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P Puccetti

Publications and source records attributed to P Puccetti.

At least 37 records · Page 2Linked to original sources
PubMed1997

Circulating levels of IL-10 are critically related to growth and rejection patterns of murine mastocytoma cells.

Previously tumorigenic P815 tumor cells are rejected by histocompatible mice after transfection with a mutated retroviral gene, and the host is made resistant to subsequent challenge with tumorigenic (control) cells transfected with the nonmutated sequence. To functionally characterize the class I-restricted response to the tumor cell vaccine, we have assessed the in vitro (by CD8+ cells) and in vivo production of type 1 or type 2 cytokines in mice injected with either type of transfected P815 derivative. IL-12 and IL-10 were selectively or preferentially expressed by the regressor mice, and this correlated with the detection of functional type 1 reactivity in vivo (i.e., delayed-type hypersensitivity). Other cytokines were produced by the regressor mice only in vitro (IFN-gamma) or were not detected at all with either type of tumor recipient (IL-4). By means of monoclonal antibody-mediated neutralization or enhancement of endogenous cytokine levels, IL-10 was found to serve an important role in the growth and rejection patterns of the transfected P815 derivatives. In addition to previous evidence for an IL-12 requirement in promoting anti-P815 reactivity, these data establish IL-10 as an important cytokine in permitting optimal expression of this reactivity, which apparently develops in the absence of a strong bias toward a type 1 or type 2 cytokine response.

Animals↗
PubMed1997

Neutrophil production of IL-12 and IL-10 in candidiasis and efficacy of IL-12 therapy in neutropenic mice.

Neutrophil defects predispose to severe fungal infections, yet the immunomodulatory role of these cells is poorly defined. The contribution of neutrophils to the early cytokine balance governing Th1 and Th2 cell development was examined in mice with candidiasis. Neutrophils secreted IL-12 and IL-10, correlating with the respective development of self-limiting (Th1-associated) and progressive (Th2-associated) disease. Exogenous IL-12 was effective in protecting neutropenic hosts susceptible to infection. These results suggest that 1) neutrophils, via their ability to release cytokines, play an active role in determining the qualitative development of the T cell response, and 2) their early role in anticandidal immunity can be replaced by exogenous IL-12.

Animals↗
PubMed1997

A tumor-associated and self antigen peptide presented by dendritic cells may induce T cell anergy in vivo, but IL-12 can prevent or revert the anergic state.

Ag-specific CD8+ cell responses, including delayed-type hypersensitivity in vivo and IFN-gamma production in vitro, are initiated by host immunization with P815AB, a self peptide bearing CTL epitopes and expressed by murine mastocytoma cells. Using P815AB-pulsed dendritic cells (DC) and monitoring class I-restricted skin test reactivity in DC-primed mice, we have previously shown that the development of a Th1-like response to P815AB requires T helper effects, such as those mediated by coimmunization with class II-restricted (helper) peptides or by the use of rIL-12. The adjuvanticity of IL-12 was suggested to involve improved recognition of class II-restricted epitopes of P815AB. In the present study, we provide evidence for the occurrence of I-A(d)-restricted epitopes in the tumor peptide. We also show that in the absence of helper peptide or rIL-12, P81 5AB not only failed to initiate CD8+ cell responses in vivo and in vitro, but resulted in a transient state of functional unresponsiveness, characterized by a profound inability of CD4+ cells to produce IL-2 in vitro. Ag-specific T cell anergy was also observed after neutralization of endogenous IL-12 at the time of priming with P815AB plus helper peptide. All of these effects were reversed by rIL-12, which was added to DC cultures and administered to the DC-recipient mice. Anergy induction may thus contribute to P81 5AB unresponsiveness in vivo. IL-12 may act to prevent or revert anergy to this tumor-associated and self peptide.

Animals↗
PubMed1997

An immunoregulatory role for neutrophils in CD4+ T helper subset selection in mice with candidiasis.

Granulocytes may serve immunoregulatory and effector roles in different limbs of the immune response to infection. Using live vaccine strain or virulent challenge in mucosal or systemic infection of mice with Candida albicans, we examined the effect of mAb-mediated depletion of neutrophils on the course of primary and secondary challenge and on development of CD4+ cell-dependent immunity. We obtained evidence of deleterious effects of neutrophil depletion occurring at the time of infection under all conditions of testing, both in naive and in previously immunized mice. In contrast, neutrophil depletion appeared to benefit the hosts late in the course of an overwhelming systemic infection. In an attempt to correlate neutrophil function with the nature of the T cell response, we tested the ability of neutrophils to produce cytokines associated with functionally distinct CD4+ Th cell responses to Candida. We found that neutrophils were endowed with the capacity to secrete IL-12 and IL-10 in vitro in response to the yeast. Neutrophil ablation early in the course of Th1-associated, self-limiting infection appeared to change the qualitative development of the T cell response, and rendered mice susceptible to infection. In addition to long recognized contributions to acute anti-candidal responses, these data suggest an important role for neutrophils both in initiation and in expression of Candida-specific immunity.

Animals↗
PubMed1997

Interleukin-12 in infectious diseases.

Interleukin-12 (IL-12) is a potent immunoregulatory cytokine that is crucially involved in a wide range of infectious diseases. In several experimental models of bacterial, parasitic, viral, and fungal infection, endogenous IL-12 is required for early control of infection and for generation and perhaps maintenance of acquired protective immunity, directed by T helper type 1 (Th1) cells and mediated by phagocytes. Although the relative roles of IL-12 and gamma interferon in Th1-cell priming may be to a significant extent pathogen dependent, common to most infections is that IL-12 regulates the magnitude of the gamma interferon response at the initiation of infection, thus potentiating natural resistance, favoring Th1-cell development; and inhibiting Th2 responses. Treatment of animals with IL-12, either alone or as a vaccine adjuvant, has been shown to prevent disease by many of the same infectious agents, by stimulating innate resistance or promoting specific reactivity. Although IL-12 may enhance protective memory responses in vaccination or in combination with antimicrobial chemotherapy, it is yet unclear whether exogenous IL-12 can alter established responses in humans. Continued investigation into the possible application of IL-12 therapy to human infections is warranted by the role of the cytokine in inflammation, immunopathology, and autoimmunity.

Animals↗
PubMed1997

Initiation of T-helper cell immunity to Candida albicans by IL-12: the role of neutrophils.

The Th1/Th2 paradigm of acquired immunity is proving essential for a better understanding of immunoregulation in candidiasis and perhaps other fungal infections, conditions that may be life-threatening in humans and difficult to control by chemotherapy alone, especially in neutropenic and severely immunocompromised patients. In its basic conception applied to Candida infection in mice, this paradigm calls for: (i) an association between Th1 responses and the onset/maintenance of phagocyte-dependent immunity, critical for opposing infectivity of the commensal, focusing an infection, and clearing the yeast from infected tissue; (ii) the ability of the yeast to activate Th2 response as an evasive strategy; (iii) the reciprocal regulation of Th1 and Th2 responses, resulting in a dynamic balance between these two types of reactivity. This balance, in concert with a variety of environmental factors, may regulate the status of the yeast as a commensal or pathogen in the mucosal tissue of colonized humans, but may also determine the outcome of deep-seated systemic infections once hematogenous dissemination of the yeast has occurred. An important corollary of this hypothesis may be the possible combined effects on Th immunity of Candida carriage/infection and various disease states. While immune deficiency or dysregulation, resulting in an altered cytokine balance as may occur in AIDS, can reasonably be expected to increase local infectivity of the yeast, it is even more intriguing that antifungal chemotherapy will resolve some of the unusual skin (atopic dermatitis-like) disorders frequently observed in this clinical setting, patients with AIDS. Besides, an immunopathologic role for Candida has been suggested for atopic dermatitis, atopy, and other conditions, overtly associated or not with Candida. Thus, the Th cell dichotomy to Candida may have important implications not only for regulation of the balance between commensalism and infection, but may also contribute to the onset or dominance of Th2 responses in other disease states. A similar example, although with different effects, may be provided by the temporary improvement seen in atopic dermatitis patients in concomitance with acute severe infections, an effect that has been proposed to result from transient down-regulation of the predominant Th2 cell reactivity. With a view to either controlling Candida infections or opposing Candida-related immunopathology, the promotion of yeast-specific Th1 responses appears to be a promising immunotherapeutic approach. This, in principle, could be achieved by subtraction of Th2 cytokines or by administration of Th1-promoting cytokines. However, our initial studies with exogenous IL-12 were unsuccessful, suggesting that the recombinant cytokine: (i) is unable to oppose Th2 differentiation driven in vivo by IL-4/IL-10; (ii) may induce endogenous IL-10 production as a regulatory response, and (iii) may potentate local inflammatory responses in gastrointestinal infection or even trigger IFN-gamma-dependent mechanisms of fungal septic shock.. More recent studies seem to provide encouraging results, at least under specific conditions of testing. In acute candidemia, neutrophils appear to be a major source of the directive cytokines, IL-12 and IL-10, thus contributing to the selection of Th1 and Th2 cell responses to LVS or virulent infection, respectively. Neutrophils may also be an important source of IL-10 released in response to challenge with exogenous IL-12. As a result, the Th1-promoting role of IL-12 may be largely unopposed (by IL-10 induction) in neutropenic mice, which would otherwise succumb to LVS challenge. These animals are, in fact, cured by replacement therapy with IL-12 and acquire durable, Th1-associated anticandidal protection. These findings may be very important for immunotherapy of fungal infections in humans. Neutropenic patients are those at the highest risk for developing systemic candidal infections. (ABSTRACT TRUNCATED)

Animals↗
PubMed1997

Hydroxyurea induces the gene expression and synthesis of proinflammatory cytokines in vivo.

The anticancer agent hydroxyurea (HU) was previously found to cause dose-dependent adrenal activation in the rat. The increased secretion of corticosterone (CORT) that results appeared to protect animals against HU toxicity, which was dramatically enhanced in adrenalectomized (ADX) rats. Similarities with the endocrine and toxicological profiles of proinflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor (TNF) led us to suggest that these effects of HU might be mediated by an increased synthesis of these cytokines. The goal of this study was therefore to demonstrate that HU induces the gene expression and synthesis of proinflammatory cytokines in vivo. Intact and ADX rats were treated with HU, mRNA was extracted from spleen cells 2 and 24 hr after treatment and message levels for IL-1 alpha, IL-2, IL-4, IL-6, TNF alpha and interferon-gamma were evaluated using the reverse transcriptase-polymerase chain reaction technique. In some experiments, circulating levels of CORT and TNF were also measured. We found that transcripts of the proinflammatory cytokines, TNF, IL-6 and (though less clearly) IL-1 alpha, were expressed in the majority of intact rats treated with HU but were absent or less evident in most controls. In contrast, gene expression of IL-2, IL-4 and interferon-gamma was not influenced by drug treatment. Adrenalectomy markedly enhanced the effects of HU. Twenty-four hours after administration of the drug, the expression of TNF and IL-6 mRNAs was still higher in ADX rats compared with intact animals. Parallel measurements of plasma CORT levels revealed that gene expression of IL-1 alpha and, to a lesser extent, TNF was inversely related to levels of circulating CORT. Adrenalectomy per se caused a significant increase in plasma TNF levels compared with intact controls. Hydroxyurea elicited significant increases in circulating TNF in both ADX and intact rats. These findings lend support to our working hypothesis and provide an explanation for both the rise in glucocorticoid secretion induced by HU in intact rats and the increase in lethality observed in animals with disruptions of the hypothalamo-pituitary-adrenal axis.

Adrenalectomy↗
PubMed1996

IL-12 is both required and sufficient for initiating T cell reactivity to a class I-restricted tumor peptide (P815AB) following transfer of P815AB-pulsed dendritic cells.

Delayed-type hypersensitivity responses, mediated by CD8+ cells and detected by skin test assay, occur in sensitized mice in response to challenge with a class I-restricted synthetic peptide related to a poorly immunogenic tumor rejection Ag, P815AB, of murine mastocytoma cells. Efficient priming for this response, which requires functional CD4+ cells and production of IFN-gamma in the host, is achieved by transfer of dendritic cells (DC) pulsed in vitro with a physical mixture of P815AB and T helper peptides, such as a class II-restricted synthetic peptide of tetanus toxin. We now show that the adjuvant effect of the T helper peptide was associated with the appearance of early and late IL-12 transcripts in the spleens of DC recipient mice, correlated with a late IFN-gamma response, and was negated by serologic ablation of endogenous IL-12 at the time of cell transfer. rIL-12, administered in vivo to the DC recipient mice, could substitute for the T helper peptide in initiating skin test reactivity following transfer of DC pulsed with P815AB alone, leading to Ag-specific production of IFN-gamma by CD4+ and CD8+ T cells. In vitro and in vivo cell depletion experiments suggested the following: 1) the exogenous IL-12 required both CD4+ and CD8+ cells for activity; 2) the immune response initiated by IL-12 relied on later production of IL-12 by the host; and 3) the early adjuvanticity of the exogenous IL-12 involved improved recognition of class II-restricted epitopes of this otherwise poorly immunogenic tumor peptide.

Adjuvants, Immunologic↗
PubMed1996

Evidence for tumor necrosis factor alpha 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.

Animals↗
PubMed1996

Impaired neutrophil response and CD4+ T helper cell 1 development in interleukin 6-deficient mice infected with Candida albicans.

To define the role of interleukin (IL)6 in Candida albicans infection, IL-6 deficient mice were assessed for susceptibility to systemic or gastrointestinal infection, as well as for parameters of elicited T helper cell (Th) immunity. IL-6-deficient mice were more susceptible than wild-type mice to either type of infection caused by virulent C. albicans. In response to systemic challenge with a live vaccine strain of yeast, IL-6-deficient mice failed to mount Th1-associated protective immunity, but the resulting Th2-biased response could be redirected to the Th1 phenotype by IL-10 neutralization. Severe impairment of the macrophage and neutrophil response to infection was observed in IL-6-deficient mice, but administration of IL-6 would increase both neutrophil response and resistance to infection. IL-6 seems to oppose the Th2-promoting role of IL-10 in candidiasis, its early regulatory activity involving effects on neutrophil function.

Animals↗
PubMed1996

A retroviral peptide encoded by mutated env p15E gene is recognized by specific CD8+ T lymphocytes on drug-treated murine mastocytoma P815.

Highly immunogenic ("xenogenized") tumour variants appear after treatment of murine mastocytoma P815 with the triazene derivative DTIC, a phenomenon associated with the appearance of structurally abnormal p15E env proteins in the variant cells. In the present study, we have isolated and sequenced several p15E cDNA gene fragments amplified by means of polymerase chain reaction (PCR) from parental (P815) and xenogenized (P815/DTIC) tumour cells. Compared to known p15E sequences in parental cells, one p15E sequence from xenogenized cells presented three distinct nucleotide changes, one of which was apparently unique to P815/DTIC DNA and cDNA upon single-nucleotide primer extension assay. One major histocompatibility complex (MHC) class I-binding peptide, corresponding to a putative mutation in the p15E sequence, was tested in parallel with the parental peptide for recognition by P815/DTIC-specific cytotoxic T cells in vitro. The results suggested that the amino acid substitution at the relevant position of the p15E protein may produce an antigenic T cell epitope. By skin test assay of mice primed with either the synthetic peptide or P815/DTIC cells, evidence was obtained that the mutated peptide is immunogenic in vivo, and that the neoepitope is expressed by P815/DTIC cells. In accordance with previous data in the L5178Y/DTIC tumour model system, these findings reinforce the notion that xenogenization of tumour cells may result in the expression of class I-binding mutated peptides of retroviral origin.

Amino Acid Sequence↗
PubMed1995

TGF-beta is important in determining the in vivo patterns of susceptibility or resistance in mice infected with Candida albicans.

Resistance and susceptibility of mice to systemic infection with the fungus Candida albicans are associated with the preferential expansion of Th1 and Th2 cells, respectively. In this study, endogenous production of TGF-beta was found to be increased soon after infection of healer mice with a live vaccine strain of the fungus, but down-regulated in nonhealer mice with virulent yeast challenge. Although not affecting the outcome of primary challenge, serologic ablation of TGF-beta in the former animals abrogated development of acquired resistance and resulted in impaired production of IL-12/IFN-gamma and higher expression of IL-4/IL-10 at the time of reinfection with virulent yeast. A CD4+ population expressing the memory phenotype, CD44highMEL-14low, which appeared to be expanded by yeast infection of nonhealer mice, was similarly increased in the healer mice by anti-TGF-beta treatment. In vitro rTGF-beta impaired the candidacidal function of IFN-gamma-activated macrophages. Yet in nonhealer mice infected with virulent C. albicans, administration of rTGF-beta delayed progression of the disease, which was concomitant with the detection of lower levels of IL-4. In addition to previous evidence for an obligatory role of IFN-gamma and IL-12 in Candida-driven Th1 cell differentiation in vivo, the present data establish TGF-beta as a third cytokine, the presence of which may be required for optimal Th1 development leading to long-lived anticandidal resistance.

Animals↗
PubMed1995

Multiple point mutations in an endogenous retroviral gene confer high immunogenicity on a drug-treated murine tumor.

Exposure in vivo of murine L5178Y lymphoma cells to cytoreductive triazene derivatives leads to the generation of immunogenic variant lines expressing new transplantation Ags recognized by CTL. In one such clonal variant (clone D), at least one subset of T cell neoepitopes are provided by proteins previously shown by serology to be products of endogenous retroviral env sequences. We report here on characterization of PCR-amplified gp70 env genes in clone D. Relative to known gp70 sequences in parental cells and in current databases, one gp70 sequence presented four distinct nucleotide changes, two of which were apparently unique to clone D DNA and cDNA upon differential hybridization analysis. Transfection experiments with the entire gp70 gene or subgenic fragments encompassing a single putative mutation showed that products of the mutated env gene or fragments may confer immunogenicity in vivo and susceptibility in vitro to lysis by clone D-primed, H-2Kd- or H-2Ld-restricted CTL. By skin test assay of mice primed with either clone D or three mutated synthetic peptides, evidence was obtained that amino acid substitutions at the relevant positions of the gp70 protein may produce immunogenic T cell epitopes and that these epitopes are expressed in vivo by clone D. These data point to the role of mutated retroviral tumor peptides as rejection Ags in histocompatible hosts.

Amino Acid Sequence↗
PubMed1995

Interleukin-4 and -10 exacerbate candidiasis in mice.

Neutralization of endogenous interleukin (IL)-4 or IL-10 in mice with Candida albicans infection initiates or accelerates development of a T helper (Th)1-associated protective response. Here, we report the effect of IL-4 and IL-10 administration on the course of systemic or gastrointestinal (GI) candidiasis and on the development of Th immunity using yeast/host combinations that result either in Th1-associated self-limiting infection (healer mice) or in Th2-associated progressive disease (nonhealer mice). Treatment with IL-4 or IL-10 greatly exacerbated the course of systemic infection in nonhealer mice and rendered healer mice, inoculated with attenuated yeast cells, susceptible to infection. Under the latter conditions of yeast challenge and IL-4/IL-10 administration, the development of a fatal disease was associated with inhibition of IL-12 production and detection of progressive Th2 cell dominance. In contrast, in healer mice allowed to resolve their infections and to develop long-lived anti-candidal resistance, the expression of this acquired resistance was not impaired by IL-4 and/or IL-10, as shown by the outcome of reinfection with virulent yeast cells. In the GI model of infection, both IL-4 and IL-10 were found to exacerbate the course of infection and to induce the appearance of CD4+ T cells producing high levels of IL-4 and IL-10 in Peyer's patches. These findings demonstrate that exogenous IL-4 and IL-10 may greatly affect the development of Th responses to C. albicans in vivo, but do not modify the expression of established and predominant Th1 cell reactivity.

Animals↗
PubMed1995

CD8+ cell activation to a major mastocytoma rejection antigen, P815AB: requirement for tum- or helper peptides in priming for skin test reactivity to a P815AB-related peptide.

Delayed-type hypersensitivity (DTH) responses, mediated by CD8+ cells and detected by skin test assay, occur in sensitized mice in response to challenge with class I-restricted antigenic peptides of mutagenized (tum-) P815 mastocytoma cells. In contrast, a nonapeptide related to a tumor rejection antigen, P815AB, failed in this study to elicit DTH after sensitization of mice with irradiated tumor cells or adoptive transfer of P815AB-pulsed dendritic cells. Unresponsiveness, however, could be overcome by immunization with tumor cells co-expressing P815AB and tum- antigens. When used for cell pulsing in vitro, a mixture of P815AB and tum- peptides was also highly effective in inducing anti-P815AB reactivity, as was the combined use of P815AB and class II-restricted peptides of tetanus toxin or Plasmodium berghei circumsporozoite protein. While the effector phase of the CD8+ cell-mediated DTH to P815AB was unaffected by the ablation of CD4+ cells, the same treatment, or neutralization of IFN-gamma, negated the induction of reactivity if it occurred at the time of sensitization. Thus, defective activation of CD4+ cells may contribute to the poor immunogenicity of P815AB. Besides providing an insight into the mechanisms of anti-tumor protection induced by tum- cells, these data offer useful information for the design of vaccination strategies against identified tumor antigens.

Amino Acid Sequence↗
PubMed1995

T helper cell dichotomy to Candida albicans: implications for pathology, therapy, and vaccine design.

Acquired immunity to Candida albicans is believed to prevent mucosal colonization of adult immunocompetent individuals from progressing to symptomatic infection. Resistance to disease appears to correlate with the detection of delayed-type hypersensitivity responses in vivo and a T helper type 1 (Th1) cytokine secretion profile in vitro. Cellular immunodeficiency, particularly HIV infection, greatly increases the risk of mucosal infection, confirming that CD(4+)-cell-directed immunity is effective locally in controlling infectivity of the yeast. While Th1-type CD4+ cell activation resulting in phagocyte-dependent immunity clearly represents an important mechanism of anticandidal resistance, clinical observations suggest that Th2-type CD4+ cell reactivity may be triggered by Candida antigens in several disease states, including symptomatic infections and immunopathology. This may imply that a Th1-type pattern of reactivity characterizes the saprophytic yeast carriage and resistance to disease by healthy humans, whereas Th2-type responses would be mostly associated with pathology. Moreover, Candida-specific T helper responses, namely humoral and cell-mediated immunity, appear to be reciprocally regulated, as typically occurs in experimental models of parasitic and retroviral infection, where the Th1/Th2 paradigm of acquired immunity has been best characterized. Recent studies, besides providing direct evidence for the occurrence of cross-regulatory Th1 and Th2 responses in mice with candidiasis, emphasize the potential of cytokine/anticytokine therapy for recruiting Candida-specific responses toward protective, Th1-type CD4+ cell reactivity. At the same time, these studies call attention to the possible consequences of C. albicans infection for immunopathology, allergy, and coinfection.

Animals↗