Comprehensive germline mutation analysis and clinical profile in a large cohort of Brazilian xeroderma pigmentosum patients.

BACKGROUND: Xeroderma pigmentosum (XP) patients present a high risk of developing skin cancer and other complications at an early age. This disease is characterized by mutations in the genes related to the DNA repair system. OBJECTIVES: To describe the clinical and molecular findings in a cohort of 32 Brazilian individuals who received a clinical diagnosis of XP. METHODS: Twenty-seven families were screened for germline variants in eight XP-related genes. RESULTS: All patients (N = 32) were diagnosed with bi-allelic germline pathogenic or potentially pathogenic variants, including nine variants previously undescribed. The c.2251-1G>C XPC pathogenic variant, reported as the founder mutation in Comorian and Pakistani patients, was observed in 15 cases in homozygous or compound heterozygous. Seven homozygous patients for POLH/XPV variants developed their symptoms by an average age of 7.7 years. ERCC2/XPD, DDB2/XPE and ERCC5/XPG variants were found in a few patients. Aside from melanoma and non-melanoma skin tumours, a set of patients developed skin sebaceous carcinoma, leiomyosarcoma, angiosarcoma, mucoepidermoid carcinoma, gastric adenocarcinoma and serous ovarian carcinoma. CONCLUSIONS: We reported a high frequency of XPC variants in 32 XP Brazilian patients. Nine new variants in XP-related genes, unexpected non-skin cancer lesions and an anticipation of the clinical manifestation in POLH/XPV cases were also described.

Germline large genomic alterations on 7q in patients with multiple primary cancers.

Patients with multiple primary cancers (MPCs) are suspected to have a hereditary cancer syndrome. However, only a small proportion may be explained by mutations in high-penetrance genes. We investigate two unrelated MPC patients that met Hereditary Breast and Ovaria Cancer criteria, both presenting triple negative breast tumors and no mutations in BRCA1, BRCA2 and TP53 genes. Germline rearrangements on chromosome 7q, involving over 40 Mb of the same region, were found in both patients: one with mosaic loss (80% of cells) and the other with cnLOH (copy-neutral loss of heterozygosity) secondary to maternal allele duplication. Five children tested had no alterations on 7q. The patients shared 330 genes in common on 7q22.1-q34, including several tumor suppressor genes (TSGs) previously related to breast cancer risk and imprinted genes. The analysis of the triple negative BC from one patient revealed a mosaic gain of 7q translated for over-expressed cancer-related genes. The involvement of TSGs and imprinted genes, mapped on 7q, has the potential of being associated to MPC risk, as well as cancer progression. To our knowledge, this is the first description of patients with MPCs that harbor constitutive large alterations on 7q.

Fas ligand triggers pulmonary silicosis.

We investigated the role of Fas ligand in murine silicosis. Wild-type mice instilled with silica developed severe pulmonary inflammation, with local production of tumor necrosis factor (TNF)-alpha, and interstitial neutrophil and macrophage infiltration in the lungs. Strikingly, Fas ligand-deficient generalized lymphoproliferative disease mutant (gld) mice did not develop silicosis. The gld mice had markedly reduced neutrophil extravasation into bronchoalveolar space, and did not show increased TNF-alpha production, nor pulmonary inflammation. Bone marrow chimeras and local adoptive transfer demonstrated that wild-type, but not Fas ligand-deficient lung macrophages recruit neutrophils and initiate silicosis. Silica induced Fas ligand expression in lung macrophages in vitro and in vivo, and promoted Fas ligand-dependent macrophage apoptosis. Administration of neutralizing anti-Fas ligand antibody in vivo blocked induction of silicosis. Thus, Fas ligand plays a central role in induction of pulmonary silicosis.

Comparative analysis of syngeneic and allogeneic mixed lymphocyte reaction of 'naturally' activated and resting T cells.

'Naturally' activated (NA) or resting T lymphocytes obtained from the spleen of normal BALB/c mice were compared in their capacity to mount a syngeneic mixed lymphocyte reaction (SMLR). Both T-cell subsets were able to proliferate and secrete IL-3/GM-CSF in SMLR cultures. IL-2 was present in 'resting' T-cell SMLR supernatants, and barely detectable in NA T-cell SMLR supernatants. Both NA and 'resting' T-cell SMLRs were inhibited with anti-class II, anti-CD4, or anti-IL-2R MoAbs. NA T cells exhibited a background proliferative and secretory activity in the absence of syngeneic accessory cells. This autonomous activity was susceptible to anti-CD4, but poorly inhibited with anti-class II MoAbs. Both NA and 'resting' T lymphocytes displayed strong responsiveness to allogeneic stimuli. The analysis of the relative frequency of proliferating cells in the SMLR (BALB/c), or allo-MLR (B10, B10.A, B10.D2) from NA or 'resting' T cells indicated an enrichment for syngeneic reactivity among NA T lymphocytes. The meaning of these results for NA T-cell function and repertoire is discussed.

Naturally activated and resting T cells differ in their activation requirements for growth and secretory activities.

"Naturally activated" (NA) and "small resting" (SR) T lymphocytes were stimulated with anti-CD3 or anti-thy 1 monoclonal antibodies (mAbs). Both proliferation and secreted IL-2, IL-3/GM-CSF activities were found in NA T cell, but not in SR T cell cultures. SR T cells could be fully activated by anti-CD3 only if PMA or IL-2 was added to the cultures. NA T cell proliferation induced with anti-CD3 was blocked with anti-IL-2 or anti-IL-2R mAbs. The combination of anti-CD3 and rec IL-4 was not effective in promoting SR T cell proliferation. IL-4 plays a minor role in NA T cell activation with anti-CD3, as assayed with neutralizing anti IL-4 mAbs. No differences in the proliferative and secretory activities were found when NA or SR T cells were stimulated with Con A. Both NA and SR T cells responded when stimulated with the calcium ionophone A23187 plus PMA. Only NA T cells responded to A23187 alone. The mechanisms and the possible physiologic relevance of this differential responsiveness behavior are discussed.

Do self-heart-reactive T cells expand in Trypanosoma cruzi-immune hosts?

Anti-heart T-cell activity was evaluated by a lymph node cell proliferative assay in isogenic strains of mice immunized with several Trypanosoma cruzi epimastigote and trypomastigote antigenic preparations. In addition, chronically infected animals were boosted with trypomastigote antigens and their lymph node cells were tested by in vitro proliferative responses. Our results indicated that (i) use of allogeneic sources of heart antigens may induce alloreactive responses in T. cruzi-immune T cells, (ii) specific autoimmune T-cell reactivity against self-heart constituents could not be demonstrated after immunization of the host with T. cruzi, and (iii) a proportion of chronically infected mice showed a small but detectable level of auto-anti-heart T-cell reactivity. These results argue against the notion that T. cruzi epitopes cross-reactive with self-heart tissue play a role in initiating T-cell-mediated autoimmunity. Anti-heart autoreactive T cells, generated in a proportion of the animals, may result from heart lesions associated with the infection process.

Stage-specific distinctions in potassium channel blocker control of T-lymphocyte activation.

Effects of four known blockers of T-lymphocyte potassium channels [verapamil, quinine, 4-aminopyridine (4-AP) and tetraethylammonium (TEA)], were studied on polyclonal T-cell activation induced by two plant mitogens (phytohemmaglutinin; PHA and concanavalin A; ConA), a mitogenic anti-Thy 1.2 monoclonal antibody (mAb G7) and phorbol ester (phorbol myristate acetate; PMA). Potassium channel blockers blocked all four modes of T-cell activation in a dose-dependent fashion with the same rank order of potency (verapamil greater than quinine greater than 4-AP greater than TEA). Kinetic studies of the timing of potassium channel blocker effect, indicated that, while 4-AP and TEA interfere only with early events of T-cell activation, verapamil and quinine can also interfere with later steps of T-cell mitogenesis. This notion was confirmed by studies of interleukin 2(IL-2)-directed activated T-cell growth. Verapamil and quinine blocked this late step in different types of activated T-cells with the same potency they blocked induction of resting T-cell mitogenesis. On the other hand, 4-AP and TEA, at maximal inhibitory doses for resting T-cells, showed little or no effect at IL-2-directed growth. Kinetic studies of the timing of quinine effect showed that the target of quinine action on activated T-cells is critically involved in IL-2 signalling within the first 2-4 h of IL-2 addition. These studies suggest that, besides the voltage-gated potassium channel previously described, a second target for verapamil and quinine action controls IL-2-derived signals to activated T-cells.

Analysis of isolated and combined effects of calcium ionophore and phorbol ester on T lymphocyte activation.

Isolated and combined effects of the calcium ionophore A23187 and of the protein kinase C activator phorbol myristate acetate (PMA) on T cell activation parameters were analysed on unprimed Balb/c lymph node T lymphocytes (LNL). High doses of PMA were mitogenic for resting T cells, but non-mitogenic doses of PMA induced T cell proliferation in combination with A23187, which was non-mitogenic by itself. Mitogenesis induced by a combination of A23187 and PMA (A23187/PMA) showed the following characteristics: it was not abolished after extensive depletion of accessory cells; purified L3T4+, but not Lyt2+ T cells responded in the absence of accessory cells; mitogenesis was completely blocked by a mixture of two monoclonal antibodies directed to the murine interleukin 2 (IL-2) receptor (7D4/3C7mAbs); cyclosporin A, dibutyril cyclic AMP, and T cell K+ channel blockers quinine and verapamil all blocked mitogenesis. A marked synergism between A23187 and PMA was noted in induction of T cell enlargement, IL-2 release, and induction of IL-2 responsiveness. No synergism was noted in IL-2 receptor expression, A23187 and PMA being able to induce IL-2 receptors alone. Calcium ionophore induced IL-2 receptor expression, but failed to induce IL-2 release and IL-2 responsiveness. Addition of A23187/PMA to the IL-2-dependent CTL-L clone did not result in cell proliferation. Addition of A23187/PMA to Con A-activated T cell blasts leads to a vigorous proliferative response. This response is blocked by 7D4/3C7 mAbs, indicating a role for endogenously produced IL-2 in this case. The results indicate that T cell mitogenesis by A23187/PMA is IL-2-dependent, and suggest a critical role for protein kinase C in IL-2 release and induction of IL-2 responsiveness. In addition, the data suggest distinct, but co-operative pathways of IL-2 receptor induction, controlled by elevated Ca2+ alone and by protein kinase C. Subsequent intracellular events of T cell activation by A23187/PMA may be quite similar to those triggered by Con A, since both kinds of stimulation are blocked by agents such as cyclosporin A, dbcAMP and K+ channel blockers.

Purinergic modulation of T-lymphocyte activation: differential susceptibility of distinct activation steps and correlation with intracellular 3',5'-cyclic adenosine monophosphate accumulation.

The mechanism by which purinergic agonists modulate murine T-lymphocyte activation and proliferation was investigated. Adenosine and other compounds such as ATP and 2-chloroadenosine (ClAdo) were found to block T-cell mitogenesis induced by concanavalin A (Con A) in a dose-dependent fashion. The nonmetabolizable adenosine analog ClAdo was the most potent agent capable of inhibiting T-cell mitogenesis. Extracellular addition of the permeable cAMP analog dibutyryl cyclic AMP (dbcAMP) also led to a dose-dependent blockade of T-cell mitogenesis, although with less efficiency when compared to ClAdo. Addition of IL-2-enriched fluids failed to reverse blockade of T-cell mitogenesis by ClAdo or dbcAMP. ClAdo blocked T-cell enlargement induced after 20 hr of culture with Con A. We analyzed the effect of micromolar concentrations of ClAdo on interleukin-2 (IL-2) production, expression of IL-2 receptors (7D4 and 3C7 surface antigens), and induction of IL-2 responsiveness after in vitro cultivation with Con A. ClAdo inhibited both IL-2 secretion and induction of IL-2 responsiveness up to control levels in the same dose range it inhibited T-cell mitogenesis. However, cell surface expression of IL-2 receptors was not affected. Short incubations of resting splenic T cells with ClAdo led to a dose-dependent accumulation of cyclic AMP in responding cells. This effect was markedly reduced by the purinergic antagonist 3-isobutyl-1-methylxanthine (IBMX) but was not prevented by the adenosine uptake blocker dipyridamole. ClAdo elicited cAMP accumulation in the same dose range it inhibited T-cell activation events. Extracellular administration of dbcAMP to splenic T cells stimulated by Con A mimicked the effects of ClAdo on T-cell activation parameters, as revealed by a dose-dependent blockade of both IL-2 secretion and IL-2 responsiveness induction, without affecting IL-2 receptor expression. Short incubations of Con A-activated T-cell blasts with ClAdo also led to a dose-dependent accumulation of cAMP. We then analyzed the effect of purines and dbcAMP on IL-2-mediated activated T-cell growth. Purines caused a dose-dependent inhibition of IL-2-mediated T-cell proliferation and ClAdo was the most potent purinergic agonist tested. The effect of ClAdo on Con A-induced T blasts was shifted to the right, if compared to earlier T-cell activation steps.(ABSTRACT TRUNCATED AT 400 WORDS)