Elevated atherogenic index following oral administration of quaternized polyamine nanogels.

Quaternized polyamine nanogels possessing poly(ethylene glycol) (PEG)-tethered chains as the surface layer were prepared by redox-initiated emulsion polymerization of 2-(N,N-diethylamino)ethyl methacrylate (EAMA) in the presence of vinylbenzyl-ended poly(ethylene glycol) (PEG-CH2PhCHCH2), followed by quaternization with methyl iodide (QNG-I). QNG-I absorbed taurocholic acid regardless of environmental pH, because of the fixed positive charge of QNG-I. Oral administration of polyamine nanogels into mice tended to cause intestinal retention, with accumulation of up to 70% of the initial dose. Levels of low-density lipoprotein cholesterol (LDL-C) in hyperlipidemic mice effectively decreased following oral administration of QNG-I. Interestingly, oral administration of QNG-I increased the levels of high-density lipoprotein cholesterol (HDL-C), resulting in an extremely high atherogenic index. Iodide counter-anions in QNG-I played an important role in the increase in HDL-C levels.

A highly sensitive chemiluminescence assay for superoxide detection and chronic granulomatous disease diagnosis.

Reactive oxygen species (ROS) produced by neutrophils are crucial for defense against infectious diseases, and the adequate measurement of ROS levels is an important way to evaluate the possibility of infections. The fluorescent probe dihydrorhodamine 123 has been applied exclusively to the measurement of ROS thus far. We developed a novel method for detecting ROS, which utilizes the chemiluminescent probes Luminol and Diogenes. The new method quantitatively detects ROS produced by as few as 10 to 10(4) neutrophils. Furthermore, this method can detect ROS levels in one microliter of whole blood or ROS produced by Epstein-Barr immortalized B lymphocytes. This method will be valuable for prompt diagnosis of neonatal chronic granulomatous diseases in which neutrophils aberrantly produce superoxide.

Influence of advanced age on Mycobacterium bovis BCG vaccination in guinea pigs aerogenically infected with Mycobacterium tuberculosis.

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only tuberculosis (TB) vaccine currently available, but its efficacy against adult pulmonary TB remains controversial. BCG induces specific immune responses to mycobacterial antigens and may elicit protective immunity against TB. TB remains a major public health problem, especially among the elderly, yet the efficacy of BCG in the elderly is unknown. We investigated the ability of BCG vaccination to prevent TB in young (6-week-old), middle-aged (18-month-old), and old (60-month-old) guinea pigs. BCG-Tokyo vaccination reduced the growth of Mycobacterium tuberculosis H37Rv in all three groups. By use of an enzyme-linked immunospot (ELISPOT) assay, antigen-specific gamma interferon (IFN-γ)-producing cells were detected in the 60-month-old guinea pigs after a booster vaccination with BCG-Tokyo. Our findings suggest that BCG-Tokyo has a protective effect against tuberculosis infection regardless of age.

Cell adhesion markedly increases lucigenin-enhanced chemiluminescence of the phagocyte NADPH oxidase.

Lucigenin-enhanced chemiluminescence (LECL) is widely used for the detection of reactive oxygen species released from various cells and mitochondria. However, the LECL response varies depending on cell species and assay conditions at least in part by unknown factors. Here we report that cell adhesion is an important factor for increasing LECL of tetradecanoylphorbol acetate (TPA)-stimulated human neutrophils. More than 90% LECL remained even after complete removal of the cell suspension 10 min after TPA stimulation, and approximately 22.5% of neutrophils were adhered to the reaction tube. These results indicate that LECL by an adhering neutrophil is approximately 45x higher than that by a non-adhering neutrophil. LECL by leukocyte adhesion deficiency neutrophils was one-fifth of that by normal neutrophils and completely disappeared when the cell suspension was removed, confirming that LECL depends highly on cell adhesion. The oxidase activity of adhering neutrophils measured after permeabilization with Renex 30 together with NADPH addition was similar to that of non-adhering neutrophils, indicating that lucigenin and cell adhesion do not enhance the oxidase activity. Based on these findings, we propose that a mixture of adhering and non-adhering neutrophils can be used for simultaneous screenings of adhering activity and the oxidase activity of neutrophils.

Novel two-parameter flow cytometry (MIL4/SSC followed by MIL4/CT7) allows for identification of five fractions of guinea pig leukocytes in peripheral blood and lymphoid organs.

Though the guinea pig has been an extremely useful animal model for a variety of diseases, the tools necessary to undertake a full-scale immunological analysis of the guinea pig have been lacking. For instance, traditional two-parameter forward/side scatter (FSC/SSC) flow cytometry, though effective in human and other animal models, is unable to adequately identify the distinct fractions of guinea pig peripheral blood leukocytes (PBL). We introduce here a new flow cytometric technique (MIL4/SSC followed by MIL4/CT7) which redresses this lack by identifying and characterizing five distinct fractions of PBL: neutrophils, lymphocytes, monocytes, eosinophils plus basophils, and the novel MIL4(-)SSC(large)CT7(high) population. The MIL4(-)SSC(large)CT7(high) cells possess cytoplasmic inclusion bodies of variable size that were positive for periodic acid Schiff (PAS). Their cell surface stained positive for the helper/inducer lymphocyte markers, T cell markers, CD45, Thy-1, asialo GM1 and FcR, but negative for B cell markers, such as membrane-type IgM, CD8 and MHC class II. The novel flow cytometric technique also allowed us to establish that the five leukocyte fractions were found in PBL, splenocytes, thymocytes and lymph node cells. Cells which were positive for inclusion bodies comprised 16.6% of splenocytes, 9.9% of PBL and 4.3% of liver cells, but were comparatively rare in lymph node cells, thymocytes, and BM cells. The novel flow cytometric technique introduced here will allow a better understanding of the response of each type of guinea pig leukocyte and thereby shed light on the diseases with which they are associated.

A rabbit ear flap perfusion experiment to evaluate the percutaneous absorption of drugs.

A rabbit ear flap single-pass perfusion system was examined as an experimental method for studying the relationship between the physiological conditions of tissues and drug disposition after topical applications. Tyrode solutions containing bovine serum albumin (BSA) and sucrose or flurbiprofen (FP), used as a model drug, were perfused through the vessel in the ear flap to evaluate the physiological conditions prior to the application of FP to the skin surface. The extracellular volume and distribution properties of FP in the perfused ear were similar to those in an in vivo experimental system. In addition, the perfused ear flap exhibited a pharmacological response to bradykinin (BK). The amount of FP in the outflow Tyrode solution containing BSA after application to the skin surface of the perfused ear decreased with the addition of BK, while that in the tissues under the application site increased. FP binds to BSA, which leaked from the intravascular space, and could be retained in the tissues under the application site. The protein binding also affected the redistribution of FP to other tissues in the ear flap after application to the skin. The rabbit ear perfusion system is a useful method for studying the percutaneous absorption of drugs especially variations in the disposition of drugs in oedematous tissues.