ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1996.tb05654.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

ABSTRACTExtracorporeal photochemotherapy (photopheresis), an immunomodulatory therapy that targets circulating T helper lymphocytes, has been applied to the management of human immunodeficiency virus (HIV) disease. Any therapy that exerts its actions on CD4+T cells has the potential of exacerbating HIV infection. Therefore, it was necessary to observe immune function during treatment. Because cytotoxic T lymphocytes (CTL) and natural killer cells are thought to play an important role in the response against HIV infection, we examined the effect of photopheresis on HIV cytolytic activity. The study group consisted of seven patients with late‐stage HIV disease who had not received any previous treatment for HIV infection. Patients were treated exclusively with photopheresis on two consecutive days each month for 14–32 months (average, 25 months). Peripheral lymphocytes, collected at various points during treatment, were used as effectors in aWr release assay. Epstein‐Barr virus (EBV)‐transformed autologous B cell lines transfected with recombinant vaccinia vectors that expressed the HIV env (gp120, gp41) and gag (p24) proteins were used as target cells. All seven patients demonstrated relatively constant levels of cytolysis (>10% above controls) during treatment in the context of stable CD4+T cell counts and a stable clinical status. These results suggest that extracorporeal photochemotherapy did not impair the cytolytic response to HIV infection and may have enhanced it in some p

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1996.tb05655.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

ABSTRACTInactivation of the blood‐borne parasiteTrypanosoma cruziby UVA and 4′‐aminomethyl‐4,5′,8‐trimethylpsor‐alen (AMT) was studied in the blood components fresh frozen plasma (FFP) and platelet concentrate (PC). The AMT was utilized at a concentration of 50 μg/mL and the inactivation procedure included the flavonoid rutin (at 0.35 mM), a quencher of type I and type II photo‐reactants, which we have previously found to maintain platelet integrity during this treatment regimen. Within both FFP and PC, complete inactivation of the infective form ofT. cruzi, the trypomastigote, was achieved at a UVA (320–400 nm radiation) fluence of 4.2 J/cm2. We note that while the infectivity of the parasite is eliminated at 4.2 J/cmZthe trypomastigote motility continues for at least 16 h post‐treatment and is inhibited only after much higher light doses. Isolation of total DNA from the parasite cells after treatment in the presence of3H‐AMT indicated that at the lethal UVA fluence about 0.5 AMT adducts per kilobase pairs occurred. These results suggest that this psoralen plus UVA methodology, which shows promise in enhancing the viral safety of PC, may in addition eliminate bloodborneT. cruzi, the causative a

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1996.tb05656.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

ABSTRACTPsoralen plus UVA (320–400 nm radiation; PUVA) is a highly effective therapy for cutaneous diseases caused by skin infiltration with normal or neoplastic T‐lympho‐cytes. In comparing the effects of pharmacologically relevant, low‐dose PUVA treatment on growth of human keratinocytes, peripheral blood leukocytes (PBMC), and T‐lymphocyte cell lines, we determined that PBMC or T‐lymphocytes were>50‐fold more sensitive to cytotoxic effects of PUVA, while antiproliferative effects were produced by similar PUVA levels in all cell types. Low doses of PUVA (10 ng/mL 8‐methoxypsoralen and 1–2 J/cm2) were highly cytotoxic for phytohemagglutinin‐activated normal lymphocytes or transformed T‐lymphocytes as assessed by two viability assays and by flow cytofluo‐rometry. Altered lymphocyte morphology, nuclear fragmentation, TUNEL+ nuclei or nuclear fragments, and the appearance of a sub‐G, DNA peak indicated that cell death occurred by apoptosis, beginning about 1 day after PUVA treatment and continuing for several days thereafter. From assessment of cell cycle progression in mi‐mosine‐synchronized cells, PUVA treatment markedly slowed cell cycle progression, eventually producing cell cycle arrest and apoptotic entry. We propose that the probable basis for disease remissions (psoriasis, cutaneous T‐cell lymphoma) produced by PUVA treatment is through selective cytotoxic effects on clonal T‐lymphocyte populations that

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1996.tb05657.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

ABSTRACTWhereas previous studies have indicated that DNA damage as a result of 8‐methoxypsoralen (8‐MOP) and UVA treatment leads to cell death, this study establishes the minimum concentrations of 8‐MOP and UVA necessary to induce apoptosis in human T‐lymphocytic and mono‐cytic cell lines. In order to assess apoptosis, we used fluorescent microscopy to examine changes in light scattering as well as internucleosomal DNA fragmentation. Generation of a dose response curve showed that the minimum combination of UVA and 8‐MOP that was necessary to induce greater than background levels of apoptosis within 24 h of treatment was 0.5 J/cm2UVA and 12.5 ng/mL of 8‐MOP. A striking observation was that UVA alone at doses 1.0 J/cm2, but not 8‐MOP alone (6300 ng/mL), induced significant apoptosis in the Sup‐T1 cell line within 24 h. Although the percentage of apoptotic Sup‐T1 cells induced by UVA alone was not as great as that of 8‐MOP and UVA in combination, a highly significant correlation between the product of the concentration of 8‐MOP (ng/mL) times the dose of UVA (J/ cm2) and the percentage of apoptotic cells was observed. This correlation provides an important tool for studying the relationship of UVA‐induced DNA damage to apoptosis induction. Moreover, it will provide a means by which early events in the apoptotic

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1996.tb05658.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

ABSTRACTPhotopheresis is an extracorporeal form of photochemo‐therapy with 8‐methoxypsoralen (8‐MOP) and UVA (PUVA). Patients ingest 8‐MOP and then a psoralen‐rich buffy coat is obtained by centrifugation and mixed with saline. This mixture is recirculated through a UVA radiation field and then reinfused. Photopheresis appears to be effective for several T cell‐mediated disorders, because the treatment results in a specific immune response against the pathogenic clone of T cells involved. With PUVA therapy, the whole body of the patient is exposed to UVA, after ingestion of 8‐MOP. Upon UVA exposure 8‐MOP binds to, amongst others, DNA and induces DNA monoadducts and interstrand cross‐links. As a result of these photoadducts photocarcinogenicity is a risk in PUVA. In PUVA for psoriasis, it proved that angular furocoumarins, although almost incapable of inducing DNA cross‐links (less carcinogenic), are still effective. In order to determine if monoadducts induced by photopheresis could also be effective we used, specifically, 4,6,4′‐trimethylangelicin (TMA). In this report, we compare the photodegradation of both TMA and 8‐MOP under conditions relevant to thein vivosituation, as well as the effect both compounds have on the viability of rat lymphocytes as measured with the 3–(4,5‐dimethylthia‐zol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay. We show that TMA did not induce immunosuppressionin vivo, even after extensive irradiation. In addition a dose dependency of 8‐MOPNVAversusthe induced immune suppression was carried out. It was shown that there is a

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1996.tb05659.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1996.tb05660.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1996.tb05661.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

ABSTRACTNeutral red is a lysosomal probe and a biological pH indicator. In aqueous solutions, the protonated (NRW) and neutral (NR) forms of monomeric neutral red exhibit distinct absorption maxima (535 and 450 nm, respectively) but have the same fluorescence with a maximum at 637 nm and a quantum yield of 0.02. The similarity of the fluorescence spectra at acidic and basic pH suggests deprotonation of cationic species in the first singlet excited state. The NR fluorescence strongly depends on the solvent polarity as shown by addition of increasing amounts of water to pure dioxane, which gradually shifts the fluorescence maximum from 540 nm in pure dioxane to 637 nm in water. The fluorescence quantum yield increases from 0.17 in dioxane to 0.3 upon addition of 7% water and then decreases, reaching 0.02 in pure water. Immediately after incubation of human skin fibroblasts with neutral red, excitation with 435 nm light produces a fluorescence whose maximum is recorded at 575 nm. This fluorescence is located in the perinuclear region and originates from large fluorescent intracytoplasmic spots, suggesting staining of the endoplasmic reticulum‐Golgi complex. At longer times, this fluorescence is shifted to 606 nm, suggesting slow diffusion of the lysosomotropic dye toward the more hydrated and acidic interior of ly‐sosomes. Addition of a lysosomotropic detergent to cells previously incubated with neutral red shifts the fluorescence to the blue. Thus, in complex biological systems, this probe cannot be a good pH indicator but is a very sensitive probe of lysosomal rnicroenvironrne

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1996.tb05662.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY

摘要:

AbstractFourier transform multipixel spectroscopy was applied to subcellular localization of endogenous protoporphyrin (endo‐PP) in single living B16 melanoma cells during photosensitization. Continuous fluorescence spectra for each pixel were recorded using a Sagnac interferometer coupled to a charge‐coupled device camera. Multiple frames of data were acquired for each pixel composing the image, then they were stored as interferometric data and resolved as spectra for every pixel (103‐−4 × 103point pixels in a single cell). The net result was the intensity I (x, y, λ), for each pixel of the image (x, y), at any wavelength (λ). The present study demonstrates the application of Fourier transformed multipixel spectroscopy for spectral imaging of melanoma cells incubated with 5‐aminolevulinic acid (ALA). The fluorescence image of ALA‐treated cells revealed endo‐PP all over the cytosol with a vesicular distribution, which represent mitochondria and endoplasmic reticulum compartments. Two main spectral fluorescence peaks were demonstrated at 630 and 670 nm, of monomeric and aggregated protoporphyrin, with intensities that differed from one sub‐cellular site to another. Photoirradiation of the cells induced point‐specific subcellular fluorescence spectrum changes and demonstrated photoproduct formation. Spectral‐image reconstruction revealed the subcellular distribution of porphyrin species in single photosensitized cells. Multipixel spectroscopy of exogenous protoporphyrin revealed an endosomal‐lysosomal compartment in aggregated states, whereas monomeric porphyrin species were localized mainly on the outer membrane. Photo‐products could be visualized at sites of formation i

ISSN:0031-8655    

DOI:10.1111/j.1751-1097.1996.tb05663.x

出版商:Blackwell Publishing Ltd    

年代:1996

数据来源: WILEY