization of the right and left bronchial arteries, he underwent segmentectomy of the lingula and was discharged without complications. Two months after the operation, bronchoscopic examination showed that the racemose hemangioma had shrunk and the swelling in the surrounding mucosa had decreased. If a submucosal small nodule is observed during bronchoscopy, biopsy should be performed after bronchial arteriography, and if the nodule is diagnosed as racemose hemangioma, bronchial artery embolization should be performed.     

PSEUDOMEMBRANOUS COLITIS COMPLICATING ULCERATIVE COLITIS

Clostridium difficile toxin (CD toxin) causes antibiotic‐associated colitis, or pseudomembranous colitis (PMC). Although CD toxin is sometimes found in the stools of patients with ulcerative colitis (UC), UC is rarely complicated by PMC. We report herein a case of PMC complicating UC, and present a review of the literature. A 71‐year‐old woman was diagnosed as having UC of the left colon, and treated with prednisolone and mesalazine. Later, however, lumbar spinal stenosis was also detected. After surgery for lumbar spinal stenosis, she suffered postoperative infection of the lumbar region. After 3‐week treatment with antibiotics, she developed diarrhea, bloody stools, and abdominal pain. Colonoscopy revealed PMC of the cecum, ascending colon, sigmoid colon, and rectum. Stools were positive for CD toxin. As cefotiam hydrochloride, levofloxacin hydrate (LVFX), and prednisolone were suspected as the causative agents, she was treated with 1.5 g vancomycin (VCM) daily for 2 weeks without ceasing LVFX. Her symptoms improved, and colonoscopy confirmed resolution of PMC. The possibility of PMC should be considered in UC patients treated with antibiotics, immunosuppressive agents or corticosteroids who complain of gastrointestinal symptoms. These patients should be thoroughly investigated by several modalities, including colonoscopy and CD toxin testing.     

Molecular design of biodegradable polymeric micelles for temperature-responsive drug release.

We designed thermo-responsive and biodegradable polymeric micelles for an ideal drug delivery system whose target sites are where external stimuli selectively release drugs from the polymeric micelles. The thermo-responsive micelles formed from block copolymers that were composed both of a hydrophobic block and a thermo-responsive block. Poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) showing a lower critical solution temperature (LCST) around 40 degrees C was synthesized for the thermo-responsive block, while biodegradable poly(D,L-lactide), poly(epsilon-caprolactone), or poly(D,L-lactide-co-epsilon-caprolactone) was used for the hydrophobic block. By changing both the block lengths of the poly(D,L-lactide)-containing block copolymers, physical parameters such as micelle diameter and critical micelle concentration were varied. On the other hand, the choice of the hydrophobic block was revealed to be critical in relation to both on the thermo-responsive release of the incorporated anti-cancer drug, doxorubicin, and the temperature-dependent change of the hydrophobicity of the micelles' inner core. One polymeric micelle composition successfully exhibited rapid and thermo-responsive drug release while possessing a biodegradable character.     

Intermittent pressure overload triggers hypertrophy-independent cardiac dysfunction and vascular rarefaction

For over a century, there has been intense debate as to the reason why some cardiac stresses are pathological and others are physiological. One long-standing theory is that physiological overloads such as exercise are intermittent, while pathological overloads such as hypertension are chronic. In this study, we hypothesized that the nature of the stress on the heart, rather than its duration, is the key determinant of the maladaptive phenotype. To test this, we applied intermittent pressure overload on the hearts of mice and tested the roles of duration and nature of the stress on the development of cardiac failure. Despite a mild hypertrophic response, preserved systolic function, and a favorable fetal gene expression profile, hearts exposed to intermittent pressure overload displayed pathological features. Importantly, intermittent pressure overload caused diastolic dysfunction, altered beta-adrenergic receptor (betaAR) function, and vascular rarefaction before the development of cardiac hypertrophy, which were largely normalized by preventing the recruitment of PI3K by betaAR kinase 1 to ligand-activated receptors. Thus stress-induced activation of pathogenic signaling pathways, not the duration of stress or the hypertrophic growth per se, is the molecular trigger of cardiac dysfunction.