Insight into topological and functional relationships of cytochrome c oxidase subunit I of Saccharomyces cerevisiae by means of intragenic complementation

AbstractIn yeast, revertants were selected from four respiratory deficient mutants carrying mutations in the cytochrome c oxidase subunit I gene. Intragenic second site mutations revealed amino acids which are functionally complementary to the original mutated position and may be in topological interaction with it. The results provide additional data in favour of the model proposed for the structure of the binuclear centre in proton-motive oxidases

Early Functional Effects of Clostridium difficile Toxin A on Human Colonocytes

International audienceBackground & Aims: Previous in vitro studies have able to secrete interleukin 8 (IL-8) under appropriate shown that Clostridium difficile toxin A is able to distimulation. 7-9 Because IL-8 plays a key role in neutrorectly affect the intestinal epithelial barrier function. phil recruitment to sites of inflammation, 10 this finding The aim of this study was to examine the early effects supports the concept that the epithelium plays a central of toxin A on mucin exocytosis and determine whether role in triggering the inflammatory reaction associated this toxin can induce the production of the chemokine with various pathogens, including Clostridium difficile interleukin 8 (IL-8) from human colonic epithelial cells. toxins. However, the issue of whether this mechanism Methods: Two model systems were used: the HT29could apply to normal colonocytes has remained unset-Cl.16E colonic goblet cell line and primary cultures of tled because of the inability to maintain such cells in human normal colonocytes. Results: Toxin A exerted a culture. rapid and dose-related inhibition of stimulated mucin C. difficile produces two exotoxins: toxin A and toxin exocytosis without altering baseline (constitutive) mu-B. Toxin A has biological effects evidenced by the fact cin exocytosis from HT29-Cl.16E cells. Toxin A was that it induces a severe inflammatory enteritis in intestialso able to induce the secretion of IL-8 from both nal loop models 11 and activates granulocytes. 12 In addi-HT29-Cl.16E cells and primary cultures of human normal colonocytes, as early as 2-3 hours of incubation

High-scale expansion of melanoma-reactive TIL by a polyclonal stimulus: predictability and relation with disease advancement

International audienceThe rationale of treating melanoma patients by infusion with tumor-infiltrating leukocytes (TIL) is to perform an adoptive therapy through injection of tumor-specific T cells. Nonetheless, methods currently used for ex vivo TIL expansion have not been evaluated for their efficacy to expand TAA-specific T cells. We have addressed this question here, using a culture method in which high TIL growth was induced by a polyclonal T cell stimulus. Intracellular cytokine assays were performed to measure the proportion of T cells responding to autologous tumor cells among the lymphocytes from lymph node biopsies (TIL) of 26 patients with stage III melanoma. The data show that TIL from 18 of these patients contained detectable amounts of tumor-specific T cells before expansion. Although they decreased somewhat in percent abundance during expansion, they were still present afterwards, ranging from 0.3 to 13.8%. Since a median number of 1.7 x 10(10) TIL was obtained from these patients (starting from 3.6 x 10(6) TIL), a total amount of tumor-reactive cytokine-secreting TIL of between 2.8 x 10(6) and 1.12 x 10(9) was obtained in each case from 18 patients. The TIL populations from 8 patients did not contain tumor-reactive T cells: neither before expansion, nor after expansion. Lack of tumor-reactive TIL only occurs for patients bearing several tumor-invaded lymph nodes (40%), but not for those having a single invaded lymph node. Therefore, high numbers of tumor-reactive T cells can be produced, through a polyclonal TIL stimulation, from most early stage III melanoma patients but from only about half of the patients with a more disseminated disease. For this last group, the possibility of getting tumor-reactive TIL can be predicted by checking the presence of these cells before expansion

The doubling potential of T lymphocytes allows clinical-grade production of a bank of genetically modified monoclonal T-cell populations

International audienceBackground aims. To produce an anti-leukemic effect after hematopoietic stem cell transplantation we have long considered the theoretical possibility of using banks of HLA-DP specific T-cell clones transduced with a suicide gene. For that application as for any others, a clonal strategy is constrained by the population doubling (PD) potential of T cells, which has been rarely explored or exploited. Methods. We used clinical-grade conditions and two donors who were homozygous and identical for all HLA-alleles except HLA-DP. After mixed lymphocyte culture and transduction, we obtained 14 HLA-DP–specific T-cell clones transduced with the HSV-TK suicide gene. Clones were then selected on the basis of their specificity and functional characteristics and evaluated for their doubling potential. Results. After these steps of selection the clone NAT-DP4[(TK)], specific for HLA-DPB1*04:01/04:02, which produced high levels of interferon-γ (IFNγ), tumor necrosis factor (TNF), interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), was fully sequenced. It has two copies of the HSV-TK suicide transgene whose localizations were determined. Four billion NAT-DP4[(TK)] cells were frozen after 50 PDs. Thawed NAT-DP4[(TK)] cells retain the potential to undergo 50 additional PDs, a potential very far beyond that required to produce a biological effect. This PD potential was confirmed on 6/16 additional different T-cell clones. This type of well-defined clone can also support a second genetic modification with CAR constructs. Conclusion. The possibility of choosing rare donors and exploiting the natural proliferative potential of T lymphocytes may dramatically reduce the clinical and immunologic complexity of adoptive transfer protocols that rely on the use of third-party T-cell populations

Randomized trial of adoptive transfer of melanoma tumor-infiltrating lymphocytes as adjuvant therapy for stage III melanoma

International audienceThe aim of this study was to demonstrate the interest of using tumor-infiltrating lymphocytes (TIL) as adjuvant therapy for stage III (regional lymph nodes) melanoma. After lymph node excision, patients without any detectable metastases were randomly assigned to receive either TIL plus interleukin-2 (IL-2) for 2 months, or IL-2 only. The primary endpoint was determination of the duration of the relapse-free interval. Eighty-eight patients determined as eligible for treatment were enrolled in the study. After a median follow-up of 46.9 months, for the study population the analysis did not show a significant extension of the relapse-free interval or overall survival. However, a significant interaction (P<0.001) was found between the treatment and the number of invaded lymph nodes. In the group with only one invaded lymph node, the estimated relapse rate was significantly lower (P adjusted =0.0285) and the overall survival was increased (P adjusted =0.039) in the TIL+IL-2 arm compared with the IL-2 only arm. No differences between the two arms, either as regards the duration of disease-free survival or overall survival, were noted in the group with more than one invaded lymph node whatever the number of invaded lymph nodes. Treatment was compatible with normal daily activity. This study demonstrates for the first time that the efficiency of TIL in stage III melanoma (AJCC) is directly related to the number of invaded lymph nodes, indicating that tumor burden might be a crucial factor in the efficacy and/or in vitro expansion of T cells specific for autolo-gous tumor antigen, a finding which could be of value in future vaccine development for the treatment of melanoma

Long-term follow-up of patients treated by adoptive transfer of melanoma tumor-inWltrating lymphocytes as adjuvant therapy for stage III melanoma

International audienceThe first analysis of our clinical trial on interest of using tumor-infiltrating lymphocytes (TIL) as adjuvant therapy for stage III (regional lymph nodes) melanoma was published in 2002 [5]. The aim of this paper is to update clinical results of 7 years of follow-up after the last treated patient. In the trial conducted between December 1993 and January 1999, patients without any detectable metastases after lymph node excision were randomly assigned to receive either TIL plus interleukin-2 (IL-2) for 2 months, or IL-2 only. The duration of the relapse-free interval was the primary objective. Eighty-eight patients were enrolled in the study. Currently, the last analysis performed in June 2006, after a median follow-up of 114.8 months, did not show change of non-signiWcant extension of the relapse-free interval or overall survival. However, this second analysis strengthens our first hypothesis about the relationship between number of invaded lymph nodes and TIL treatment eVectiveness. In the group with only one invaded lymph node, the estimated relapse rate was signiWcantly lower (P adjusted = 0.0219) and the overall survival was increased (P adjusted = 0.0125) in the TIL+IL-2 arm compared with the IL-2 only arm. No diVerences between the two arms, either with regard to the duration of disease-free survival (P adjusted = 0.38) or overall survival (P adjusted = 0.43), were noted in the group with more than one invaded lymph node, whatever the number of invaded lymph nodes. Treatment was compatible with normal daily activity. This study, with a very long follow up (median of almost 10 years), postulates for the Wrst time relationship between TIL eYciency in stage III melanoma (AJCC) and number of invaded lymph nodes, indicating that tumor burden might be a crucial factor in the production of an eVective in vitro expansion of T cells speciWc for autologous tumor antigen, a finding which could be of value in future vaccine development for the treatment of melanoma

NO-dependent and NO-independent IL-1 production by a human colonic epithelial cell line under inflammatory stress

1. The present study was designed to investigate, in an in vitro model of the human intestinal barrier, the ability of epithelial cells to produce interleukin-1 (IL-1), the cellular mechanisms involved in IL-1 release, and the intracellular signalling pathways involved in IL-1 up-regulation during inflammatory stress. 2. This study was based on the human colonic epithelial cell line HT29-Cl.16E, maintained as polarized monolayers on filters mounted in culture chambers and treated with various proinflammatory cytokines (interferon γ (IFNγ), tumour necrosis factor α (TNFα), IL-1β) alone or in combination. 3. IL-1 production, restricted to IL-1α, was induced by the combination of IFNγ/TNFα. When IL-1β was added to IFNγ/TNFα, it led to an additional production of IL-1α. IL-1α release was associated with cell damage, as shown by the correlation between lactate dehydrogenase (LDH) release and extracellular IL-1 production, and was not accounted for by a secretory mechanism. 4. Both IFNγ/TNFα and IFNγ/TNFα/IL-1β induced inducible nitric oxide synthase (iNOS) expression as shown by quantitation of NO(2)(−)/NO(3)(−) by use of the Griess reagent, quantitation of cells scoring positive with an anti-iNOS antibody and detection of mRNAs coding for iNOS by RT–PCR. The use of N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of NOS, led to the demonstration of two distinct signalling pathways in IL-1 production by HT29-Cl.16E cells, one dependent on NO (L-NMMA-sensitive) under treatment with IFNγ/TNFα/IL-1β, and the other independent of NO (L-NMMA-insensitive) under treatment with IFNγ/TNFα. 5. Moreover, we examined whether a redox-based mechanism could be responsible for the apparent discrepancy between NO production and NO implication in IL-1 production under IFNγ/TNFα and IFNγ/TNFα/IL-1β treatments. Experiments with cysteine, which acts as a powerful reductant, suggest that the nitrosonium character of NO is involved in the NO-dependent pathway in IL-1 production