Efficient induction of CD8 T-associated immune protection by vaccination with mRNA transfected dendritic cells

Dendritic cells are excellent targets for antigen-specific immune intervention. Here we attempted to introduce a CD8 T cell-dependent epitope into dendritic cells for presentation on major histocompatibility complex class I and induction of immunity. Murine bone-marrow-derived dendritic cells were subjected to electroporation with mRNA transcribed in vitro from a plasmid encoding lymphocytic choriomeningitis virus glycoprotein or enhanced green fluorescent protein under the control of a T7 promotor. The transfection efficiency of dendritic cells was 22 to 40%. Maturation was not inhibited by the electroporation. Dendritic cells electroporated with the appropriate antigen induced cell number-dependent in vitro proliferation in CD8 T cells expressing a transgenic receptor recognizing the 33 to 41 sequence of lymphocytic choriomeningitis virus glycoprotein in association with H-2Kb/Db, indicating correct synthesis, processing, and presentation of the epitope. Naive C57BL/6 mice vaccinated with electroporated dendritic cells and challenged with lymphocytic choriomeningitis virus were protected. Vaccination induced epitope-specific T cells as assessed by tetramer staining in blood and spleen. These results indicate that targeting dendritic cells with antigen-encoding mRNA can induce antigen-specific CD8 T cell responses as well as protective anti-viral immunity in vivo. Targeting dendritic cells with antigen-encoding mRNA may find wider application for immune intervention in disorders such as autoimmunity and cancer.     

Tat-induced deregulation of neuronal differentiation and survival by nerve growth factor pathway

HIV-1 enters the brain at the early stage of infection and resides primarily in a limited number of macrophages/microglia and astrocytes. Infection of these cells, however, may not explain the massive neuronal pathology which is seen in AIDS-associated dementia, suggesting a role for factors released from HIV-1 infected cells that trigger a cascade of events leading to neurodegeneration. Our results indicate that Tat, the potent regulatory protein of HIV-1 which is secreted by infected cells and can affect neighboring uninfected cells by transcellular means, can influence multiple biological events that lead to neuronal injury. These findings demonstrate that treatment of neuronal cells with Tat affects MAPK/ERK1/2 activity, the downstream central component of the nerve growth factor (NGF) signaling pathway. Furthermore, our data indicate that treatment of cells with Tat severely decreases expression of p35, a neuron-specific activator of cdk5, a cyclin dependent kinase that phosphorylates several neuronal proteins including neurofilament, and plays an important role in neuronal differentiation and survival. In parallel, Tat can bind to the cellular protein, Puralpha, which associates with cdk5. Further, results from Puralpha knockout animals revealed a decrease in p35 activity, pointing to the importance of Puralpha association with cdk5 in the activity of cdk5:p35 complex. These data demonstrate the cooperativity between HIV-1 Tat and the Puralpha in deregulation of the NGF signal transduction pathway in neuronal cells.     

Modification of insulin-like growth factor 1 receptor, c-Src, and Bcl-XL protein expression during the progression of Barrett's neoplasia

Oncogenes, growth factors, cell surface receptors, and cell-cycle and apoptotic regulatory proteins have been implicated in the growth regulation and progression of Barrett's-associated neoplasia. Among these, insulin-like growth factor 1 receptor (IGF1-R) and c-Src are reported to be key regulators of mitogenesis and tumorigenesis. In addition, c-Src may exert its transforming capability by inducing increased expression of IGF1-R on the neoplastic cells. Bcl-X(L), a member of the Bcl-2 family, blocks apoptosis and has been reported to increase in Barrett's-associated neoplasia. To study the modifications in IGF1-R, c-Src, and Bcl-X(L) protein expression during the progression of Barrett's-associated neoplasia, we analyzed 34 resected gastroesophagectomy specimens by immunohistochemistry using antibodies to human IGF1-R, c-Src, and Bcl-X(L). In these cases, we found 22 intestinal (Barrett's) metaplasias (IMs), 25 low-grade dysplasias (LGDs), 28 high-grade dysplasias (HGDs), 34 invasive adenocarcinomas (CAs), and 19 lymph node metastases. High IGF1-R cytoplasmic staining was present in 14 of 19 (74%) node metastases, in 28 of 34 (82%) CAs, in 18 of 28 (64%) HGDs, in 13 of 25 (52%) LGDs, and in 5 of 22 (23%) IMs. Strong and diffuse c-Src expression was identified in 17 of 19 (89%) node metastases, in 29 of 34 (85%) Cas, in 26 of 28 (93%) HGDs, in 18 of 25 (72%) LGDs, and in 9 of 22 (41%) IMs. Bcl-X(L) cytoplasmic staining was evident in 12 of 19 (63%) node metastases, in 20 of 34 (59%) Cas, in 20 of 28 (71%) HGDs, in 15 of 25 (60%) LGDs, and in 6 of 22 (27%) IMs. In 11 cases, c-Src activity was measured by kinase assay and reflected the immunohistochemical results. Our data indicate that expression levels of IGF1-R, c-Src, and Bcl-X(L) proteins are coordinately elevated in Barrett's-associated neoplasia. These findings indicate important roles of these growth regulatory proteins in the malignant progression of Barrett's-associated neoplasia.     

Color Doppler sonography and scintigraphy of the testis: a prospective, comparative analysis in children with acute scrotal pain

OBJECTIVE: To compare testicular color Doppler sonography with testicular scintigraphy in differentiating between surgical and nonsurgical conditions of the pediatric testis, and to evaluate the role of testicular color Doppler sonography in the pediatric population. MATERIALS AND METHODS: Forty-six children (age range, 1 day to 18 years; median age, 11 years) with acute scrotal pain were evaluated with both scintigraphy and color Doppler sonography by two separate groups of radiologists who had no knowledge of the results of the other modality. The final radiologic diagnosis was classified as a surgical condition, nonsurgical condition, or indeterminate and was compared with the patient's surgical diagnosis or clinical diagnosis, which was established by response to treatment and follow-up. RESULTS: Sonography correctly diagnosed 11 of 14 surgical conditions and 31 of 32 nonsurgical conditions. There was one indeterminate sonogram. There were no false-positive examinations, and there were three false-negative examinations (sensitivity = 78.6% [95% CI, 66.7-90.5%], specificity = 96.9% [95% CI, 94.3-99.5%], accuracy = 91.3%). Color flow was demonstrated in the asymptomatic testis in 34 of 44 boys. Scintigraphy correctly diagnosed 11 of 14 surgical conditions and 29 of 32 nonsurgical conditions. There were two indeterminate scintigrams. There were two false-positive examinations and two false-negative examinations (sensitivity = 78.6% [95% CI, 66.7%-90.5%], specificity = 90.6% [95%CI, 82.2%-99.0%], accuracy = 87.0%). CONCLUSIONS: Color Doppler sonography and scintigraphy show similar sensitivity for the diagnosis of testicular torsion. A small number of false-negative cases can occur with either modality. The two studies may provide complementary information in indeterminate cases.     

Adjacent QT dispersion: A good predictor of ventricular arrhythmias after myocardial infarction

The 12-lead surface electrocardiogram adjacent QTc dispersion, which is the maximum difference of corrected QT interval between two adjacent leads, is a simple method to determine regional variation in repolarization and refractoriness. The aim of this study is to evaluate adjacent QTc dispersion as a marker of susceptibility to ventricular arrhythmias after myocardial infarction. A total of 135 consecutive patients with acute myocardial infarction were enrolled in the study. Adjacent QTc, measured by lens magnifier, was calculated on the first, second and third days after acute myocardial infarction. On the second day after acute myocardial infarction, adjacent QTc dispersion was significantly greater in patients with ventricular arrhythmias (P < 0.001). Adjacent QTc dispersion on the first and fifth day after acute myocardial infarction was not associated with development of ventricular arrhythmias. On the second day after acute myocardial infarction, adjacent QTc dispersion is a simple and feasible method for prediction of ventricular arrhythmias.     

In vitro effects of ascorbate and Trolox on the biocompatibility of dental restorative materials

Previous in vitro studies on the cytotoxicity of eight dental restorative materials including composites, compomers, resin-modified glass ionomer cements and glass ionomer cements have demonstrated a depletion of intracellular glutathione in gingival fibroblasts incubated with eluates of these materials and a protective effect of N-acetylcysteine. In the present study, we investigate the effects of two other antioxidants: ascorbate and Trolox. It was found that Trolox reduced the cytotoxicity induced by resin-based biomaterial eluates. In contrast, ascorbate increased in a dose-dependent manner the toxic effect of all eluates except for Z100 MP and Tetric flow (composites). The effect of D-mannitol was studied for GC FUJI II and was found to neutralize the additional toxic effect of ascorbate. Ascorbate increased the depletion of intracellular glutathione of these dental material eluates (between 17% and 24%, depending on the material). Quantification of metal ions in the dental material eluates showed the presence of significant amounts of aluminum and iron in GC FUJI II > photac fil > GC FUJI II LC > F2000. The mechanism of this increased cytotoxicity could be explained by the Fenton reaction resulting from the pro-oxidant effect of ascorbate in the presence of iron (transition metal ions) and/or aluminum.