Systemic thrombin inhibition by Hirulog does not alter medial smooth muscle cell proliferation and inflammatory activation after vascular injury in the rabbit.

The study evaluated the role of thrombin in activation of vascular smooth muscle cells early after vascular injury. The direct thrombin inhibitor Hirulog (10 mg/kg SQ tid) or vehicle was administered to rabbits over 3 days following balloon injury to the abdominal aorta and the right iliac artery. Hirulog treatment yielded marked systemic anticoagulation as evidenced by an about 3.5-fold prolongation of quantitative thrombin time one hour after an injection, but with a reduction to almost baseline levels at the end of the dosing interval. After 3 days, proliferating cells in the right iliac artery were enumerated. The expression of intercellular adhesion molecule 1, macrophage-colony stimulating factor, tumor necrosis factor alpha, and interleukin-1beta as markers for inflammatory activation of the vessel wall was examined by immunohistochemistry and graded semiquantitatively. Mitotic indices did not differ between control and Hirulog-treated animals. There was also no difference in the expression of markers of inflammatory activation between both groups. In conclusion, thrombin inhibition by Hirulog administration does not reduce acutely (within 3 days) vascular smooth muscle cell proliferation or inflammatory activation after angioplasty. Thrombin inhibitors may therefore limit restenosis in the rabbit by acting later or via other, unknown pathways. The lack of effect of the thrombin inhibitor on the cellular events during the early phase of the response to balloon injury may explain the failure of such strategies to reduce restenosis in recent clinical trials despite effects towards acute thrombotic complications. Together, these results suggest that acute thrombin generation is not a crucial stimulus for early smooth muscle cell proliferation and inflammatory activation after vascular injury.

ELISA methods for the analysis of antibody responses induced in multiple sclerosis patients treated with recombinant interferon-beta.

Upon treatment with protein therapeutics, a subset of patients will typically develop antibodies against the drug. These anti-drug antibodies can be of concern because they have the potential to alter the drug's therapeutic activity. In the case of relapsing-remitting multiple sclerosis (RRMS) patients receiving recombinant interferon-beta (IFN-beta), those receiving BETASERON (IFN-beta-1b; E. coli expressed, non-glycosylated, des-Met-1, Cys17Ser recombinant IFN-beta) have a higher incidence of IFN-beta specific antibodies compared to those receiving AVONEX (IFN-beta-1a; mammalian cell-expressed, natural sequence, glycosylated recombinant IFN-beta). The current study reports the development and characterization of ELISAs that detect distinct components of the anti-IFN-beta response in patients' sera, and therefore can potentially be used to characterize the composition of the anti-IFN-beta antibody response. ELISAs were developed using a constant detecting reagent but a variety of IFN-beta-derived test antigens (e.g., native IFN-beta, biotinylated IFN-beta, IFN-beta peptides) and capture methods. Assays were characterized using serum samples from a small number of patients treated with recombinant IFN-beta (either BETASERON or AVONEX). Assays in which IFN-beta was captured via a specific mAb, or in which biotinylated IFN-beta was captured via streptavidin, detected serum antibodies that recognize IFN-beta in its native structural state. In contrast, assays in which IFN-beta was coated directly onto the assay plates detected antibodies that recognize forms of IFN-beta possessing a folded structure distinct from the native structure. Certain epitopes present on native IFN-beta were not represented in these assays in which the test antigen was directly coated on plastic. Antibodies specific for linear epitopes could be detected using linear peptides as test antigens; the locations of these epitopes were mapped by reference to the X-ray crystal structure of IFN-beta-1a. Together, these data show that the mode of antigen presentation employed in IFN-beta ELISAs determines which antibody specificities are detected, and can affect whether or not a given serum sample is identified as positive for anti-IFN-beta antibodies. As a consequence, screening samples in a single ELISA format presenting IFN-beta in a non-native form may lead to underestimation of the incidence of IFN-beta treated MS patients that have generated antibodies specific to the native, active form of the drug.

Organic dust exposures from compost handling: case presentation and respiratory exposure assessment.

Inhalation of dust from contaminated organic materials may result in acute respiratory tract illness. Possible mechanisms include toxic and cellular reactions to microbial and other organic products or immunologic responses after prior sensitization to an antigen. A case is presented of a 52 year old male who developed fever, myalgia, and marked dyspnea 12 hr after shoveling composted wood chips and leaves. Inspiratory crackles, hypoxemia, and bilateral patchy pulmonary infiltrates were seen. Precipitating antibody tests for the usual antigens were inconclusive. He improved over 3 days. In order to assess the environmental conditions the patient had experienced, we returned to the site to reproduce and measure respiratory exposures during hand loading of the compost. Visible clouds of fine particulate were easily generated during handling activities. Microscopic examination of these dusts indicated a predominance of spores. Endotoxin concentrations from inspirable and respirable dust samples ranged from 636 to 16,300 endotoxin units/m3. Levels of contaminants found were consistent with those associated with respiratory illness in other agricultural settings. Two respiratory disorders, hypersensitivity pneumonitis (HP) and organic dust toxic syndrome (ODTS), may occur after exposure to organic dusts containing fungal spores and endotoxins. Despite extensive clinical and environmental investigations, we were unable to differentiate these two disorders, and suggest they may represent parts of a spectrum of responses to complex organic dusts, rather than completely distinct clinical entities.

Time and concentration dependence of the dicyclohexylcarbodiimide inhibition of proton movements in the cytochrome bc1 complex from yeast mitochondria reconstituted into proteoliposomes.

The cytochrome bc1 complex was isolated from yeast mitochondria solubilized with the detergent dodecyl maltoside and reconstituted into proteoliposomes to measure electrogenic proton pumping. Optimal respiratory control ratios of 4.0, obtained after addition of the uncoupler CCCP, and H+/e- ratios of 1.6 were obtained when the proteoliposomes were prepared with egg yolk phosphatidylcholine supplemented with cardiolipin. Moreover, it was critical to remove excess dodecyl maltoside in the final concentrated preparation prior to reconstitution to prevent loss of enzymatic activity. The rate of electrogenic proton pumping, the respiratory control ratios, and the H+/e- ratios were decreased by incubation of the cytochrome bc1 complex with dicyclohexylcarbodiimide (DCCD) in a time and concentration dependent manner. Maximum inhibitions were observed when 50 nmol DCCD per nmol of cytochrome b were incubated for 30 min at 12 degrees C with the intact cytochrome bc1 complex. Under these same conditions maximum labeling of cytochrome b with [14C] DCCD was reported in a previous study [Beattie et al. (1984). J. Biol. Chem. 259, 10562-10532] consistent with a role for cytochrome b in electrogenic proton movements.