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Biomedical subjects

P Puccetti

Publications and source records attributed to P Puccetti.

At least 55 records · Page 3Linked to original sources
PubMed1995

Anticancer drug toxicity via cytokine production: the hydroxyurea paradigm.

Our previous observations on the toxic effects of hydroxyurea (HU) in adrenalectomized (ADX) rats prompted us to suggest that these effects might be mediated by an increased synthesis of proinflammatory cytokines. This study was conducted to determine whether HU stimulates cytokine gene expression in vivo. The polymerase chain reaction (PCR) technique was used to assess levels of mRNA for interleukin-1 alpha (IL-1 alpha), tumor necrosis factor (TNF) and interleukin-4 (IL-4) in spleen cells from intact and ADX rats treated with HU or vehicle. In ADX rats, expression of proinflammatory-cytokine mRNA was markedly increased by HU, but no expression of these genes was seen in intact animals after treatment. In the latter rats, cytokine-gene expression seemed to be down-regulated by HU-induced elevations in plasma corticosterone levels, since IL-1 alpha and TNF transcripts could be detected only after corticosterone levels had returned to normal (24 h after treatment). Interestingly, IL-4 mRNA could not be detected in either treated or untreated ADX rats, indicating that expression of this gene is closely related to circulating levels of corticosterone. These findings strongly suggest that the increased toxicity displayed by HU in ADX animals is mediated by stimulation of cytokine synthesis in vivo.

Adrenalectomy↗
PubMed1995

Platelet radiolabelling with 111 Indium for in vivo studies: a methodological reappraisal.

Two ligands widely used for 111In labeling of human platelets, oxine and tropolone, were compared as to the degree of their influence on platelet function. The labelling efficiency was assessed as a function of experimental variables. A good preservation of platelet function as evaluated by ex vivo aggregation was obtained with both techniques. The extent of tracer detached from platelets was evaluated in vitro and in vivo: in normal subjects 111In was virtually all bound to platelets. A significant reduction in radiolabelling efficiency and a decreased percentage of 111In bound to platelets in vivo was observed in patients with platelet counts < 150,000/mL.

Blood Platelets↗
PubMed1995

A TH1-TH2-like switch in candidiasis: new perspectives for therapy.

An imbalance in TH1-type and TH2-type responses may allow Candida albicans to modify the host response to favor its own persistence. This hypothesis has important consequences for allergy, autoimmunity and co-infection, and also highlights a potential role for cytokine and anti-cytokine therapy in Candida-related pathology.

Animals↗
PubMed1995

T helper cell type 1 (Th1)- and Th2-like responses are present in mice with gastric candidiasis but protective immunity is associated with Th1 development.

The relative contributions of local T helper cell type 1 (Th1)- and Th2-like responses to the course of primary and secondary gastrointestinal (GI) candidiasis were examined in adult immunocompetent BALB/c mice. Both Th1 cytokines, such as interferon-gamma (IFN-gamma), and the Th2 cytokines, interleukin (IL)-4 and IL-5, were produced by CD4+ cells from Peyer's patches (PP) and mesenteric lymph nodes at a time when the fungus was cleared from the stomach and intestine. Augmentation of antigen-specific Th2-like responses by treatment with cholera toxin did not modify the course of disease. In contrast, treatment with soluble IL-4 receptor, which increased Th1 cells, was associated with enhanced yeast clearance. In addition, IFN-gamma but not IL-4 mRNA was present in PP and spleen CD4+ cells in mice resistant to subsequent GI inoculation. Activation of Th1- but not Th2-like responses may be responsible locally for controlling GI candidiasis and generating protective immunity.

Animals↗
PubMed1994

IL-12 is both required and prognostic in vivo for T helper type 1 differentiation in murine candidiasis.

In murine models of systemic candidiasis, healing and nonhealing patterns of disease are associated with preferential expansion of Th1 and Th2 cells, respectively. As previous studies have shown IL-12 to be expressed transcriptionally in healer mice and to be required for Th1 development in vitro, this cytokine might play a role in Candida-driven Th1 cell differentiation in vivo. In the present study, IL-12-neutralizing Abs or recombinant IL-12 were administered to mice with healing or progressive candidiasis, respectively, and the animals were monitored for mortality, resistance to reinfection, serum levels of specific Abs, and IFN-gamma, IL-4, and IL-10 message/protein expression by CD4+ cells. In self-limiting infection by a yeast vaccine strain, neutralization of IL-12 ablated development of acquired anticandidal resistance and led to appearance of Candida-specific IgE and IL-4-producing cells. In mice with progressive systemic disease as well as in a mucosal infection model, administration of IL-12 did not result in therapeutic activity under conditions of yeast infection that would instead be resolved by serologic ablation of IL-4 or IL-10. Yet, in systemically infected mice cured by anti-IL-4 or anti-IL-10 therapy, the emergence of a Th1 response correlated with the detection of high levels of circulating IL-12 and splenic IL-12 transcripts. Although exogenous IL-12 may not be sufficient for Th conversion in the presence of an overwhelming IL-4/IL-10 response, endogenous production of IL-12 may be both required and prognostic for Th1 differentiation in vivo.

Animals↗
PubMed1994

Neutralization of IL-10 up-regulates nitric oxide production and protects susceptible mice from challenge with Candida albicans.

In contrast to several inbred strains of mice that develop Th1-associated anticandidal protection, DBA/2 mice are highly susceptible to systemic infection with Candida albicans cells of the attenuated variant PCA-2, and fatal outcome is observed in concurrence with sustained CD4+ cell production in vitro of IL-4 and IL-10. These Th2 cytokines were previously shown to inhibit nitric oxide (NO) production and yeast killing by activated macrophage cultures. We now show that macrophages from DBA/2 mice, either intact or infected with PCA-2, have lower capacity than resistant strains to synthesize NO in response to IFN-gamma. However, when treated with anti-IL-10 Abs at the time of infection, DBA/2 mice survived challenge and displayed increased production of NO in vitro after IFN-gamma activation. Cure was associated with the onset of footpad responses and durable protection, and higher frequencies of IFN-gamma-secreting cells were found in splenic CD4+ lymphocytes that expressed lower levels of IL-4 and IL-10 mRNA. Therefore, in DBA/2 mice, IL-10 contributes significantly to the selection of a Th2 response and lethality after PCA-2 challenge. An IL-10-induced defect in the activation and/or expansion of IFN-gamma-producing Th1 cells, IL-10 suppression of yeast killing, and the relative inability of DBA/2 macrophages to produce adequate levels of candidacidal NO may all contribute to the abnormal susceptibility of these mice to candidiasis.

Animals↗
PubMed1994

Interleukin-12 but not interferon-gamma production correlates with induction of T helper type-1 phenotype in murine candidiasis.

By means of polymerase chain reaction-assisted mRNA amplification, we have monitored message levels of interleukin (IL)-12 in splenic macrophages and of interferon-gamma (IFN-gamma), IL-4, and IL-10 in CD4+ and CD8+ T cells using Candida albicans/host combinations that result either in a T helper type-1 (Th1)-associated self-limiting infection ("healer mice") or in a Th2-associated progressive disease ("nonhealer mice"). The timing and pattern of message detection did not differ qualitatively by the expression of IFN-gamma or IL-10 mRNA in CD4+ and CD8+ cells from healer (i.e. PCA-2 into CD2F1) vs. nonhealer (i.e. CA-6 into CD2F1 or PCA-2 into DBA/2) mice. In contrast, IL-4 mRNA was uniquely expressed by CD4+ cells from nonhealer animals. IL-12p40 was readily detected in macrophages from healer mice but was detected only early in infection in mice with progressive disease. Cytokine levels were measured in sera, and antigen-driven cytokine production by CD4+ and CD8+ cells was assessed in vitro, while IFN-gamma-producing cells were enumerated in CD4- CD8- cell fractions. Overall, our results showed that (i) antigen-specific secretion of IFN-gamma protein in vitro by CD4+ cells occurred only in healing infection; (ii) IL-4- and IL-10-producing CD4+ cells would expand in nonhealer mice in the face of high levels of circulating IFN-gamma, likely released by CD4- CD8- lymphocytes; (iii) a finely regulated IFN-gamma production correlated in the healer mice with IL-12 mRNA detection, and IL-12 was required in vitro for yeast-induced development of IFN-gamma-producing CD4+ cells. Although the mutually exclusive production of IL-4/IL-10 and IFN-gamma by early CD4+ cells may be the major discriminative factor of cure and noncure responses in candidiasis, IL-12 rather than IFN-gamma production may be an indicator of Th1 differentiation.

Animals↗
PubMed1994

Use of a skin test assay to determine tumor-specific CD8+ T cell reactivity.

We have observed delayed-type hypersensitivity (DTH) reactions in immunized mice challenged subcutaneously with class I-binding peptides related to rejection antigens recognized by cytotoxic T lymphocytes on mutagenized (tum-) variants of mastocytoma P815. As observed by skin test in virally infected mice challenged with viral peptides, the intrafootpad injection of tum- peptides resulted in a dose-dependent DTH that peaked at approximately 24 h. The response was mediated by CD8+ cells and could be induced by previous vaccination of mice with live tumor cells, intrasplenic deposition of the eliciting peptide, or adoptive transfer with peptide-pulsed syngeneic dendritic cells. These sensitization procedures resulted in an immunologically specific footpad reaction detectable for up to 2-6 months after priming. The evaluation by DTH in cancer patients of long-lived CD8+ anti-tumor T cell responses following local challenge with tumor-specific peptides may be of great interest in human immunotherapy trials involving immunization against identified tumor antigens.

Animals↗
PubMed1994

Endogenous retroviral gp70 genes of the murine lymphoma L5178Y: analysis of restriction fragment polymorphism upon induction of drug-mediated immunogenicity.

Highly immunogenic ("xenogenized") tumor variants appear after treatment of murine lymphoma L5178Y with the triazene derivative DTIC, this phenomenon being associated with the appearance of structurally abnormal gp70 env proteins in the cell variants. In the present study, we have isolated and sequenced several PCR-amplified gp70 cDNA genes from L5178Y cells. One of the resulting clones was used as a probe in Southern and Northern analysis of parental and xenogenized cells. The results indicated that xenogenization of tumor cells is associated with changes in retroviral env sequences detectable at the genomic level.

Animals↗
PubMed1994

Cure of murine candidiasis by recombinant soluble interleukin-4 receptor.

Neutralization of interleukin (IL)-4 by specific antibody exerts therapeutic activity in a murine model of systemic candidiasis characterized by strong T helper type 2 (Th2) responses. To investigate whether recombinant soluble IL-4 receptor (sIL-4R) could be used to block IL-4 action in vivo, mice treated with pharmacologic doses of sIL-4R at the time of infection were examined for progression of disease, development of footpad responses, serum IgE levels, and cytokine production in vitro by CD4+ lymphocytes. Following sIL-4R treatment, persistent ablation of circulating IL-4 detected by ELISA was associated with a cure rate of > 90% in otherwise lethally infected mice, onset of durable protection, and a shift from a predominant Th2 to a Th1 pattern of reactivity. In addition, when administered to genetically susceptible adult mice with gastrointestinal yeast colonization, the sIL-4R stimulated Th1-associated anticandidal resistance.

Animals↗
PubMed1994

Tolerance to staphylococcal enterotoxin B initiated Th1 cell differentiation in mice infected with Candida albicans.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that specifically activates T cells bearing V beta 8 T-cell receptor domains, which eventually leads to a long-lasting state of clonal anergy accompanied by selective cell death in the targeted CD4+ subset. Because the superantigen is known to promote Th1 cell differentiation in vitro, we have investigated the effect of SEB treatment on the course of Th2-associated progressive disease in mice infected systemically with Candida albicans. On the basis of the kinetics of SEB-induced changes in CD4+ cells and production in sera of interleukin 4 (IL-4), IL-10, and gamma interferon, we obtained evidence that V beta 8+ cell anergy concomitant with infection abolished the early IL-4/IL-10 response of the host to the yeast, ultimately leading to a state of resistance characterized by gamma interferon secretion in vitro by antigen-specific CD4+ cells. In contrast, SEB administered near the time of challenge resulted in accelerated mortality. Significant resistance to infection was also afforded by exposure of mice to a retrovirally encoded endogenous superantigen. These data suggest that CD4+ V beta 8+ T cells play an important role in vivo in the initiation of a Th2 response to C. albicans and that suppression of their activity may alter the qualitative development of the T-cell response and the outcome of infection.

Animals↗
PubMed1993

Immunogenic properties of retroviral protein P15E from drug-treated murine mastocytoma P815.

A triazene-xenogenized tumor sub-line was derived from the mouse mastocytoma cell line P815 following several transplant generations in vivo on DTIC. The highly immunogenic P815/DTIC variant line expressed new CTL-defined antigens. Novel antigens were also detected by antibodies in immunoprecipitation and by Western blot analysis. Upon immunoprecipitation of metabolically labeled cells, one such variant-specific 20-kDa antigen was shown to be related to retroviral envelope protein p15E. When injected intrasplenically into recipient mice, the electroblotted nitrocellulose-bound 20-kDa antigen resulted in increased frequency in CTL precursors to P815/DTIC cells. In addition to previous data in the L5178Y/DTIC tumor-model system, these data suggest that expression of aberrant, retrovirus-related proteins may be a common finding in different parental tumors xenogenized by triazene treatment.

Animals↗
PubMed1993

Accelerated hematopoietic recovery and protective effect of the cyclooxygenase inhibitor indomethacin in bacterial infection of neutropenic mice.

The effects of indomethacin administration on Pseudomonas aeruginosa infection were investigated in neutropenic mice. Cyclophosphamide-treated mice received the drug at 2.5 to 12 mg/kg according to different regimens, to be challenged with a lethal intraperitoneal inoculum of P. aeruginosa 5 days after myelosuppression. A single exposure of the neutropenic mice to 7 mg/kg indomethacin during the first 6 to 48 hr after myelosuppression was found to optimally restore the animals' antibacterial resistance, both in terms of survival of infected mice and clearance of the organisms from the peritoneal cavity. However, when administered 24 hr before challenge, the same drug dosage had no effect in enhancing survival. Cure was associated with accelerated hematopoietic recovery, as revealed by peripheral blood leukocyte counts, spleen weight and cellularity, cellular response to infection in the peritoneal cavity, and enumeration in vitro of bone marrow and splenic granulocyte-macrophage colony-forming cells. Following indomethacin administration, a rapid burst in the levels of colony-stimulating activity was detected in the bloodstream, and exposure of splenic macrophages or marrow cells to indomethacin in vitro was found to result in enhanced expression of transcripts specific for granulocyte-macrophage colony-stimulating factor. These data support the notion that the administration of cyclooxygenase inhibitors may be useful in promoting hematopoiesis and reducing the risk of opportunistic infections in myelosuppressed hosts.

Animals↗
PubMed1993

CD4+ subset expression in murine candidiasis. Th responses correlate directly with genetically determined susceptibility or vaccine-induced resistance.

Previous work has shown that in hybrid (BALB/cCr x DBA/2Cr)F1 mice the development of a fatal disseminated disease by systemic infection with virulent Candida albicans is associated with the detection of strong Th2-like responses. However, a predominant Th1-like response and long-lived antifungal protection are induced by vaccinating these mice with live blastospores of attenuated C. albicans strains. When injected into DBA/2Cr mice, one such live vaccine strain was found in the present study to result in a progressive disease characterized by strong Th2 responses. Elevated serum IgG1, IgA, and IgE responses, weak or absent footpad reactions, sustained production in vitro of Th2 (IL-4 and IL-10) but not Th1 (IL-2 and IFN-gamma) cytokines by CD4+ cells, and eosinophilia were all detected in DBA/2 mice after infection with the attenuated vaccine. This was in marked contrast with the development of strong Th1 responses and persistent anticandidal protection in similarly infected, H-2-compatible BALB/cCr mice. Therefore, our data suggest that the type of Th response that predominates in mice after C. albicans infection correlates with genetically determined susceptibility or vaccine-induced resistance. Moreover, the genetic control of this resistance may not be associated with the H-2 complex.

Animals↗
PubMed1993

Interleukin-4 and interleukin-10 inhibit nitric oxide-dependent macrophage killing of Candida albicans.

Mouse peritoneal and splenic macrophages treated with interferon-gamma (IFN-gamma) and infected with the yeast Candida albicans expressed high fungicidal activity in vitro that correlated with increased nitrite concentrations in culture supernatants. Both effects were reduced by an inhibitor of nitric oxide (NO) synthesis which, in vivo, impaired the animals' ability to mount a footpad reaction and clear the fungus from infected organs. Because T helper type-2 (Th2) cytokines in candidiasis are known to limit the expression of protective Th1 functions, we tested the effect of interleukin (IL)-4 and IL-10 on candidacidal activity and NO production of IFN-gamma-activated macrophages. Fungal killing and NO secretion were inhibited, in a dose-dependent manner, by the two cytokines either separately or in combination. Impaired candidacidal activity was also demonstrable in the presence of monoiodoacetic acid, an inhibitor of phagocytosis. These data demonstrate that NO is involved in macrophage killing of C. albicans and support the notion that regulation of Th1 effector function by IL-4 and IL-10 might involve modulation of NO synthesis.

Animals↗
PubMed1993

Cell-mediated immunity to chemically xenogenized tumors--VI. The effect of cell treatment with retroviral env antisense oligonucleotides.

The occurrence of aberrant, retrovirus-related proteins is a common finding in immunogenic clones of the triazene-xenogenized L5178Y lymphoma line (L5178Y/DTIC). In clone D-cells, newly expressed 80 kDa antigens related to xenotropic murine leukemia virus env gene products induce specific humoral and cell-mediated responses and possess biologic activity in vivo. To further clarify the relationship between immunogenic properties of clone D and retroviral gene expression, tumor cells were treated in vitro with antisense oligonucleotides complementary to xenotropic and/or polytropic env sequences of murine leukemia virus. The cells were then assayed for expression of antigens recognized by humoral and cell-mediated responses with specificity for clone D. The results demonstrated that inhibition of env mRNA translation adversely affected the expression of immunogenic determinants in the xenogenized tumor cells.

Animals↗
PubMed1993

Mucosal and systemic T helper cell function after intragastric colonization of adult mice with Candida albicans.

In 85% of adult DBA/2 mice inoculated intragastrically with Candida albicans, significant numbers of yeast cells were recovered from the gastrointestinal tract for up to 4 weeks, with the animals eventually clearing infection in the absence of systemic disease despite the occurrence of localized, self-limiting foci of mucosal involvement in their stomachs. Two major findings in colonized mice were defective production of IgA, interleukin (IL)-4, and IL-5 by Peyer's patches lymphocytes and increased numbers of interferon-gamma-producing T cells in mesenteric lymph nodes and spleens. Relatively low levels of circulating antibodies of T helper type 2 (Th2)-dependent isotypes were also found in colonized mice, which exhibited strong footpad responses and increased resistance to systemic reinfection. Unlike systemic challenge, gastrointestinal colonization of adult immunocompetent DBA/2 mice with C. albicans appears to be an effective stimulus for the systemic development of protective Th1 responses.

Animals↗