Evaluation of the drug lymphocyte stimulation test (DLST) with shosaikoto]

BACKGROUND: Our aim is to evaluate the significance of DLST by Shosaikoto. METHODS: We clinically evaluated 3 cases of drug-induced pneumonia assumed to be caused by Shosaikoto, and we performed DLST of Shosaikoto for healthy controls, and compared the data with drug-induced pneumonia cases of Shosaikoto. RESULTS: As clinical characteristics of 3 cases, 2 cases were positive for hepatitis C virus antibody, and 1 case was positive for DLST of Shosaikoto. The observed chest high-resolution CT (HRCT) findings showed hypersensitivity pneumonia (HP) pattern in all 3 cases. Prognosis was good in all 3 cases. DLST of Shosaikoto was positive in 27.5% of healthy controls. Stimulation index (S.I.) of DLST in drug-induced pneumonia cases increased depending on drug dilution density, compared to that of healthy controls. CONCLUSION: DLST of Shosaikoto showed high false-positive rate. However, we may be able to distinguish the true-positive cases with the false-positive cases by comparing the S.I. of DLST according to drug dilution density.     

Influence of insulin immobilization to thermoresponsive culture surfaces on cell proliferation and thermally induced cell detachment

Temperature-responsive culture dishes immobilized with insulin have been fabricated and studied to shorten cell culture periods by facilitating more rapid cell proliferation. Cells are recovered as contiguous cell sheets simply by temperature changes. Functionalized culture dishes were prepared by previously reported electron beam grafting copolymerization of N-isopropylacrylamide (IPAAm) with its carboxylate-derivatized analog, 2-carboxyisopropylacrylamide (CIPAAm), having similar molecular structure to IPAAm but with carboxylate side chains to tissue culture polystyrene dishes. Insulin was then immobilized onto culture dishes through standard amide bond formation with CIPAAm carboxylate groups. Adhesion and proliferation of bovine carotid artery endothelial cells (ECs) were examined on these insulin-immobilized dishes. Insulin immobilization was shown to promote cell proliferation in serum-supplemented medium. Increasing the grafted CIPAAm content on the tissue culture surfaces reduces cell adhesion and proliferation, even though these surfaces contained increased amounts of immobilized insulin. This result implies that a discrete balance exists between the amount of CIPAAm-free carboxylate groups and immobilized insulin for optimum cell proliferative stimulation. Cells grown on the insulin-immobilized surfaces can be recovered as contiguous cell monolayers simply by lowering culture temperature, without need for exogenous enzyme or calcium chelator additions. In conclusion, insulin-modified thermoresponsive culture dishes may prove useful for advanced cell culture and tissue engineering applications since they facilitate cell proliferation, and cultured cells can be recovered as viable contiguous monolayers by merely reducing culture temperature.     

Deposition of complement protein C3b on mixed self-assembled monolayers carrying surface hydroxyl and methyl groups studied by surface plasmon resonance

Since complement activation is recognized as a common response of the host defense system when an artificial medical device is applied to a patient, great effort has been devoted to studies on the interaction of the complement system with artificial materials. However, some uncertainties remain, partially because of the lack of well characterized surfaces and suitable analytic methods for study of the surface phenomena that occur on artificial materials under physiologic conditions. In this study, we employed self-assembled monolayers (SAMs) and the surface plasmon resonance (SPR) technique to study interactions of the serum complement with well characterized surfaces. Self-assembled monolayers carrying various concentrations of hydroxyl groups were prepared using 11-mercapto-1-undecanol (C11-OH) and one of n-nonanethiol, n-dodecanethiol, and n-hexadecanethiol. The amount of NHS deposition on the SAMs increased with increasing C11-OH content of the SAMs, and the amount of anti-C3b antibody immobilization formed on the NHS deposition layers increased with increasing C11-OH content of the SAMs. These results clearly demonstrate that a large amount of C3b, produced through the activation of the complement system, binds covalently to and is adsorbed by hydroxyl-group-rich surfaces. The combination of SAMs and the SPR technique is suitable for studying the interaction of the complement system with solid surfaces, and the results should give basic information needed for a rational design of biocompatible surfaces on synthetic materials.     

Evidence of allosteric conformational changes in the antibody constant region upon antigen binding

We have addressed the question of whether antigen binding induces a conformational change in the heavy chain constant (C(H)) domain of antibodies using staphylococcal protein A or streptococcal protein G as probes, since these proteins are known to bind to IgG domains such as C(H)1 and C(H)2-C(H)3 domains. Biosensor assays on interactions between these proteins and mouse IgG specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) or their enzymatic fragments conducted in the presence or absence of the hapten, NP-epsilon-aminocaproic acid (NP-Cap), showed that the binding of IgG to these proteins was inhibited by the binding of NP-Cap. The results of isothermal titration calorimetry also revealed that the association constant for the interaction of protein A with IgG2b decreased by the addition of NP-Cap. These results suggested that antigen binding induced conformational changes in binding sites for protein G or protein A located at C(H)1 and C(H)2-C(H)3 domains, respectively.     

Depletion of mitochondrial DNA in HIV-1-infected patients and its amelioration by antiretroviral therapy

Mitochondrial DNA (mtDNA) of peripheral blood mononuclear cells (PBMCs) collected from Human immunodeficiency virus 1 (HIV-1)-infected patients and healthy controls were measured longitudinally using real-time polymerase chain reaction to evaluate the effects of antiretroviral agents on mtDNA synthesis in vivo and to assess the value of monitoring mtDNA in PBMCs to predict adverse events amongst these patients. MtDNA levels in PBMCs were significantly decreased in treatment-naive HIV-1-infected patients compared with healthy people. MtDNA levels were not only significantly correlated with CD4(+) T-cell count, but also inversely correlated with HIV-1 viral load. MtDNA levels in untreated patients and healthy controls were stable during the period of observation. On the other hand, amongst patients treated with regimens containing AZT/3TC or d4T/3TC, mtDNA increased during treatment and recovered to levels comparable to healthy controls. In contrast, mtDNA decreased immediately after the initiation of an AZT/ddC-containing regimen. We did not find a correlation between mtDNA levels and changes in clinical parameters. There was no significant difference in mtDNA levels between patients with and those without lipoatrophy. Furthermore, there was no obvious difference in mtDNA levels amongst those patients exhibiting signs and symptoms of peripheral neuropathy. In conclusion, the decrease in mtDNA levels in PBMCs amongst HIV-1-infected patients and its amelioration by antiretroviral therapy may suggest the influence of direct effects on mitochondria or mtDNA by HIV-1 infection. Further investigations are needed to elucidate the mechanisms contributing to decreased mtDNA and the value of mtDNA measurement in the care of HIV-1-infected individuals.     

Effects of vitamin A on proliferation of human distal airway epithelial cells in culture

Using a serum-free culture method, we investigated the effects of vitamin A on the proliferation of human distal airway epithelial cells. Outgrowth of epithelial cells from lung tissue explants was enhanced by treatment with all-trans retinol at concentrations of 10^–8 to 10^–7 M. The colony-forming activity of cells harvested from the primary culture and replated onto Swiss 3T3 fibroblastic feeders was, in contrast, significantly reduced by 10^–7 M to 10^–5 M retinol. When the primary cells were harvested and subcultured on Primaria plates, population expansion was also inhibited by retinol at 10^–10 to 10^–6 M. We further investigated the cells to determine whether there was any difference in sensitivity to the growth-inhibitory effects of vitamin A between cells from the primary culture incubated with and without retinol. The population increase in cells harvested from the primary culture was inhibited equally in retinol-treated and non-treated cells by subsequent treatment with retinol or retinoic acid, this inhibition being dose-dependent. DNA synthetic activity was also inhibited. Interestingly, both the growth rate and the colony-forming efficiency on feeders were greater in the subculture of cells from the retinol-treated primary culture than in those non-treated. When the cells in the secondary subculture were treated with retinoic acid and replated again, they showed a greater population increase rate than those non-treated. Our results showed that human distal airway epithelial cells isolated from lung tissue were sensitive to the growth-inhibitory effect of vitamin A, but the proliferative potential in some fraction of the epithelial cell population was possibly enhanced by vitamin A treatment.     

Persistence of archetypal JC virus DNA in normal renal tissue derived from tumor-bearing patients.

JC virus DNAs derived from the urine of nonimmunosuppressed individuals generally contain an archetypal regulatory region which may have generated various regulatory regions of JC virus from from the brain with progressive multifocal leukoencephalopathy (PML). In this study, we examined whether JC virus persisting in normal human kidney tissue contains the archetypal regulatory region. Renal medulla, cortex, and tumor from 32 patients bearing renal tumors were screened for JC virus DNA by blot hybridization. Viral DNA was detected in the medulla in 13 cases (41%), in the cortex in 2 cases (6%), but not at all from the tumor. A number of viral DNA-positive specimens (8 from the medulla and 2 from the cortex) were used to amplify and sequence viral regulatory regions by polymerase chain reaction. Structures of the regulatory regions from all the specimens were, with a few nucleotide variations, identical with that of the archetypal region which was previously detected in the JC virus DNA from urine. This finding supports the hypothesis that the JC virus associated with PML evolved from the archetypal JC virus during persistence in human hosts. Furthermore, we present evidence that renal JCV is replicating and that progeny virions are excreted into the urine.