Preclinical evaluation of autologous dendritic cells transfected with mRNA or loaded with apoptotic cells for immunotherapy of high-risk neuroblastoma

Children with high-risk neuroblastoma (NB) have a poor clinical outcome. The purpose of the present study was to evaluate different strategies for immunotherapy of high-risk NB based on vaccination with antigen-loaded dendritic cells (DCs). DCs are professional antigen-presenting cells with the ability to induce antitumor T-cell responses. We have compared DCs either loaded with apoptotic tumor cells or transfected with mRNA from the NB cell line HTB11 SK-N-SH, for their capacity to induce T-cell responses in vitro. Monocyte-derived DCs from healthy donors were loaded with tumor-derived antigens in the form of apoptotic cells or mRNA, matured and used to prime autologous T cells in vitro. After 1 week, T-cell responses against antigen-loaded DCs were measured by ELISPOT assay. DCs loaded with apoptotic NB cells or transfected with NB-cell mRNA were both able to efficiently activate autologous T cells. Both T cells of the CD8+ and CD4+ subset were activated. T cells activated by NB mRNA transfected DCs extensively crossreacted with DCs loaded with apoptotic NB cells and vice versa. The results indicate that loading of DCs with apoptotic NB cells or transfection with tumor mRNA represent promising strategies for development of individualized cancer vaccines/cancer gene therapy in treatment of NB.     

Papular mucinosis of the breast after radiation therapy

Localized papular mucinosis is a type of mucinosis induced by several different causes. However, to the best of our knowledge, prior radiation therapy has not been reported to be related to papular mucinosis. We present a case of a 47‐year‐old woman who had undergone an operation for a breast carcinoma 2 years earlier and received local radiotherapy in the affected breast. Currently, she presents multiple erythematous papules that are caused by abundant dermal mucin deposits. We discuss some potential differential diagnoses.     

mRNA-based electrotransfection of human dendritic cells and induction of cytotoxic T lymphocyte responses against the telomerase catalytic subunit (hTERT).

Dendritic cells (DCs) are recognised as the most potent antigen-presenting cells for induction of cellular immune responses, and vaccination with DCs pulsed with antigens has emerged as a promising strategy for generating protective immunity in mammals. We have developed a transfection method that uses in vitro synthesised mRNA and square-wave electroporation for transient expression in DCs and other cell types. The method is highly efficient and produces almost complete transfection of cells in culture. When using mRNA encoding the enhanced green fluorescence protein (EGFP), highest expression in DCs occurred on the second day after transfection and produced a 76-fold increase in mean fluorescence above background. High levels of expression were maintained for at least 5 days post-transfection. In comparison, square-wave electroporation of DCs with EGFP plasmid DNA yielded 15% transfected cells and a 28-fold increase of mean fluorescence. DCs transfected with mRNA encoding the telomerase catalytic subunit (hTERT) acquired strong telomerase activity and were capable of eliciting a hTERT-specific cytotoxic T lymphocyte (CTL) response in vitro.     

Morphology Prediction in HDPE/PA‐6/EVOH Ternary Blends: Defining the Role of Elasticity Ratio

The morphology of uncompatibilized HDPE/PA‐6/EVOH ternary blends was predicted using static and dynamic phenomenological models wherein PA‐6 to EVOH component ratio and temperature were chosen as influential parameters. The type of morphology and particle size in HDPE/PA‐6/EVOH blends are then examined by scanning electron microscopy and image analysis techniques. The core–shell structure was observed for all ternary samples, in which PA‐6 domains were encapsulated by a thin layer of EVOH within HDPE matrix. Among various criteria, that is, viscosity ratio, interfacial tension, and elasticity ratio, the elasticity ratio has been found to be the most influential parameter for investigation of core–shell size alteration with temperature and component ratio.     

Hyperhomocysteinemia reduction in ethanol-fed rabbits by oral betaine

Betaine has been shown to protect the body by increasing S-adenosyl methionine levels in experimental animals. Therefore, we investigated the preventive effect of betaine on ethanol-induced hyperhomocysteinemia in a rabbit animal model. A total of 32 New Zealand white rabbits were divided into four equal groups (control, ethanol, betaine, and betaine and ethanol) with different dietary regimens and were followed up for 3?months. The day before the beginning, the 30th, 60th, and 90th days of the treatment, blood samples were collected from the marginal ear vein. Plasma total homocysteine was determined by an Axis? Homocysteine EIA kit, and a SimulTRAC-SNB Radioassay kit ([⁵⁷Co]/folate [¹²⁵I]) was used to determine folate and vitamin B₁₂ in the serum. Ethanol-induced hyperhomocysteinemia was manifested by a decrease in the levels of vitamin B₁₂ and folate with a concomitant increase in the level of total homocysteine (tHcy) for the ethanol group only. While vitamin B₁₂ showed no significant difference in the betaine and ethanol group on the 90th day compared to the control group, the concentration of folate differed significantly (p?<?0.05). There was a significant difference only on the 90th day between the ethanol and the other groups for tHcy (p?<?0.05); however, no significant difference was observed between tHcy and gender. Taken together, the present study indicates that the protective effect of betaine is related to the ability to promote the betaine homocysteine methyl transferase pathway by providing the methyl group and subsequently increase B₁₂ and folate. Therefore, this investigation recommends the use of the beneficial properties of betaine as a pretreatment method for alcoholics.     

Increased submucosal factor XII la-positive dendrocytes in oral lichen planus

Factor XIIIa +“dendrocytes”, normal residents of the submucosa and dermis. are a morphologically and phenotypically distinctive subset of the monocyte-macrophage system. Because these cells arc believed to participate in the regulation of immune responses, we postulated that they may play a role in the pathogenesis of lichen planus, a condition of immune dysregulation. Tissue sections of oral lichen planus were evaluated immunohistochemically for evidence of differences in dendrocyte populations in lesional and non-lesional areas from the same patient. In addition to factor XIIIa, sections were stained for antigens (CD68, S-100 protein, CD36) that may be expressed by other cells that occasionally exhibit dendritic profiles. CDI8 (found on leukocytes and dendrocytes) and its ligand ICAM-I (intercellular adhesion molecule) were also identified in sections to determine if these antigens are operative in lichen planus. Results showed that XIIIa + dendrocytes were significantly increased in number (and size) in lichen planus. The mean number of dendrocytes in connective tissue subjacent to basement membrane (0.064 mm²) was 27 in lichen planus as compared to 10 in adjacent unaffected tissue. Similar increases were also evident in connective tissue deep to this zone (mean of 20 dendrocytes vs. mean of 8). CD68+ macrophages were also abundant in the lichen planus infiltrate, and S-100 + connective tissue cells were frequently seen. CD36 + dendritic cells were seen in relatively small numbers in the same sites where dendrocytes were found. ICAM-I + connective tissue dendritic cells of undetermined lineage were also evident in the diseased areas. Endothelial cells in and around the infiltrate were strongly reactive to anti-ICAM-1. Basal and parabasal keratinocytes overlying the lymphoid infiltrates were also strongly ICAM-1 +. It appears that XIIIa + dendrocytes have a role in the immunologic mechanisms of lichen planus, and that these cells may be acting in concert with other immunocompetent cells, such as macrophages and S-100 + epithelial dendritic cells, to activate T lymphocytes. ICAM-I expression by dendrocytes. endothelial cells, and keratinocytes may facilitate leukocyte migration to, and retention in. the disease focus.     

Injuries to the hand inflicted by rotary snowcutters

In the period January 1, 1975, through December 31, 1986, 28 patients were treated for hand injuries caused by small home rotary snowcutters. These injuries were characterized by extensive lacerations and contusions, particularly on the dorsal side of the hand and the fingers, with concomitant extensor tendon ruptures, comminuted and complicated fractures, and traumatic amputations of fingers of the dominant hand. The index and middle finger were nearly always injured; the thumb was usually totally spared. The frequency of postoperative complications was low after delayed primary surgical treatment. In this series the mean permanent medical disablement was found to be 12%, ranging from 0 to 50%. The present observations, when made available, alarmed national authorities. In turn, manufacturers were required to equip rotary snowcutters with motorquenchers. This device will probably effectively prevent these injuries in the future.     

Co-expression of p16(INK4A) and laminin 5 gamma2 by microinvasive and superficial squamous cell carcinomas in vivo and by migrating wound and senescent keratinocytes in culture

The high frequency of mutation, deletion, and promoter silencing of the gene encoding p16(INK4A) (p16) in premalignant dysplasias and squamous cell carcinomas (SCC) of epidermis and oral epithelium classifies p16 as a tumor suppressor. However, the point during neoplastic progression at which this protein is expressed and presumably impedes formation of an SCC is unknown. Induction of p16 has been found to be responsible for the senescence arrest of normal human keratinocytes in culture, suggesting the possibility that excessive or spatially abnormal cell growth in vivo triggers p16 expression. We examined 73 skin and oral mucosal biopsy specimens immunohistochemically to test this hypothesis. p16 was not detectable in benign hyperplastic lesions, but instead was expressed heterogeneously in some dysplastic and carcinoma in situ lesions and consistently at areas of microinvasion and at superficial margins of advanced SCCs. p16-positive cells in these regions coexpressed the gamma2 chain of laminin 5, identified previously as a marker of invasion in some carcinomas. Normal keratinocytes undergoing senescence arrest in culture proved to coordinately express p16 and gamma2 and this was frequently associated with increased directional motility. Keratinocytes at the edges of wounds made in confluent early passage cultures also coexpressed p16 and gamma2, accompanying migration to fill the wound. These results have identified the point during neoplastic progression in stratified squamous epithelial at which the tumor suppressor p16 is expressed and suggest that normal epithelia may use the same mechanism to generate non-dividing, motile cells for wound repair.