Bacterial ghosts (BGs)—Advanced antigen and drug delivery system

Bacterial ghosts (BGs) are empty bacterial envelopes of Gram-negative bacteria produced by controlled expression of cloned gene E, forming a lysis tunnel structure within the envelope of the living bacteria. BGs are devoid of cytoplasmic content and possess all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat. BGs are ideally suited as an advanced drug delivery system (ADDS) for toxic substances in tumor therapy. The inner space of BGs can be loaded with either single components or combinations of peptides, drugs or DNA which provides an opportunity to design new types of (polyvalent) drug delivery vehicles. Uptake of BGs loaded with Doxorubicin (Dox) by CaCo2 cells led to effective Dox release from endo-lysosomal compartments and accumulation in the nucleus. Viability and proliferative capacity of the cells were significantly decreased (2–3 orders of magnitude) after internalization of Dox loaded BGs as compared to cells incubated with free Dox. The same effect was observed with leukemia cells. Melanoma cells also revealed a high capability to internalize BGs. These results indicate that BGs are able to target a range of types of cancer. BGs have also been investigated as DNA delivery vectors. Studies show DNA loaded BGs are efficiently phagocytosed and internalized by both professional APCs and tumor cells with up to 82% of cells expressing the plasmid-encoded reporter gene. Our studies with BGs as an ADDS system contribute (i) to optimize drug delivery for the treatment of cancer; (ii) define specific conditions for selection and preparation of BG formulations; (iii) and provide a background for the clinical application of BGs in cancer therapy.     

Anaphylaxis to isosulfan blue and cross-reactivity to patent blue V: case report and review of the nomenclature of vital blue dyes.

BACKGROUND: Blue dyes used for lymphatic mapping in sentinel lymph node biopsy cause intraoperative anaphylactic reactions in up to 2.7% of patients. With increasing implementation of this technique, the incidence of anaphylaxis to these dyes can be expected to increase. In the literature, the chemically often unrelated and inconsistently designated dyes have been confused, adding to other inconsistencies in the nomenclature. OBJECTIVE: To demonstrate the nomenclature, chemical and physiologic differences, and allergenicity of the various blue dyes used in a medical context. METHODS: We describe a patient with an intraoperative grade IV anaphylactic reaction to isosulfan blue. Immediate-type hypersensitivity was proved by positive skin test reactions and CD63 expression to isosulfan blue and cross-reactivity to patent blue V. RESULTS: A review of the literature clarified the exact nomenclature of the blue dyes and the possible pitfalls of confusing nomenclature in the context of structurally closely related dyes with different allergenic properties. For the detection of type I hypersensitivity, intracutaneous tests are valuable tools. An IgE-mediated mechanism has been shown recently. In most cases, sensitization exists without known previous exposure in a medical context. This may be due to the widespread use of such dyes in objects of everyday life. Preoperative antiallergic medication use does not prevent anaphylactic reactions but apparently reduces their severity. CONCLUSION: For better comparison and precision, the Chemical Abstracts Service number of the respective dye should always be given.     

Propagation of hepatitis A virus in human embryo fibroblasts

human diploid fibroblasts and human primary liver cell carcinoma cells (PLC/PRF/5) were infected with hepatitis A virus (HAV) adapted to growth in cell culture or derived directly from human stool. Viral antigen was expressed in PLC/PRF/5 cells 28 days after infection with cell culture-adapted HAV, and 50 days after infection with virus from human stool. In human fibroblasts the periods until first expression of viral antigen were 90 and 210 days, respectively. During further passages of HAV in fibroblasts the time of first appearance of antigen decreased to about 28 days. Biophysical properties of HAV extracted from infected fibroblasts were comparable to those of HAV derived directly from human stool. Immunofluorescence studies showed that the antigen was located exclusively within the cytoplasm of the infected fibroblasts. Kinetics of antigen production indicated that an equilibrium between virus multiplication and cell metabolism was reached in persistently infected fibroblasts.     

IFN-gamma-enhanced allergen penetration across respiratory epithelium augments allergic inflammation

BACKGROUND: Respiratory allergen contact is the critical event in the elicitation and boosting of allergen-specific immune responses, as well as in the induction of immediate and late inflammatory reactions. OBJECTIVE: We sought to investigate the influence of various factors of allergic inflammation on the integrity and barrier function of respiratory epithelium for allergens. METHODS: We cultured the human bronchial epithelial cell line 16HBE14o- in a transwell culture system as a surrogate of intact respiratory epithelium and used purified iodine 125-labeled recombinant major birch pollen allergen (rBet v 1) to study the extent, kinetics, and factors influencing transepithelial allergen penetration. RESULTS: Culture supernatants from activated allergen-specific T H 1 clones decreased transepithelial resistance. A screening of various factors (histamine, IFN-gamma, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-8, IL-12, and TNF-alpha) identified IFN-gamma as a potent factor capable of reducing epithelial barrier properties and enhancing transepithelial allergen penetration. Increased submucosal allergen concentrations caused by IFN-gamma-mediated reduction of epithelial barrier function provoked a more than 7-fold augmentation of histamine release from sensitized basophils. CONCLUSION: These results demonstrate that the T H 1 cell-derived cytokine IFN-gamma facilitates allergen penetration through the respiratory epithelium and thereby can aggravate allergic inflammation.     

Role of the Salmonella pathogenicity island 1 effector proteins SipA, SopB, SopE, and SopE2 in Salmonella enterica subspecies 1 serovar Typhimurium colitis in streptomycin-pretreated mice

Salmonella enterica subspecies 1 serovar Typhimurium (serovar Typhimurium) induces enterocolitis in humans and cattle. The mechanisms of enteric salmonellosis have been studied most extensively in calf infection models. The previous studies established that effector protein translocation into host cells via the Salmonella pathogenicity island 1 (SPI-1) type III secretion system (TTSS) is of central importance in serovar Typhimurium enterocolitis. We recently found that orally streptomycin-pretreated mice provide an alternative model for serovar Typhimurium colitis. In this model the SPI-1 TTSS also plays a key role in the elicitation of intestinal inflammation. However, whether intestinal inflammation in calves and intestinal inflammation in streptomycin-pretreated mice are induced by the same SPI-1 effector proteins is still unclear. Therefore, we analyzed the role of the SPI-1 effector proteins SopB/SigD, SopE, SopE2, and SipA/SspA in elicitation of intestinal inflammation in the murine model. We found that sipA, sopE, and, to a lesser degree, sopE2 contribute to murine colitis, but we could not assign an inflammation phenotype to sopB. These findings are in line with previous studies performed with orally infected calves. Extending these observations, we demonstrated that in addition to SipA, SopE and SopE2 can induce intestinal inflammation independent of each other and in the absence of SopB. In conclusion, our data corroborate the finding that streptomycin-pretreated mice provide a useful model for studying the molecular mechanisms of serovar Typhimurium colitis and are an important starting point for analysis of the molecular events triggered by SopE, SopE2, and SipA in vivo.     

Human chromosome 16 conservation in primates

A study was made of the organization of the chromosome orthologous to HSA16 in primates using a panel of 8 BAC probes spanning human chromosome 16. The probes were used in FISH experiments on great apes and on representatives of the Old World monkeys, New World monkeys, and lemurs. The domestic cat was used as an outgroup. The results indicate that 16p and 16q were separate chromosomes in a primate ancestor. They fused in a Catarrhini ancestor giving rise to the present day form found in HSA, great apes, and Old World monkeys. Several rearrangements were found in New World monkeys.