Claude Capron

CD4+CD25+ regulatory T cells inhibit natural killer cell functions in a transforming growth factor–β–dependent manner

Tumor growth promotes the expansion of CD4+CD25+ regulatory T (T reg) cells that counteract T cell–mediated immune responses. An inverse correlation between natural killer (NK) cell activation and T reg cell expansion in tumor-bearing patients, shown here, prompted us to address the role of T reg cells in controlling innate antitumor immunity. Our experiments indicate that human T reg cells expressed membrane-bound transforming growth factor (TGF)–β, which directly inhibited NK cell effector functions and down-regulated NKG2D receptors on the NK cell surface. Adoptive transfer of wild-type T reg cells but not TGF-β −/− T reg cells into nude mice suppressed NK cell–mediated cytotoxicity, reduced NKG2D receptor expression, and accelerated the growth of tumors that are normally controlled by NK cells. Conversely, the depletion of mouse T reg cells exacerbated NK cell proliferation and cytotoxicity in vivo. Human NK cell–mediated tumor recognition could also be restored by depletion of T reg cells from tumor-infiltrating lymphocytes. These findings support a role for T reg cells in blunting the NK cell arm of the innate immune system.

Retinoic Acid Metabolism and Mechanism of Action: A Review

Retinoids are vitamin A (retinol) derivatives essential for normal embryo development and epithelial differentiation. These compounds are also involved in chemoprevention and differentiation therapy of some cancers, with particularly impressive results in the management of acute promyelocytic leukemia (APL). Although highly effective in APL therapy, resistance to retinoic acid (RA) develops rapidly. The causes of this resistance are not completely understood and the following factors have been involved: increased metabolism, increased expression of RA binding proteins, P-glycoprotein expression, and mutations in the ligand binding domain of RARalpha. RA exerts its molecular actions mainly through RAR and RXR nuclear receptors. In addition to the nuclear receptor based mechanism of RA action, covalent binding of RA to cell macromolecules has been reported. RA derives from retinol by oxidation through retinol and retinal dehydrogenases, and several cytochrome p450s (CYPs). RA is thereafter oxidized to several metabolites by a panel of CYPs that differs for the different RA isomers. Phase II metabolism, mainly glucuronidation, is also observed. The role RA metabolism plays in the expression of its biological actions is not completely understood: in several systems, metabolism decreases RA activity, whereas in other systems metabolism appears involved in its action. In addition, several RA metabolites have shown activity and cannot be classified as only catabolites. Therapy of cancer with retinoids is still in its infancy, but the use of new analogues with improved pharmacological properties, along with combination with other drugs, could undoubtedly improve the management of several cancers in the future.

A mutation of human cytochrome c enhances the intrinsic apoptotic pathway but causes only thrombocytopenia.

We report the first identified mutation in the gene encoding human cytochrome c (CYCS). Glycine 41, invariant throughout eukaryotes, is substituted by serine in a family with autosomal dominant thrombocytopenia caused by dysregulated platelet formation. The mutation yields a cytochrome c variant with enhanced apoptotic activity in vitro. Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome c activity is not a critical regulator of most physiological apoptosis.

Exposure of human megakaryocytes to high shear rates accelerates platelet production

Platelets originate from megakaryocytes (MKs) by cytoplasmic elongation into proplatelets. Direct platelet release is not seen in bone marrow hematopoietic islands. It was suggested that proplatelet fragmentation into platelets can occur intravascularly, yet evidence of its dependence on hydrodynamic forces is missing. Therefore, we investigated whether platelet production from MKs could be up-regulated by circulatory forces. Human mature MKs were perfused at a high shear rate on von Willebrand factor. Cells were observed in real time by videomicroscopy, and by confocal and electron microscopy after fixation. Dramatic cellular modifications followed exposure to high shear rates: 30% to 45% adherent MKs were converted into proplatelets and released platelets within 20 minutes, contrary to static conditions that required several hours, often without platelet release. Tubulin was present in elongated proplatelets and platelets, thus ruling out membrane tethers. By using inhibitors, we demonstrated the fundamental roles of microtubule assembly and MK receptor GPIb. Secretory granules were present along the proplatelet shafts and in shed platelets, as shown by P-selectin labeling. Platelets generated in vitro were functional since they responded to thrombin by P-selectin expression and cytoskeletal reorganization. In conclusion, MK exposure to high shear rates promotes platelet production via GPIb, depending on microtubule assembly and elongation.

Human cytochrome P450s involved in the metabolism of 9-cis- and 13-cis-retinoic acids

The purpose of this work was to identify the principal human cytochrome P450s (CYPs) involved in the metabolism of the retinoic acid (RA) isomers, 9-cis- and 13-cis-RA, by using a combination of techniques including human liver microsomes (correlation of activity and inhibition), and lymphoblast microsomes expressing a single CYP. Concerning the 9-cis-RA, 4-OH- and 4-oxo-9-cis-RA were formed with human liver microsomes, and their formation correlated with activities linked to CYPs 3A4/5, 2B6, 2C8, 2A6, and 2C9. The use of lymphoblast microsomes expressing a single human CYP identified CYPs 2C9>2C8>3A7 as the most active in the formation of 4-OH-9-cis-RA. With regard to 13-cis-RA, specific P450 activities linked to CYPs 2B6, 2C8, 3A4/5, and 2A6 were correlated with the formation of 4-OH- and 4-oxo-13-cis-RA. Microsomes expressing a single CYP identified CYPs 3A7, 2C8, 4A11, 1B1, 2B6, 2C9, 2C19, 3A4 (decreasing activity) in the formation of 4-OH-13-cis-RA. The use of CYP-specific inhibitors in human liver microsomes disclosed that the formation of the 4-OH-9-cis-RA was best inhibited by sulfaphenazole (72%) and quercetin (66%), whereas ketoconazole and troleandomycin inhibited its formation by 55 and 38%, respectively; the formation of 4-OH-13-cis-RA was best inhibited by troleandomycin (54%) and ketoconazole (46%), whereas quercetin was a weak inhibitor (14%). In conclusion, adult human CYPs 2C9, 2C8, 3A4 have been identified as active in the 9-cis-RA metabolism, whereas CYPs 3A4 and 2C8 were active in 13-cis-RA metabolism. The fetal form CYP3A7 was also identified as very active in either 9-cis- or 13-cis-RA metabolism. The role of these human CYPs in the biological response or resistance to RA isomers remains to be determined.

A major role of TGF-beta1 in the homing capacities of murine hematopoietic stem cell/progenitors.

Transforming growth factor-beta1 (TGF-beta1) is a pleiotropic cytokine with major in vitro effects on hematopoietic stem cells (HSCs) and lymphocyte development. Little is known about hematopoiesis from mice with constitutive TGF-beta1 inactivation largely because of important embryonic lethality and development of a lethal inflammatory disorder in TGF-beta1(-/-) pups, making these studies difficult. Here, we show that no sign of the inflammatory disorder was detectable in 8- to 10-day-old TGF-beta1(-/-) neonates as judged by both the number of T-activated and T-regulator cells in secondary lymphoid organs and the level of inflammatory cytokines in sera. After T-cell depletion, the inflammatory disease was not transplantable in recipient mice. Bone marrow cells from 8- to 10-day-old TGF-beta1(-/-) neonates showed strikingly impaired short- and long-term reconstitutive activity associated with a parallel decreased in vivo homing capacity of lineage negative (Lin(-)) cells. In addition an in vitro-reduced survival of immature progenitors (Lin(-) Kit(+) Sca(+)) was observed. Similar defects were found in liver cells from TGF-beta1(-/-) embryos on day 14 after vaginal plug. These data indicate that TGF-beta1 is a critical regulator for in vivo homeostasis of the HSCs, especially for their homing potential.

Oxygen tension level and human viral infections

The role of oxygen tension level is a well-known phenomenon that has been studied in oncology and radiotherapy since about 60 years. Oxygen tension may inhibit or stimulate propagation of viruses in vitro as well as in vivo. In turn modulating oxygen metabolism may constitute a novel approach to treat viral infections as an adjuvant therapy. The major transcription factor which regulates oxygen tension level is hypoxia-inducible factor-1 alpha (HIF-1α). Down-regulating the expression of HIF-1α is a possible method in the treatment of chronic viral infection such as human immunodeficiency virus infection, chronic hepatitis B and C viral infections and Kaposi sarcoma in addition to classic chemotherapy. The aim of this review is to supply an updating concerning the influence of oxygen tension level in human viral infections and to evoke possible new therapeutic strategies regarding this environmental condition.

Effects of endoplasmic reticulum stressors on maturation and signaling of hemizygous and heterozygous wild-type and mutant forms of KIT

Gain of function mutations of KIT are frequent in some human tumors, and are sensible to tyrosine kinase inhibitors. In most tumors, oncogenic mutations are heterozygous, however most in vitro data of KIT activation have been obtained with hemizygous mutation. This study aimed to investigate the maturation and activation of wild‐type (WT) and mutant (M) forms of KIT in hemizygous and heterozygous conditions. WT and two types of exon 11 deletions M forms of human KIT were expressed in NIH3T3 cell lines. Membrane expression of KIT was quantified by flow cytometry. Quantification of glycosylated forms of KIT and phosphorylated forms of AKT and ERK were performed by western blot. Simultaneous activation of WT KIT and treatment with endoplasmic reticulum (ER) inhibitors, tunicamycin or brefeldin A induced a complete inhibition of membrane expression of the 145 kDa form of KIT. By contrast activation or ER inhibitors alone, only partly inhibited this form. ER inhibitors also inhibited KIT activation‐dependent phosphorylation of AKT and ERK1/2. Brefeldin A induced a complete down regulation of the 145 kDa form in hemizygous M, and induced an intra‐cellular accumulation of the 125 kDa form in WT but not in hemizygous M. Heterozygous cells had glycosylation and response to ER inhibitors patterns more similar to WT than to hemizygous M. Phosphorylated AKT was reduced in hemizygous cells in comparison to WT KIT cells and heterozygous cells, and in the presence of brefeldin A in all cell lines. Effects of ER inhibitors are significantly different in hemizygous and heterozygous mutants. Differences in intra‐cellular trafficking of KIT forms result in differences in downstream signaling pathways, and activation of PI3K/AKT pathway appears to be tied to the presence of the mature 145 kDa form of KIT at the membrane surface.

Oxygen and viruses: a breathing story.

The effect of oxygen on virus replication is complex, and the role of hypoxia-inducible factor 1α (HIF-1α) in the metabolism of virus-infected cells remains uncertain. Solid tumours are hypoxic, and some viruses use this low oxygen tension level to facilitate their replication in tumour cells, thereby causing cell lysis. In addition, the interactions between viruses and HIF-1α may stimulate a trained immunity. However, the evolutionary basis for the oxygen regulatory mechanism of virus replication is ill-defined and requires further investigation.