Required early complement activation in contact sensitivity with generation of local C5-dependent chemotactic activity, and late T cell interferon gamma: a possible initiating role of B cells

Complement (C) is an important component of innate immunity, and was also shown recently to participate in induction of acquired B cell humoral immunity. In this study, we present evidence that C also participates in acquired T cell immunity. We found that C was involved in early events of the efferent elicitation phase of contact sensitivity (CS), and delayed-type hypersensitivity (DTH). Thus, CS and DTH were inhibited by administration of a C-blocker, soluble recombinant C receptor-1 (sCR1), when given 30 min before, but not 3 h after local antigen challenge. Among C components, local C5 were thought crucial to elicitation of CS, since local administration of anti-C5 monoclonal antibodies or locally injected C-depleting cobra venom factor also inhibited CS and DTH. These findings were consistent with our previous finding of the importance of C5 for CS elicitation, using congenitally C5-deficient mice. To dissect the mechanism of C dependence in CS, we demonstrated that locally increased early macrophage chemotactic activity (probably C5a) in evolving CS skin extracts, as well as late elaboration of IFN-gamma, were both inhibited by anti-C treatment. In addition, histological analysis showed that leukocyte recruitment into CS ear sites was similarly C-dependent. Furthermore, an initiating role of B cell-derived C-fixing immunoglobulin was suggested by demonstration of impaired CS responses in B cell-deficient mice. In summary, these results suggest that C was activated locally, perhaps via a B cell product, in an important early component of the stepwise events necessary to elicit CS, leading to local production of C5-dependent macrophage chemotactic activity and later IFN-gamma, and subsequently leading to cell infiltration, for development of T cell-dependent CS.     

Lower extremity salvage after radical resection of malignant tumors in the groin and lower abdominal wall

The development of primary human brain tumors, particularly glioblastoma multiforme (GBM), has been associated with a number of molecular and chromosomal abnormalities. In this study, a novel tumor suppressor locus was identified and localized after the transfer of a human chromosome 4 into U251 human GBM cells. Hybrid clones containing a transferred neomycin-resistance tagged chromosome 4 revealed an inability to form tumors in nude mice and a greatly decreased efficiency of soft agarose colony formation. As a control, clones containing a transferred chromosome 2 were generated, and these retained the tumorigenic phenotype of the parental U251 cells. The presence of the transferred chromosomes was demonstrated by gain of polymorphic loci and FISH analyses. Several suppressed hybrid clones were shown to contain spontaneously reduced versions of the transferred chromosome 4. A common region of the fragmented chromosome 4 was retained among these clones that included the epidermal growth factor locus at 4q24-26 and several adjacent markers. The identification of a common fragment in the suppressed clones suggests the presence of a tumor suppressor gene or genes in this region, involved in glioma oncogenesis.     

Gastrin-releasing peptide1-27, unlike bombesin, does not reduce sham feeding in rats

We compared the potencies of systemic administration of bombesin (BN) and its mammalian homologue gastrin-releasing peptide (GRP) to decrease sham feeding in rats. Bombesin (at doses of 8, 16 and 32 micrograms/kg, intraperitoneally) inhibited sham feeding by 37% (p < 0.001), 58% (p < 0.001) and 65% (p < 0.001), respectively, confirming previous results. Gastrin-releasing peptide (16, 32, and 64 micrograms/kg) failed to affect sham feeding. Bombesin (16 micrograms/kg) and gastrin-releasing peptide (32 micrograms/kg) inhibited real feeding by 64% (p < 0.001) and 44% (p < 0.004), respectively. Pregastric food stimulation is not sufficient for the inhibitory action of GRP.     

Defibrillation efficacy with endocardial electrodes is influenced by reductions in cardiac preload

Several studies were undertaken to develop three-dimensional (3-D) cell culture models that allow conditions closer to the in vivo situation. To this end, alginate gels were tested as a 3-D cell culture model that might be useful in the study of the effects of UVA on human dermal fibroblasts. Cell culture in alginate gels and the irradiation conditions were optimized. Results showed that optimized cultures in alginate gels experienced considerable cell death on UVA irradiation compared to the classical monolayer cell culture. Viability tests (cell counting and neutral red assay) were performed to show that only UVA-irradiated alginate gels were responsible for this cytotoxicity. The implication of oxygen species in the phototoxicity induced by ultraviolet light has already been described; for this reason we investigated whether oxygen species were involved in the cytotoxicity induced by alginate upon UVA irradiation. It appeared that superoxide anion is not implicated.     

Cell cycle analysis and synchronization of pluripotent hematopoietic progenitor stem cells

Hematopoietic stem cells purified from mouse bone marrow are quiescent with less than 2% of Lin- Hoechst(low)/Rhodamine(low) (Lin- Ho(low)/Rho(low)) and 10% to 15% of Lin-/Sca+ cells in S phase. These cells enter proliferative cycle and progress through G1 and into S phase in the presence of cytokines and 5% heat-inactivated fetal calf serum (HI-FCS). Cytokine-stimulated Lin- Ho(low)/Rho(low) cells took 36 to 40 hours to complete first division and only 12 hours to complete each of 5 subsequent divisions. These cells require 16 to 18 hours to transit through G0/G1 period and 28 to 30 hours to enter into mid-S phase during the first cycle. Up to 56% of Lin- Rho(low)/Ho(low) cells are high-proliferative potential (7 factor-responsive) colony-forming cells (HPP-CFC). At isolation, HPP-CFC are quiescent, but after 28 to 30 hours of culture, greater than 60% are in S phase. Isoleucine-deprivation of Lin- Ho(low)/Rho(low) cells in S phase of first cycle reversibly blocked them from entering into second cycle. After the release from isoleucine-block, these cells exhibited a G1 period of less than 2 hours and entered into mid-S phase by 12 hours. Thus, the duration of G1 phase of the cells in second cycle is 4 to 5 times shorter than that observed in their first cycle. Similar cell cycle kinetics are observed with Lin-/Sca+ population of bone marrow cells. Stem cell factor (SCF) alone, in the presence of HI-FCS, is as effective as a cocktail of 2 to 7 cytokines in inducing quiescent Lin-/Sca+ cells to enter into proliferative cycle. Aphidicolin treatment reversibly blocked cytokine-stimulated Lin-/Sca+ cells at G1/S boundary, allowing their tight synchrony as they progress through first S phase and enter into second G1. For these cells also, SCF alone is sufficient for their progression through S phase. These studies indicate a very short G1 phase for stem cells induced to proliferate and offer experimental approaches to synchronize murine hematopoietic stem cells.