Regulation of Fas ligand-induced apoptosis by TNF.

Fas ligand (FasL, CD95L) expression helps control inflammatory reactions in immune privileged sites such as the eye. Cellular activation is normally required to render lymphoid cells sensitive to FasL-induced death; however, both activated and freshly isolated Fas(+) lymphoid cells are efficiently killed in the eye. Thus, we examined factors that might regulate cell death in the eye. TNF levels rapidly increased in the eye after the injection of lymphoid cells, and these cells underwent apoptosis within 24 h. Coinjection of anti-TNF Ab with the lymphoid cells blocked this cell death. Furthermore, TNFR2(-/-) T cells did not undergo apoptosis in the eyes of normal mice, while normal and TNFR1(-/-) T cells were killed by apoptosis. In vitro, TNF enhanced the Fas-mediated apoptosis of unactivated T cells through decreased intracellular levels of FLIP and increased production of the pro-apoptotic molecule Bax. This effect was mediated through the TNFR2 receptor. In vivo, intracameral injection of normal or TNFR1(-/-) 2,4,6-trinitrophenyl-coupled T cells into normal mice induced immune deviation, but TNFR2(-/-) 2,4,6-trinitrophenyl-coupled T cells were ineffective. Collectively, our results provide evidence of a role for the p75 TNFR in cell death in that TNF signaling through TNFR2 sensitizes lymphoid cells for Fas-mediated apoptosis. We conclude that there is complicity between apoptosis and elements of the inflammatory response in controlling lymphocyte function in immune privileged sites.     

Terminal regions of mouse nebulin: sequence analysis and complementary localization with N-RAP

The regions of mouse nebulin extending from the ends of the super repeats to the C-terminus and N-terminus were cloned and sequenced. Comparison of the mouse sequence with the previously published human sequence shows that the terminal regions of nebulin are highly conserved. The four phosphorylation motifs and SH3 domain found at the C-terminus of mouse nebulin are identical to those found in human nebulin, with the exception of four conservative substitutions. The modules linking this C-terminal region to the super repeats have deletions relative to both fetal and adult human nebulins that correspond to integral numbers of modules, making the mouse C-terminal simple repeat region among the shortest observed to date. The N-terminal region and the C-terminal modules were expressed in Escherichia coli and used for antibody production. Immunofluorescent labeling of these regions of nebulin in isolated myofibrils demonstrates that they are located near the center of the sarcomere and near the Z-line, respectively. Immunogold labeling with antibodies raised against the N-terminal nebulin sequence localizes this region in the A-band near the tips of the thin filaments. Nebulin localization is complementary to that of N-RAP, another muscle-specific protein containing nebulin-like super repeats; nebulin is exclusively found in the sarcomeres, while N-RAP is confined to the terminal bundles of actin filaments at the myotendinous junction. Cell Motil. Cytoskeleton 3:211-222, 2000 Published 2000 Wiley-Liss, Inc.     

Cutting edge: restoration of the ability to generate CTL in mice immune to adenovirus by delivery of virus in a collagen-based matrix

Viruses are commonly used for the delivery of genes coding for tumor-associated Ags to elicit tumor-specific immune responses. The success of viral vectors has been limited in preclinical and clinical trials in part because of antiviral immunity. We investigated the ability of a collagen-based matrix (Gelfoam; Pharmacia and Upjohn, Kalamazoo, MI) to improve CTL activation by recombinant adenovirus. The data show that coinjection of Gelfoam with type 5 adenovirus recombinant for prostate-specific Ag (Ad5-PSA) enhanced CTL activation. Ad5-PSA priming in Gelfoam also abrogated the inhibitory effects of adenoviral immunity on CTL activation in mice naive to PSA but immune to adenovirus. Finally, Gelfoam enhanced immunization in a self-Ag model using type 5 adenovirus recombinant for membrane-bound OVA (Ad5-mOVA) in rat insulin promoter (RIP)-mOVA-transgenic mice. Thus, Gelfoam enhances CTL activation by recombinant viral vectors in a setting where preformed Ab to the virus is present and also in a tolerant self-Ag model.     

The emerging role of platelets in adaptive immunity

Platelets' foremost role in survival is hemostasis. However, a significant quantity of research has demonstrated that platelets are an integral part of inflammation and can also be potent effector cells of the innate immune response. CD154, a molecule of vital importance to adaptive immune responses, is expressed by activated platelets and has been implicated in platelet-mediated modulation of innate immunity and inflammatory disease states. Recent studies in mice extend the role of platelet CD154 to the adaptive immune response demonstrating that platelets can enhance antigen presentation, improve CD8 T cell responses, and play a critical function in normal T-dependent humoral immunity. The latter studies suggest that the current paradigm for the B cell germinal center response should be modified to include a role for platelets.     

Using the composite variables of reproductive morphology, histology and steroid hormones to determine age and size at sexual maturity for the thorny skate Amblyraja radiata in the western Gulf of Maine

Age at size was linked to sexual maturity in 77 male and 93 female thorny skates Amblyraja radiata from the western Gulf of Maine using three criteria: 1) gross reproductive morphology, 2) histology and 3) steroid hormone concentrations. Age-bias plots and the coefficient of variation from vertebral band counts suggested that the ageing method represented a non-biased and precise approach to the age assessment of A. radiata . Maturity ogives for males, based on data gathered for clasper length, circulating testosterone concentrations and proportion of mature spermatocysts within the testes, predicted that 50% maturity occurred at a total length ( L _T) of 865 mm and c. 10·90 years of age. For females, maturity ogives, based on data gathered for ovary mass, shell gland mass, follicle size and circulating oestradiol concentrations, predicted that 50% maturity occurred at 875 mm L _T and c. 11·00 years of age. Collectively, the results suggest that analysis of several contemporaneous reproductive variables offers an accurate determination of sexual maturity in the thorny skate.     

The fibronectin attachment protein of bacillus Calmette-Guerin (BCG) mediates antitumor activity

Purpose

The receptor responsible for the attachment of bacillus Calmette-Guerin (BCG) to fibronectin, fibronectin attachment protein (FAP), has been cloned. Studies targeting FAP as an inducer of immunity in mycobacterial infections suggest that FAP is a highly immunogenic protein. In light of these findings and the need to find effective alternatives to BCG treatment for bladder cancer, we tested the ability of FAP to induce antitumor activity.

Materials and methods

The ability of FAP to bind to bladder tumor cells and the bladder wall was established using ¹²⁵I-FAP. For testing antitumor activity in vivo, mice were catheterized and 5 × 10⁴ MB-49 bladder tumor cells were implanted orthotopically on day 0. Test groups were treated with PBS only, FAP, or BCG on day 1 and day 8. A subset of mice was preimmunized with FAP prior to treatment.

Results

FAP was observed to bind to bladder tumor cells in a fibronectin-dependent manner. Attachment of FAP within the bladder followed the pattern established for BCG binding. Antitumor studies showed a significant reduction in tumor growth in FAP-treated mice that had been preimmunized with FAP. Tumor growth was not inhibited in naïve mice treated with FAP. Dose-response studies showed that FAP-induced antitumor activity is dose dependent, and experiments comparing BCG with FAP showed equivalent antitumor effects. In vitro experiments showed antigen-specific lymphocyte proliferation and a cytokine profile indicative of Th-1 polarization of the FAP-induced immune response. CD8+ T cells and natural killer cells were found to be required for the FAP-induced antitumor response.

Conclusions

FAP is an effective antitumor agent that inhibits tumor growth at a level equivalent to that observed for BCG. This protein may thus provide an alternative to BCG for treatment of superficial bladder cancer.     

低温对植物叶片中超氧物歧化酶、过氧化氢酶和过氧化氢水平的影响

番茄和鸡蛋果叶片中可提取的SOD活性不受低温的影响。在电泳谱带上SOD主同工酶带被氰化物而不被低温抑制,次同工酶带在低温下不稳定,且活性很低,它的变化不影响总的SOD活性。一些冷敏感植物叶片中CAT活性被低温抑制,而H_2O_3水平在低温下稳定或有增加,这可能使毒性更强的羟基离子(OH·)易于形成。