Antonio Giampietri

IL-9 protects mice from Gram-negative bacterial shock: suppression of TNF-alpha, IL-12, and IFN-gamma, and induction of IL-10.

IL-9 is a T cell-derived cytokine that, similar to the Th2 cytokines IL-4 and IL-10, has been implicated in the response to parasitic infections, allergy, and inflammatory processes. Because both IL-4 and IL-10 can confer protection to mice from septic shock, we investigated whether IL-9 may also be capable of conferring resistance on recipients of an otherwise lethal challenge with Pseudomonas aeruginosa. Prophylactic injections of rIL-9 appeared to be most effective in preventing the onset of a lethal shock, according to a pattern that was both dose dependent and time dependent. The protective effect of IL-9 was correlated with marked decreases in the production of the inflammatory mediators TNF-α, IL-12, and IFN-γ, as well as the induction of the anti-inflammatory cytokine IL-10. Sustained levels of IL-9-specific transcripts could be detected in the spleens of mice recovering from sublethal P. aeruginosa infection. Therefore, IL-9 may be protective in septic shock via a rather unique mechanism involving a complex modulation of inflammatory and anti-inflammatory mediators.

Drug Induced Modulation of Immune Responses in Mice: Effects of 5-(3,3-Dimethyl-1-Triazeno)-Imidazole-4-Carboxamide (DTIC) and Cyclophosphayide (CY)

Graded doses of Cyclophosphamide (Cy) or 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) were given to CD2F1 or C57Bl/6 mice. One, 45 or 60 days later the animals were tested for allograft responses, competence of producing cytotoxic lymphocytes in vitro and lethal graft-versus-host disease (GVHD) in vivo, delayed-type hypersensitivity (DTH) and humoral antibody responses against sheep red blood cells (SRBC). Both agents produced strong inhibitory effects, except for DTH, when given 1 day before the antigenic stimulus. However immunodepression lasted for at least 60 days after DTIC, whereas relatively rapid recovery of immune responsiveness was detected in mice treated with Cy. When Cy or DTIC were given to allogeneic donor mice 1 day before spleen cell transfer, immunodepressed recipients did not undergo GVHD. However when drugs were administered to recipient mice inoculated with allogeneic spleen cells, lethal GVHD occurred when Cy but not DTIC was given to the hosts. DTH responses were potentiated by Cy when the drug was given 1 day before sensitization. In contrast hypersensitivity reactions were not affected by DTIC treatment. It was concluded that DTIC is a potent and long-lasting immunodepressive agent, capable of affecting various T-cell subpopulations and possibly B lymphocytes in mice. Since the drug inhibits immune response when given before the antigenic stimulation, it was suggested that DTIC acts through a mechanism similar to that of alkylating non phase-specific agents.

Growth and rejection patterns of murine lymphoma cells antigenically altered following drug treatment in vivo.

SUMMARY Murine leukemia cells transformed by in vivo treatment with 5 - (3, 3 - dimethyl −1 - triazeno)imidazole - 4 - carboxamide (DTIC) are rejected by histocompatible recipients following inocula of 107 cells i.p. Progressive tumor growth or tumor growth and regression was monitored measuring the extent of DNA synthesis in the peritoneal cavity of mice using the [125I]5-iodo-2‘-deoxyuridine uptake method. In addition, the results were confirmed by cell count and mortality data. Comparable growth rate was found initially in both DTIC and parental lines in histocompatible hosts. Later, mice challenged with parental lines died, whereas hosts inoculated with DTIC-treated sublines rejected the tumor. On the other hand, lethal growth occurred in mice inoculated with DTIC-treated sublines when immunodepressed by cyclophosphamide given before tumor challenge, or by methotrexate given after challenge of a methotrexate-resistant DTIC-treated subline. The similarity between the growth rate of the parental and DTIC-treated lines in histocompatible hosts does not support the hypothesis of impaired “oncogenic potential” of such DTIC-treated lines. Furthermore, the growth and rejection pattern of a parental line in H-2-incompatible hosts was similar to that observed for DTIC-treated lines in histocompatible hosts, suggesting that comparable immune mechanisms were involved in both cases.

Long-term depression of two primary immune responses induced by a single dose of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC)

2 primary immune responses (anti-SRBC antibody response and allograft rejection) have been tested in mice at various time intervals after single doses of either DTIC or cyclophosphamide. The DTIC-induced immunodepression proved to be extremely long-lasting, being still detectable after 2 months.

Evidence for tumor necrosis factor α as a mediator of the toxicity of a cyclooxygenase inhibitor in Gram-negative sepsis

To investigate the effect of cyclooxygenase inhibition in experimental Gram-negative sepsis, indomethacin was administered to mice at different times (1 or 5 days, or 1 h) before sublethal infection with an intravenous inoculum of Pseudomonas aeruginosa Early indomethacin exposure did not alter the outcome of infection, yet treatment at the time of bacterial challenge resulted in a high mortality rate. Polymerase chain reaction-assisted mRNA amplification in the spleens of infected mice revealed that tumor necrosis factor alpha (TNF-alpha) messenger was selectively expressed by the drug-treated and infected mice during the 24 h preceding death. Higher TNF-alpha levels were found in sera from these mice, whose macrophages produced increased levels of nitric oxide in vitro. Both pentoxifylline, an inhibitor of TNF-alpha synthesis, and an inhibitor of nitric oxide production improved survival in the indomethacin-treated and infected mice, although no such effect followed the administration of TNF-neutralizing antibodies. These data support the notion that cyclooxygenase inhibitors may exert both positive and negative effects in Gram-negative sepsis, the latter presumably involving overproduction of TNF-alpha.

Increased immunogenicity of L1210 leukemia following short-term exposure to 5(3,3′-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) in vivo or in vitro

SummaryShort-term exposure of L1210 Ha leukemia to DTIC in vivo or in vitro resulted in the generation of leukemic cells that were moderately immunogenic for histocompatible (BALB/C × DBA/2)F1 (CD2F1) mice. In vivo treatment was carried out in the peritoneal cavity of CD2F1 host for 8–36 h. In vitro experiments were performed in glass vessels, in which tumor cells were incubated with DTIC for 2 h at 37° C. The in vitro generation of immunogenic leukemia was conditioned by the presence of mouse liver microsomes capable of producing metabolic transformation of DTIC. It follows that the increase of tumor cell immunogenicity produced in vitro and possibly in vivo by DTIC is due to (a) metabolic product (s) that has (have) not yet been identified. Somatic mutation, selection, viral activation, or other mechanisms could be responsible for the DTIC effect. The present studies suggest that similar in vivo or in vitro techniques could be used to obtain human tumor cells with higher immunogenicity.

Changes of the immunogenic properties of K36 lymphoma treated in vivo with 5(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide (DTIC)☆

Abstract Mice of AKR strain bearing syngeneic K 36 lymphoma were treated with DTIC for a number of transplant generations. The lymphoma line (K36/DTIC) became resistant to DTIC treatment and weakly immunogenic for AKR or (AKR × DBA/2)F 1 hosts. Previous findings, however, showed that DTIC-treated H-2 b or H-2 b lymphomas became DTIC-resistant as well, but acquired strong immunogenicity for histocompatible hosts. Transplantation resistance of allogeneic mice against K 36 or K36/DTIC lines injected i.p. or i.v. was also investigated. Both lines inoculated i.p. were rejected by either H- 2 -incompatible recipients, or H- 2 -compatible mice incompatible for minor histocompatibility loci (MIH). When the tumors were injected intravenously, H- 2 -compatible MIH-incompatible mice were more susceptible than H- 2 -MIH-incompatible recipients to lymphoma challenge. Moreover K36/DTIC line elicited stronger transplantation resistance than K 36 tumor. Unexpectedly H- 2 -MIH-incompatible mice homozygous for the H-2 d haplotype were partially susceptible to the i.v. challenge with K 36 lymphoma cells. However, strong transplantation resistance was found in the same hosts against K36/DTIC line. In conclusion the limited increase of tumor cell immunogenicity obtained by treatment of K 36 lymphoma with DTIC was detectable in syngeneic, hybrid and allogeneic mice.

Experimental studies of immunotoxicity of a photosensitizing agent (Photofrin II) in mice.

Immunotoxicity studies have been performed on the photosensitizing agent Photofrin II (PHFR), a porphyrin derivative used in photodynamic therapy. Hybrid CD2F1 (H-2d/H-2d) or inbred C57Bl/6 (H-2b) male mice were injected with graded doses of the agent (from 1.2 to 12 mg/Kg ip) on day -5, -3 and -1 before assays. The animals, or spleen cells collected from them on day 0 with respect to PHFR treatment, were tested for: a) competence of producing GVHD upon cell transfer into allogeneic, immunosuppressed recipients; b) graft response against challenge with allogeneic lymphoma cells; c) delayed-type hypersensitivity (DTH) against sheep red blood cells; d) in vitro response to mitogens; e) NK cell activity; f) in vitro generation of alloreactive cytotoxic T lymphocytes (CTL); g) resistance against the challenge of a sublethal dose of Pseudomonas aeruginosa. Moreover the LD50 of the drug given ip has been determined in male CD2F1 mice. The results show that PHFR, even at the highest doses used, does not affect most of the immunological parameters studied, except for a marginal inhibition of CTL generation and increment in proliferative responses to Con A or LPS. These data along with parallel studies performed by our group on human models in vitro, showing increased susceptibility of PHFR-treated tumors to NK or LAK effector cells, point out that PHFR, in the absence of systemic photoactivation, is essentially non-immunotoxic in vivo and could render tumor cells more susceptible to natural immunity.

Murine leukemia growth inhibition or enhancement following immunization with tumor cells antigenically altered by drug treatment

Summary Highly immunogenic lymphoma sublines were obtained following treatment with dimethyl-triazeno-imidazole-carboxamide (DTIC) in vivo . The hosts immune responses against parental (PL) and DTIC-treated sublines (DTS) were studied in mice presensitized with irradiated PL or DTS and challenged intraperitoneally with viable lymphoma cells. Tumor growth was monitored by evaluation of the uptake of 125 I-Iodo-deoxy-uridine ( 125 IUdR) in the peritoneal cavity of mice and by survival of recipient hosts. The results of the experiments showed that growth inhibition of PL occurred in mice sensitized with irradiated PL or DTS. On the other hand tumor inhibition or enhancement was observed in mice sensitized with irradiated DTS and challenged with viable cells of the DTIC-treated lymphomas.

In vivo treatment with recombinant interleukin-2 (IL-2) stimulates the differentiation of natural killer (NK) precursor cells

One of the most interesting problems in the field of natural immunity is represented by those regulatory mechanisms possibly involved in determining spontaneous reactivity [1, 2]. Natural killer (NK) cells have been shown to be influenced in their levels of reactivity by soluble factors such as prostaglandins and a number of lymphokines [3–8]. Interferon and interferon inducers have also been shown to be able to augment the spontaneous levels of NK activity both in vivo and in vitro in different species, including mouse and man. In addition, macrophage-like, null cell-like or T-cell-like suppressor cells have been shown to influence the levels of NK activity [9, 10].