Differential effects of injections of anti-mu and anti-delta monoclonal antibodies on B-cell populations in adult mice: regulation of xenoreactive natural antibody-producing cells

BACKGROUND: The depletion of differential B cell and xenoreactive natural antibodies (XNA) by anti-delta and anti-mu injections was analyzed in adult mice. Sequential treatment with anti-delta and then anti-mu induces a complete depletion of B cells and XNA and represents a potential approach to induce xenograft tolerance. METHODS: Adult mice were injected with anti-mu, anti-delta, anti-delta then anti-mu, or control isotype monoclonal antibodies from day 0 to day 14. The different B-cell populations were analyzed by FACS and immunohistology. Ig production was tested by ELISA. XNA were analyzed by FACS. RESULTS: Anti-mu injections induced a depletion of IgMhigh, immature B cells, marginal zone B cells, and B1 cells and an increase of IgG-XNA production. Anti-delta injections induced mature conventional IgDhigh B-cell depletion and increased IgM-XNA production. Interestingly, sequential injections of anti-delta then anti-mu induced a depletion of immature B cells, mature B cells (MZ, B2, and B1), and XNA. CONCLUSIONS: These results demonstrate that mature B-cell depletion in adult mice can be obtained by mAb injections and depends on the surface immunoglobulin cross-linking threshold. Indeed, anti-mu mAb depleted IgMhigh B cells (MZ and B1) and anti-delta, IgDhigh B cells (B2). The differential B-cell suppression shows that conventional B cells are responsible in the IgG-XNA production and MZ and B1 cells in the IgM-XNA production. Sequential repeated injections of anti-delta then anti-mu mAb depleted all B-cell populations and suppressed the whole XNA production.     

Two mutations identified in the androgen receptor of the new human prostate cancer cell line MDA PCa 2a

PURPOSE: We have characterized the androgen receptor (AR) in a new human prostate cancer cell line, MDA PCa 2a, that has recently been established from a bone metastasis of a patient whose cancer exhibited androgen-independent growth. MATERIALS AND METHODS: Androgen responsiveness of these cells was assessed by measuring the effect of DHT and R1881 on cell growth and PSA secretion. Scatchard analysis was used to characterize the affinity and abundance of AR protein. Using a PCR based strategy, genomic DNA of the entire coding region of AR gene was sequenced to identify possible mutations. RESULTS: These cells express abundant AR (Nmax = 685 +/- 149 fmol./mg. protein), but the AR binding affinity (Kd) for DHT is only 25 nM, approximately 50-fold lower affinity than the mutated AR in LNCaP prostate cancer cells (Kd = 0.5 nM) or the wildtype AR in MCF-7 breast cancer cells (Kd = 0.4 nM). Two mutations, L701H and T877A, were identified in the ligand binding domain of the AR gene. Compared with LNCaP cells, the new cell line is significantly less responsive to DHT and R1881 as well as to other androgens such as testosterone, androstenedione, and DHEA. Similar to LNCaP cells, the ligand specificity of the AR in MDA PCa 2a cells appears to be relaxed and non-androgens such as progesterone and estradiol act as agonists although with less potency than in LNCaP cells. Interestingly, in the absence of androgens, the new cell line expresses 15-fold higher baseline levels of PSA than LNCaP. CONCLUSIONS: Two mutations were identified in the AR gene of the MDA PCa 2a cell line that are likely responsible for the decreased androgen sensitivity and altered ligand specificity observed in these cells. Thus, this new cell line with partial androgen responsiveness and PSA expression can serve as a functionally relevant model system of bone metastatic prostate cancer, and can be used to investigate the role of AR mutations in prostate cancer and its progression to androgen independence.     

Transforming Growth Factor-β2 mRNA Level in Unloaded Bone Analyzed by Quantitative In Situ Hybridization

The effects of tail suspension hypokinesia on the gene expression for TGF-β₂ at different sites within bone were evaluated. TGF-β₂ mRNA signal levels were determined quantitatively by an image analysis system. The osteopenia induced by tail suspension was verified by histomorphometry. In the periosteum of nonsuspended control rats, TGF-β₂ mRNA was highly expressed in the preosteoblasts and osteoblast-rich cambial layers; very little signal was present within the middle and outer fibroblastic layers. Gene expression was significantly reduced in suspended rats, and this was evident both in terms of the number of silver grains in unit area or length of tissue and in each osteoblast and preosteoblast. Hypokinesia also reduced the expression of TGF-β₂ mRNA level in cortical and trabecular bone osteocytes, but did not adversely affect the mRNA level in chondrocytes in growth plate. The results affirm the site-specific response of TGF-β₂ gene expression in rats, and suggest that the cortical and trabecular bone osteopenia associated with hypokinesia in rats may be associated with a deficit in osteoblastic and osteocytic TGF-β₂ level. Received: 6 February 1998 / Accepted: 10 November 1998     

Simultaneous irradiation and imaging of blood vessels during pulsed laser delivery

BACKGROUND AND OBJECTIVE: Simultaneous irradiation and viewing of 10-120 microm cutaneous blood vessels were performed to investigate the effects of 2-micros 577-nm dye laser pulses. STUDY DESIGN/MATERIALS AND METHODS: A modified scanning laser confocal microscope recorded vessel response to different radiant exposures (J/cm2). Probit analysis determined the 50% probability ("threshold") radiant exposure necessary to cause embolized or partly occluding coagula, coagula causing complete blood flow stoppage, and hemorrhage. RESULTS: A statistically significant difference in the threshold radiant exposure existed for each damage category for blood vessels 10-30 microm in diameter, but not for larger vessels. For vessels over 60 microm, complete flow stoppage was unattainable; increasing laser pulse energy produced hemorrhage. In larger vessels, coagula often were attached to the superficial vessel wall while blood flowed underneath. Monte Carlo optical and finite difference thermal modeling confirmed experimental results. CONCLUSION: These results provide insight into the role of pulse duration and vessel diameter in the outcome of pulsed dye laser irradiation.