Impact of oral ibandronate 150 mg once monthly on bone structure and density in post-menopausal osteoporosis or osteopenia derived from in vivo μCT

The effect of ibandronate 150 mg/once monthly in the treatment of post-menopausal osteopenia and osteoporosis on bone micro-structure at the distal tibia and radius has not been considered to date. Seventy post-menopausal women with osteoporosis or osteopenia were recruited. All subjects received calcium and vitamin D supplementation and were randomized to either a group which took 150 mg ibandronate oral monthly or a placebo group over a 12-month period. μCT measures of the distal tibia and radius were conducted every three months, with DXA lumbar spine and hip measurements conducted only pre and post and serum markers of bone formation and resorption measured every 6 months. After 12-months no significant impact of ibandronate on the primary outcome measures bone-volume to tissue-volume and trabecular separation at the distal tibia (p ≥ 0.15) was found. Further multiple regression analyses of the primary end-points indicated a significant effect favoring the ibandronate intervention (p = 0.045). Analysis of secondary end-points showed greater increases in distal tibia cortical thickness, cortical density and total density (p ≤ 0.043) with ibandronate and no significant effects at the distal radius, but greater increases of hip DXA-BMD and lumbar spine DXA-BMD (p ≤ 0.017). Ibandronate use resulted in a marked reduction in bone turnover (p < 0.001). While ibandronate resulted in greater mineralization of bone, this effect differed from one body region to another. There was some impact of ibandronate on bone structure (cortical thickness) at the distal tibia, but not on bone-volume to tissue-volume or trabecular separation.     

Proteinase 3–dependent caspase-3 cleavage modulates neutrophil death and inflammation

Caspase-3–mediated spontaneous death in neutrophils is a prototype of programmed cell death and is critical for modulating physiopathological inflammatory responses; however, the underlying regulatory pathways remain ill defined. Here we determined that in aging neutrophils, the cleavage and activation of caspase-3 is independent of the canonical caspase-8– or caspase-9–mediated pathway. Instead, caspase-3 activation was mediated by serine protease proteinase 3 (PR3), which is present in the cytosol of aging neutrophils. Specifically, PR3 cleaved procaspase-3 at a site upstream of the canonical caspase-9 cleavage site. In mature neutrophils, PR3 was sequestered in granules and released during aging via lysosomal membrane permeabilization (LMP), leading to procaspase-3 cleavage and apoptosis. Pharmacological inhibition or knockdown of PR3 delayed neutrophil death in vitro and consistently delayed neutrophil death and augmented neutrophil accumulation at sites of inflammation in a murine model of peritonitis. Adoptive transfer of both WT and PR3-deficient neutrophils revealed that the delayed death of neutrophils lacking PR3 is due to an altered intrinsic apoptosis/survival pathway, rather than the inflammatory microenvironment. The presence of the suicide protease inhibitor SERPINB1 counterbalanced the protease activity of PR3 in aging neutrophils, and deletion of Serpinb1 accelerated neutrophil death. Taken together, our results reveal that PR3-mediated caspase-3 activation controls neutrophil spontaneous death.     

Use of Flow Cytometric CD34 Analysis to Quantify Hematopoietic Progenitor Cells

This review summarizes our experiments on flow cytometric analysis of CD34 positive mono-nuclear cells (MNC) and on colony formation of myeloid hematopoietic progenitor cells in the clonogenic assay. We examined MNC isolated by density centrifugation of bone marrow, cord blood and peripheral blood. The latter samples originated either from patients recovering from myelosuppressive treatment who received no growth factors or from patients treated with G-CSF or GM-CSF. We attempted to correlate the results obtained by CD34 analysis with the cloning efficiency determined after a 14 day culture period in the methylcellulose-based clonogenic assay. The highest cloning efficacy (60%-100%) was observed in cord blood, however, a good correlation was found in both untreated and GM-CSF treated peripheral blood samples in which a mean of 50% and 20% of the number of CD34 positive MNC gave rise to myeloid colonies. In bone marrow, the cloning efficacy was generally lower and ranged between 5% and 15%. The lowest values were observed in G-CSF treated peripheral blood in which colonies were grown from only l%-9% of the CD34⁺ MNC. Due to the variable numbers of CD34⁺ lymphoid and/or more committed myeloid precursors which form either no colonies or only clusters, there was a greater variation and a lower cloning efficiency in the latter two cell sources. In conclusion, one colour CD34 analysis of cord blood MNC and untreated or GM-CSF treated peripheral blood MNC provides reliable results with respect to the content of myeloid progenitors. Analysis of bone marrow MNC and G-CSF treated peripheral blood MNC requires two colour staining using CD34 and CD45RA. Exclusion of the CD34⁺/CD45RA⁺⁺ cell fraction will improve the correlation and give results similar to those obtained with untreated blood MNC.     

Experimental external irradiation of corneal neovascularization

The clinical effect of ionidizing radiation on ocular neovascularizations is controversial not only because of the variety of treatment modalities. The aim of our study was to investigate an experimental model which allows to evaluate radiation parameters and to study the mechanism of the inhibitory effect on neoangiogenesis. METHODS: Corneal angiogenesis was induced by use of a micropocket assay in NZW rabbits. Pellets with 500 ng bFGF in 2% methylecellulose were implanted into the stroma 2.0 mm from the limbus. Initiation of vessel growth occurred on day 3. At this time radiation was performed with different doses (single dose of 15 to 30 Gy or fractionated 5 x 5 Gy) using a 6 MeV linear accelerator. Vascular growth was quantified. RESULTS: Irradiation with a total dose of 25 Gy applied in a fractionated regimen or as single-dose irradiation on the day of surgery or on day 6 after surgery did not significantly reduce neovascular growth. In contrast, postoperative radiation therapy on day 3 was able to reduce the area of ingrowing vessels significantly (P < 0.01). In spite of the relatively high dose there were no significant side effects during the observation period of 8 weeks. CONCLUSION: Our results show that single-dose radiation (> or = 25 Gy) is sufficient to inhibit the growth of corneal neovascularizations. With this model it might be possible to investigate parameters for therapy of ocular neovascularizations as well as the underlying mechanisms.