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U Grohmann

Publications and source records attributed to U Grohmann.

At least 37 records · Page 2Linked to original sources
PubMed1998

Dendritic cells, interleukin 12, and CD4+ lymphocytes in the initiation of class I-restricted reactivity to a tumor/self peptide.

Cell-mediated immunity involving CD8+ lymphocytes is effective in mediating rejection of murine mastocytoma cells bearing P815AB, a tumor-associated and self antigen showing similarity to tumor-specific shared antigens in humans. Although this antigen may act as an efficient target for class I-restricted responses in immunized mice, neither P815AB expressed on tumor cells nor a related synthetic nonapeptide will activate unprimed CD8+ cells for in vivo reactivity, measured by skin test assay. We review evidence showing that the failure of P815AB to initiate CD8+ cell reactivity may be due to defective recruitment of accessory and Th1-like cells to the afferent phase of the response initiated by transfer of mice with dendritic cells pulsed in vitro with the P815AB peptide. Although the copresence of a T helper peptide in dendritic cell priming in vitro with P815AB may compensate for the poor generation of accessory and Th1 cells in the adoptively transferred mice, recombinant IL-12 can replace the helper peptide in both effects. Effective priming to P815AB in vivo is achieved by either exposing dendritic cells to IL-12 prior to P815AB priming or administering the recombinant cytokine in vivo. Different approaches suggest that IL-12 may act both on accessory cells to improve presentation of previously undescribed class II-restricted epitopes of P815AB and on CD4+ cells to improve recognition of such epitopes. In particular, at the CD4+ cell level, IL-12 apparently acts as an adjuvant and an inhibitor of anergy induction. These data offer useful information for developing vaccination strategies using dendritic cells and class I-restricted tumor peptides in humans.

Adjuvants, Immunologic↗
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

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

Hemisection and vital treatment of a fused tooth--literature review and case report.

Fusion and gemination of permanent teeth are developmental anomalies of the dental hard tissues which may require endodontic and surgical treatment for functional, orthodontic or aesthetic reasons. Following a review of the dental literature on tooth fusion and gemination, a case of fusion of a maxillary central incisor and a supernumerary tooth and its endodontic and surgical treatment is presented.

Child↗
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↗
PubMed1997

Dacarbazine-induced immunogenicity of a murine leukemia is attenuated in cells transfected with mutated K-ras gene.

Strong immunogenicity is induced by antitumor triazene compounds in tumor cells through a mutagenic mechanism. A highly immunogenic < > clone, isolated from a dacarbazine-treated L5178Y leukemia of DBA/2 mice, was transfected with K-ras mutated at codon 12 (i.e. ras(m12)). This transfected clone presents at least 2 mutations, one concerning K-ras gene, and the other affecting an unrelated gene, responsible for the generation of a highly immunogenic, MHC class I restricted non-self peptide. The results indicate that cells of < > clone transfected with ras(m12) were less immunogenic than cells of the same origin transfected with the vector alone. Moreover, ras(m12)-transfected cells showed lower levels of H-2K(d) gene expression with respect to those detectable in control cells. In addition, in vivo and in vitro sensitization against < > clone carrying mutated ras did not result in a strong cytotoxic T lymphocyte response against ras(m12)-transfected non immunogenic L5178y target cells. These preliminary results suggest that K-ras mutation could down-regulate the level of tumor immunogenicity, possibly acquired through a mutagenic process affecting other unrelated genes.

Animals↗
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

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

O6-alkylguanine-DNA alkyltransferase attenuates triazene-induced cytotoxicity and tumor cell immunogenicity in murine L1210 leukemia.

Methylating and chloroethylating triazene compounds (TZCs) are effective antitumor agents in murine leukemias and can induce the appearance of novel antigens in leukemic cells (chemical xenogenization). Recently, it has been shown that TZCs might have a role in the treatment of patients affected by acute myelogenous leukemias that express low levels of the DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase (OGAT). In this report, we have evaluated the role of this DNA repair enzyme in the leukemic cell response to the xenogenizing and cytotoxic properties of TZCs. OGAT-deficient murine leukemic L1210 cells were transfected with a recombinant ecotropic retrovirus containing the coding region for the human OGAT protein. Selected clones expressed the human OGAT transcript and had greatly increased OGAT activity. Compared to OGAT-deficient cells, OGAT-expressing cells were considerably more resistant to the xenogenizing properties of 1-(p-chlorophenyl)-3,3- dimethyl-triazene, measured in terms of leukemia graft rejection, and were less susceptible to the cytotoxic activity of the TZCs 8-carbamoyl-3-methyl-imidazo [5,1-d]-1,2,3,5-tetrazin-4(3H)-one and 8-carbamoyl-3-(2-chloroethyl)imidazo [5,1-d]-1,2,3,5-tetrazin-4(3H)-one. These data suggest that methylation of the O6 position of guanine is involved in the appearance of increased tumor immunogenicity after exposure to methylating TZC and that OGAT is able, at least in part, to counteract the cytotoxic effects of methylating and chloroethylating agents.

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

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↗
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

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

Untreated or drug-treated tumor cells are differentially recognized by allogeneic lymphocytes.

Murine tumor cells treated with triazene compounds (TZC), in vivo or in vitro, are capable of eliciting specific transplantation resistance in syngeneic hosts, and T-cell-mediated proliferative and cytotoxic responses, directed against novel drug-induced antigen(s). Since this phenomenon, referred to as chemical xenogenization (CX) could open up new perspectives in the immunochemotherapy of human neoplasias, it was of interest to investigate whether CX could also occur in human tumors. However, established human tumor cell lines along with fully immunocompetent autologous lymphocytes, are seldom available. Therefore studies were carried out to test whether parental or TZC-treated tumor cells could be differentially recognized by allogeneic lymphocytes. Experiments were performed in both human and murine models, using a lung adenocarcinoma line treated in vitro with TZC, or an established xenogenized mouse lymphoma, respectively. The results indicate that allogeneic cytotoxic T-lymphocytes (CTL) recognize specifically murine TZC-treated tumor cells. This was supported by the finding that antisera directed against the drug-treated cells abrogated the generation and the cytolytic activity of allogeneic CTL reactive against the TZC-treated tumor. In addition it was found that changes of the antigenic pattern of cell membrane recognizable by cloned allogeneic CTL occur in the TZC-treated human carcinoma cell line.

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↗
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↗