Droplet Digital PCR for Non-Invasive Prenatal Detection of Fetal Single-Gene Point Mutations in Maternal Plasma.

Non-invasive prenatal testing (NIPT) is based on the detection and characterization of circulating cell-free fetal DNA (ccffDNA) in maternal plasma and aims to identify genetic abnormalities. At present, commercial NIPT kits can detect only aneuploidies, small deletions and insertions and some paternally inherited single-gene point mutations causing genetic diseases, but not maternally inherited ones. In this work, we have developed two NIPT assays, based on the innovative and sensitive droplet digital PCR (ddPCR) technology, to identify the two most common ß thalassemia mutations in the Mediterranean area (ß+IVSI-110 and ß039), maternally and/or paternally inherited, by fetal genotyping. The assays were optimized in terms of amplification efficiency and hybridization specificity, using mixtures of two genomic DNAs with different genotypes and percentages to simulate fetal and maternal circulating cell-free DNA (ccfDNA) at various gestational weeks. The two ddPCR assays were then applied to determine the fetal genotype from 52 maternal plasma samples at different gestational ages. The diagnostic outcomes were confirmed for all the samples by DNA sequencing. In the case of mutations inherited from the mother or from both parents, a precise dosage of normal and mutated alleles was required to determine the fetal genotype. In particular, we identified two diagnostic ranges for allelic ratio values statistically distinct and not overlapping, allowing correct fetal genotype determinations for almost all the analyzed samples. In conclusion, we have developed a simple and sensitive diagnostic tool, based on ddPCR, for the NIPT of ß+IVSI-110 and ß039 mutations paternally and, for the first time, maternally inherited, a tool, which may be applied to other single point mutations causing monogenic diseases.

Sexual dimorphism in the cerebrospinal fluid total protein content.

Objectives Cerebrospinal fluid (CSF) is a clear, colorless body fluid filling the central nervous system. The determination of the CSF total protein (TP) content represents an important screening test of various pathologies. We aimed to address the effect of sex and age on CSF-TP content and the use of the current upper reference limits (URLs). Methods CSF-TP content was analysed in a selected population of 1,252 patients (648 women and 604 men; age 18-89 years) who underwent lumbar puncture as a part of the diagnostic work-up. Samples presenting (i) more than 5 white blood cells (WBC)/µL, (ii) discolorations and (iii) reduced glucose were not included. Results The CSF-TP content median values were significantly higher in men than in women (46 vs. 37 mg/dL) even after adjusting for age and different hospital inpatients. CSF-TP content positively correlated with age both in men and in women with a constant difference between sexes of 8.5 mg/dL. Applying the most used URLs (mainly 45 and 50 mg/dL, but also 60 mg/dL), men received a laboratory report suggestive of altered CSF-TP content more frequently than women. The use of age- and sex-calibrated CSF-TP URLs reduced, but not eliminated, this sex-gap. Conclusions Using the current URLs, a condition of "elevated CSF-TP content" may be overestimated in men or, conversely, underestimated in women, regardless of the age and of the diagnosis. These results highlighted the need to apply CSF-TP URLs values ​​normalized for both sex and age.

Non-invasive fetal sex diagnosis in plasma of early weeks pregnants using droplet digital PCR.

BACKGROUND: Fetal sex determination is useful for families at risk of X-linked disorders, such as Duchenne muscular dystrophy, adrenal hypoplasia, hemophilia. At first, this could be obtained through invasive procedures such as amniocentesis and chorionic villus sampling, having a 1% risk of miscarriage. Since the discovery of cell-free fetal DNA (cffDNA) in maternal plasma, noninvasive prenatal testing permits the early diagnosis of fetal sex through analysis of cffDNA. However, the low amount of cffDNA relative to circulating maternal DNA requires highly sensitive molecular techniques in order to perform noninvasive prenatal diagnosis. In this context we employed droplet digital PCR (ddPCR) in order to evaluate the earliest possible fetal sex determination from circulating DNA extracted from plasma of pregnant women at different gestational ages. METHODS: We identified the fetal sex on cffDNA extracted from 29 maternal plasma samples at early gestational ages, several of them not suitable for qPCR determination, using ddPCR designed for SRY gene target. RESULTS: All maternal plasma samples were determined correctly for SRY gene target using ddPCR even at very early gestational age (prior to 7 weeks). CONCLUSIONS: The ddPCR is a robust, efficient and reliable technology for the earliest possible fetal sex determination from maternal plasma.

Y-chromosome identification in circulating cell-free fetal DNA using surface plasmon resonance.

OBJECTIVE: Since the discovery of cell-free fetal DNA (cffDNA) in maternal plasma, diagnostic non-invasive prenatal methods have been developed or optimized for fetal sex determination and identification of genetic diseases. As far as fetal sex determination, this might be important for therapeutic intervention on sex-associated pathologies such as Duchenne muscular dystrophy, hemophilia and congenital adrenal hyperplasia. Surface plasmon resonance (SPR)-based biosensors might be useful for these studies, because they allow to monitor the molecular interactions in real-time providing qualitative and quantitative information, through kinetics, affinity and concentration analyses. METHODS: The Biacore™ X100 has been applied to identify Y-chromosome sequence in cffDNA obtained from plasma samples of 26 pregnant women at different gestational ages. We have performed SPR-based analysis of SRY PCR products using SRY-specific probes immobilized on the sensor chip. RESULTS: We have demonstrated that there is a statistically significant difference between samples collected by pregnancies carrying male or female fetuses. Moreover, cffDNA obtained at early gestational ages and not detectable by conventional quantitative real-time PCR can be discriminated with high accuracy and reliability using SPR-based biosensors. CONCLUSIONS: These data, in addition to their direct applicability in more extensive diagnostic trials, should be considered as the basis of future developments.

Gene Dosage of F5 c.3481C>T Stop-Codon (p.R1161Ter) Switches the Clinical Phenotype from Severe Thrombosis to Recurrent Haemorrhage: Novel Hypotheses for Readthrough Strategy.

Inherited defects in the genes of blood coagulation essentially express the severity of the clinical phenotype that is directly correlated to the number of mutated alleles of the candidate leader gene (e.g., heterozygote vs. homozygote) and of possible additional coinherited traits. The F5 gene, which codes for coagulation factor V (FV), plays a two-faced role in the coagulation cascade, exhibiting both procoagulant and anticoagulant functions. Thus, defects in this gene can be predisposed to either bleeding or thrombosis. A Sanger sequence analysis detected a premature stop-codon in exon 13 of the F5 gene (c.3481C>T; p.R1161Ter) in several members of a family characterised by low circulating FV levels and contrasting clinical phenotypes. The propositus, a 29 y.o. male affected by recurrent haemorrhages, was homozygous for the F5 stop-codon and for the F5 c.1691G>A (p.R506Q; FV-Leiden) inherited from the heterozygous parents, which is suggestive of combined cis-segregation. The homozygous condition of the stop-codon completely abolished the F5 gene expression in the propositus (FV:Ag < 1%; FV:C < 1%; assessed by ELISA and PT-based one-stage clotting assay respectively), removing, in turn, any chance for FV-Leiden to act as a prothrombotic molecule. His father (57 y.o.), characterised by severe recurrent venous thromboses, underwent a complete molecular thrombophilic screening, revealing a heterozygous F2 G20210A defect, while his mother (56 y.o.), who was negative for further common coagulation defects, reported fully asymptomatic anamnesis. To dissect these conflicting phenotypes, we performed the ProC®Global (Siemens Helthineers) coagulation test aimed at assessing the global pro- and anticoagulant balance of each family member, investigating the responses to the activated protein C (APC) by means of an APC-sensitivity ratio (APC-sr). The propositus had an unexpectedly poor response to APC (APC-sr: 1.09; n.v. > 2.25), and his father and mother had an APC-sr of 1.5 and 2.0, respectively. Although ProC®Global prevalently detects the anticoagulant side of FV, the exceptionally low APC-sr of the propositus and his discordant severe-moderate haemorrhagic phenotype could suggest a residual expression of mutated FV p.506QQ through a natural readthrough or possible alternative splicing mechanisms. The coagulation pathway may be physiologically rebalanced through natural and induced strategies, and the described insights might be able to track the design of novel treatment approaches and rebalancing molecules.

Purinergic P2Y2 receptors promote neutrophil infiltration and hepatocyte death in mice with acute liver injury.

BACKGROUND & AIMS: During progression of liver disease, inflammation affects survival of hepatocytes. Endogenous release of adenosine triphosphate (ATP) in the liver activates purinergic P2 receptors (P2R), which regulate inflammatory responses, but little is known about the roles of these processes in the development of acute hepatitis. METHODS: We induced acute hepatitis in C57BL/6 mice by intravenous injection of concanavalin A and then analyzed liver concentrations of ATP and expression of P2R. We assessed P2Y(2)R(-/-) mice and C57BL/6 wild-type mice injected with suramin, a pharmacologic inhibitor of P2YR. Toxic liver failure was induced in mice by intraperitoneal injection of acetaminophen. Hepatocyte-specific functions of P2R signaling were analyzed in primary mouse hepatocytes. RESULTS: Induction of acute hepatitis in wild-type C57BL/6 mice released large amounts of ATP from livers and induced expression of P2Y(2)R. Liver damage and necrosis were greatly reduced in P2Y(2)R(-/-) mice and C57BL/6 mice given injections of suramin. Acetaminophen-induced liver damage was reduced in P2Y(2)R(-/-) mice. Analysis of liver-infiltrating immune cells during acute hepatitis revealed that expression of P2Y(2)R in bone marrow-derived cells was required for liver infiltration by neutrophils and subsequent liver damage. Hepatic expression of P2Y(2)R interfered with expression of genes that regulate cell survival, and promoted tumor necrosis factor-α-mediated cell death, in a cell-autonomous manner. CONCLUSIONS: Extracellular ATP and P2Y(2)R have cell-type specific, but synergistic functions during liver damage that regulate cellular immune responses and promote hepatocyte death. Reagents designed to target P2Y(2)R might be developed to treat inflammatory liver disease.

Why myotoxin-containing snake venoms possess powerful nucleotidases?

The venom of the snake Bothrops asper causes muscle necrosis, pain and inflammation. This venom contains myotoxins which cause an increase in intracellular Ca(2+) concentration and release of K(+) and ATP from myotubes. ATP is a key danger molecule that triggers a variety of reactions, including activation of the innate immune response. Here, using ATP-luciferase bioluminescence imaging technique, we show for the first time in vivo, that the purified myotoxins induce rapid release of ATP, whilst the complete venom of B. asper does at a very small extent. This apparent contradiction is explained by the finding that the venom contains powerful nucleotidases that in vivo convert ATP into ADP, AMP and Adenosine. These findings indicate that high concentrations of adenosine are generated by the double action of the venom and provide the experimental basis to the suggestion that in situ generated adenosine plays an important role in envenomation via its hypotensive, paralyzing and anti-coagulant activities.

The P2X7 receptor and Pannexin-1 are both required for the promotion of multinucleated macrophages by the inflammatory cytokine GM-CSF.

The P2X(7) receptor (P2X(7)R), an ATP-gated ion channel, has been implicated in the process of cell-to-cell fusion into multinucleated macrophages (MA), but its contribution to MA fusion driven by physiological/pathological stimuli is not clearly established. Based on several lines of evidence, we demonstrate that P2X(7)R is critical for the induction of multinucleated MA by the inflammatory cytokine GM-CSF: 1) pharmacological inhibition of P2X(7)R with oxidized ATP (oATP), KN-62, and the selective antagonist A740003 abrogated GM-CSF action on rat alveolar MA and murine peritoneal MA; 2) a murine J774 P2X(7) low MA clone, selected for defective P2X(7)R function, was unresponsive; 3) MA from mice lacking P2X(7)R failed to respond to GM-CSF, in contrast to wild-type. GM-CSF also stimulated ATP-induced membrane permeabilization in J774 P2X(7) high MA and rat alveolar MA, an effect absent in the P2X(7) low MA clone and inhibited by the P2X(7) blockers oATP and KN-62. Notably, the stimulatory effects of GM-CSF on pore formation and MA fusion were both inhibited by blocking functional Pannexin-1 (Panx-1), and GM-CSF failed to stimulate MA fusion in cells from Panx-1 knockout mice. We provide further evidence that extracellular ATP release from peritoneal MA is dependent on P2X(7) but not on Panx-1 expression and that its metabolism to adenosine mediates P2X(7)-dependent MA fusion. These data demonstrate that both P2X(7) and Panx-1 are required for GM-CSF promotion of MA fusion but likely act independently through different signaling pathway(s).

P2X7 receptor drives osteoclast fusion by increasing the extracellular adenosine concentration.

Defects in bone homeostasis are a major health problem. Osteoclast differentiation and activation have a crucial role in bone remodeling in health and disease. Osteoclasts are bone-resorbing cells derived from mononuclear phagocyte progenitors. The key event in osteoclast formation is fusion of mononucleate precursors to form mature multinucleated osteclasts. Here we provide evidence of an absolute requirement for the P2X7 receptor, ATP release, and adenosine signaling in human osteoclast formation, as shown by the following findings: macrophage-colony stimulating factor/receptor activator for nuclear factor-κB ligand (M-CSF/RANKL)-stimulated fusion of human monocytes is fully prevented by an anti-P2X7 mAb, by specific P2X7 pharmacological antagonists, or by inhibition of CD39/NTPDase; fusion-competent monocytes release ATP via the P2X7 receptor; accelerated degradation of released ATP by addition of either apyrase or hexokinase strongly increases fusion; removal of extracellular adenosine by adenosine deaminase blocks, while addition of exogenous adenosine strongly potentiates, fusion; and pharmacologic stimulation of the adenosine A2A receptor increases, while selective A2A blockade inhibits, fusion. These results show that the purinergic axis plays a crucial and as yet undescribed role in osteoclast formation and reconcile previous evidence advocating a key role for either ATP or adenosine receptors in multinucleated giant cell formation.

Host genetics impact on SARS-CoV-2 vaccine-induced immunoglobulin levels and dynamics: The role of TP53, ABO, APOE, ACE2, HLA-A, and CRP genes.

Background: Development and worldwide availability of safe and effective vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) to fight severe symptoms of coronavirus disease 2019 (COVID-19) and block the pandemic have been a great achievement and stimulated researchers on understanding the efficacy and duration of different vaccine types. Methods: We investigated the levels of anti-SARS-CoV-2 antibodies (IgG) and neutralizing antibodies (NAbs) in 195 healthy adult subjects belonging to the staff of the University-Hospital of Ferrara (Italy) starting from 15 days up to 190 days (about 6 months) after the second dose of the BNT162b2 (Pfizer-BioNTech) mRNA-based vaccine (n = 128) or ChAdOx1 (AstraZeneca) adenovirus-based vaccine (n = 67) using a combined approach of serological and genomics investigations. Results: A strong correlation between IgG and NAb levels was detected during the 190 days of follow-up (r 2 = 0.807; p < 0.0001) and was confirmed during the first 90 days (T1) after vaccination (r 2 = 0.789; p = 0.0001) and 91-190 days (T2) after vaccination (r 2 = 0.764; p = 0.0001) for both vaccine types (r 2 = 0.842; p = 0.0001 and r 2 = 0.780; p = 0.0001 for mRNA- and adenovirus-based vaccine, respectively). In addition to age (p < 0.01), sex (p = 0.03), and type of vaccine (p < 0.0001), which partially accounted for the remarkable individual differences observed in the antibody levels and dynamics, interesting genetic determinants appeared as significant modifiers of both IgG and NAb responses among the selected genes investigated (TP53, rs1042522; APOE, rs7412/rs429358; ABO, rs657152; ACE2, rs2285666; HLA-A rs2571381/rs2499; CRP, rs2808635/rs876538; LZTFL1, rs35044562; OAS3, rs10735079; SLC6A20, rs11385942; CFH, rs1061170; and ACE1, ins/del, rs4646994). In detail, regression analysis and mean antibody level comparison yielded appreciable differences after genotype stratification (P1 and P2, respectively, for IgG and NAb distribution) in the whole cohort and/or in the mRNA-based vaccine in the following genes: TP53, rs1042522 (P1 = 0.03; P2 = 0.04); ABO, rs657152 (P1 = 0.01; P2 = 0.03); APOE, rs7412/rs429358 (P1 = 0.0018; P2 = 0.0002); ACE2, rs2285666 (P1 = 0.014; P2 = 0.009); HLA-A, rs2571381/rs2499 (P1 = 0.02; P2 = 0.03); and CRP, rs2808635/rs876538 (P1 = 0.01 and P2 = 0.09). Conclusion: High- or low-responsive subjects can be identified among healthy adult vaccinated subjects after targeted genetic screening. This suggests that favorable genetic backgrounds may support the progression of an effective vaccine-induced immune response, though no definite conclusions can be drawn on the real effectiveness ascribed to a specific vaccine or to the different extent of a genotype-driven humoral response. The interplay between data from the polygenic predictive markers and serological screening stratified by demogeographic information can help to recognize the individual humoral response, accounting for ethnic and geographical differences, in both COVID-19 and anti-SARS-CoV-2 vaccinations.

Trophic activity of a naturally occurring truncated isoform of the P2X7 receptor.

P2X7 is the largest member of the P2X subfamily of purinergic receptors. A typical feature is the carboxyl tail, which allows formation of a large pore. Recently a naturally occurring truncated P2X7 splice variant, isoform B (P2X7B), has been identified. Here we show that P2X7B expression in HEK293 cells, a cell type lacking endogenous P2X receptors, mediated ATP-stimulated channel activity but not plasma membrane permeabilization, raised endoplasmic reticulum Ca(2+) content, activated the transcription factor NFATc1, increased the cellular ATP content, and stimulated growth. In addition, P2X7B-transfected HEK293 cells (HEK293-P2X7B), like most tumor cells, showed strong soft agar-infiltrating ability. When coexpressed with full-length P2X7 (P2X7A), P2X7B coassembled with P2X7A into a heterotrimer and potentiated all known responses mediated by this latter receptor. P2X7B mRNA was found to be widely distributed in human tissues, especially in the immune and nervous systems, and to a much higher level than P2X7A. Finally, P2X7B expression was increased on mitogenic stimulation of peripheral blood lymphocyte. Altogether, these data show that P2X7B is widely expressed in several human tissues, modulates P2X7A functions, participates in the control of cell growth, and may help understand the role of the P2X7 receptor in the control of normal and cancer cell proliferation.

A novel recombinant plasma membrane-targeted luciferase reveals a new pathway for ATP secretion.

ATP is emerging as an ubiquitous extracellular messenger. However, measurement of ATP concentrations in the pericellular space is problematic. To this aim, we have engineered a firefly luciferase-folate receptor chimeric protein that retains the N-terminal leader sequence and the C-terminal GPI anchor of the folate receptor. This chimeric protein, named plasma membrane luciferase (pmeLUC), is targeted and localized to the outer aspect of the plasma membrane. PmeLUC is sensitive to ATP in the low micromolar to millimolar level and is insensitive to all other nucleotides. To identify pathways for nonlytic ATP release, we transfected pmeLUC into cells expressing the recombinant or native P2X7 receptor (P2X7R). Both cell types release large amounts of ATP (100-200 microM) in response to P2X7R activation. This novel approach unveils a hitherto unsuspected nonlytic pathway for the release of large amounts of ATP that might contribute to spreading activation and recruitment of immune cells at inflammatory sites.

Postnatal and non-invasive prenatal detection of ß-thalassemia mutations based on Taqman genotyping assays.

The ß-thalassemias are genetic disorder caused by more than 200 mutations in the ß-globin gene, resulting in a total (ß0) or partial (ß+) deficit of the globin chain synthesis. The most frequent Mediterranean mutations for ß-thalassemia are: ß039, ß+IVSI-110, ß+IVSI-6 and ß0IVSI-1. Several molecular techniques for the detection of point mutations have been developed based on the amplification of the DNA target by polymerase chain reaction (PCR), but they could be labor-intensive and technically demanding. On the contrary, TaqMan® genotyping assays are a simple, sensitive and versatile method suitable for the single nucleotide polymorphism (SNP) genotyping affecting the human ß-globin gene. Four TaqMan® genotyping assays for the most common ß-thalassemia mutations present in the Mediterranean area were designed and validated for the genotype characterization of genomic DNA extracted from 94 subjects comprising 25 healthy donors, 33 healthy carriers and 36 ß-thalassemia patients. In addition, 15 specimens at late gestation (21-39 gestational weeks) and 11 at early gestation (5-18 gestational weeks) were collected from pregnant women, and circulating cell-free fetal DNAs were extracted and analyzed with these four genotyping assays. We developed four simple, inexpensive and versatile genotyping assays for the postnatal and prenatal identification of the thalassemia mutations ß039, ß+IVSI-110, ß+IVSI-6, ß0IVSI-1. These genotyping assays are able to detect paternally inherited point mutations in the fetus and could be efficiently employed for non-invasive prenatal diagnosis of ß-globin gene mutations, starting from the 9th gestational week.

Autophagy-dependent anticancer immune responses induced by chemotherapeutic agents in mice.

Antineoplastic chemotherapies are particularly efficient when they elicit immunogenic cell death, thus provoking an anticancer immune response. Here we demonstrate that autophagy, which is often disabled in cancer, is dispensable for chemotherapy-induced cell death but required for its immunogenicity. In response to chemotherapy, autophagy-competent, but not autophagy-deficient, cancers attracted dendritic cells and T lymphocytes into the tumor bed. Suppression of autophagy inhibited the release of adenosine triphosphate (ATP) from dying tumor cells. Conversely, inhibition of extracellular ATP-degrading enzymes increased pericellular ATP in autophagy-deficient tumors, reestablished the recruitment of immune cells, and restored chemotherapeutic responses but only in immunocompetent hosts. Thus, autophagy is essential for the immunogenic release of ATP from dying cells, and increased extracellular ATP concentrations improve the efficacy of antineoplastic chemotherapies when autophagy is disabled.

Lack of the purinergic receptor P2X(7) results in resistance to contact hypersensitivity.

Sensitization to contact allergens requires activation of the innate immune system by endogenous danger signals. However, the mechanisms through which contact allergens activate innate signaling pathways are incompletely understood. In this study, we demonstrate that mice lacking the adenosine triphosphate (ATP) receptor P2X(7) are resistant to contact hypersensitivity (CHS). P2X(7)-deficient dendritic cells fail to induce sensitization to contact allergens and do not release IL-1ß in response to lipopolysaccharide (LPS) and ATP. These defects are restored by pretreatment with LPS and alum in an NLRP3- and ASC-dependent manner. Whereas pretreatment of wild-type mice with P2X(7) antagonists, the ATP-degrading enzyme apyrase or IL-1 receptor antagonist, prevents CHS, IL-1ß injection restores CHS in P2X(7)-deficient mice. Thus, P2X(7) is a crucial receptor for extracellular ATP released in skin in response to contact allergens. The lack of P2X(7) triggering prevents IL-1ß release, which is an essential step in the sensitization process. Interference with P2X(7) signaling may be a promising strategy for the prevention of allergic contact dermatitis.

Graft-versus-host disease is enhanced by extracellular ATP activating P2X7R.

Danger signals released upon cell damage can cause excessive immune-mediated tissue destruction such as that found in acute graft-versus-host disease (GVHD), allograft rejection and systemic inflammatory response syndrome. Given that ATP is found in small concentrations in the extracellular space under physiological conditions, and its receptor P2X(7)R is expressed on several immune cell types, ATP could function as a danger signal when released from dying cells. We observed increased ATP concentrations in the peritoneal fluid after total body irradiation, and during the development of GVHD in mice and in humans. Stimulation of antigen-presenting cells (APCs) with ATP led to increased expression of CD80 and CD86 in vitro and in vivo and actuated a cascade of proinflammatory events, including signal transducer and activator of transcription-1 (STAT1) phosphorylation, interferon-γ (IFN-γ) production and donor T cell expansion, whereas regulatory T cell numbers were reduced. P2X(7)R expression increased when GVHD evolved, rendering APCs more responsive to the detrimental effects of ATP, thereby providing positive feedback signals. ATP neutralization, early P2X(7)R blockade or genetic deficiency of P2X(7)R during GVHD development improved survival without immune paralysis. These data have major implications for transplantation medicine, as pharmacological interference with danger signals that act via P2X(7)R could lead to the development of tolerance without the need for intensive immunosuppression.

The human cathelicidin LL-37 modulates the activities of the P2X7 receptor in a structure-dependent manner.

Extracellular ATP, released at sites of inflammation or tissue damage, activates the P2X(7) receptor, which in turn triggers a range of responses also including cell proliferation. In this study the ability of the human cathelicidin LL-37 to stimulate fibroblast growth was inhibited by commonly used P2X(7) blockers. We investigated the structural requirements of the growth-promoting activity of LL-37 and found that it did not depend on helix sense (the all-d analog was active) but did require a strong helix-forming propensity in aqueous solution (a scrambled analog and primate LL-37 orthologs devoid of this property were inactive). The involvement of P2X(7) was analyzed using P2X(7)-expressing HEK293 cells. LL-37 induced proliferation of these cells, triggered Ca(2+) influx, promoted ethidium bromide uptake, and synergized with benzoyl ATP to enhance the pore and channel functions of P2X(7). The activity of LL-37 had an absolute requirement for P2X(7) expression as it was blocked by the P2X(7) inhibitor KN-62, was absent in cells lacking P2X(7), and was restored by P2X(7) transfection. Of particular interest, LL-37 led to pore-forming activity in cells expressing a truncated P2X(7) receptor unable to generate the non-selective pore typical of the full-length receptor. Our results indicate that P2X(7) is involved in the proliferative cell response to LL-37 and that the structural/aggregational properties of LL-37 determine its capacity to modulate the activation state of P2X(7).

Increased level of extracellular ATP at tumor sites: in vivo imaging with plasma membrane luciferase.

BACKGROUND: There is growing awareness that tumour cells build up a "self-advantageous" microenvironment that reduces effectiveness of anti-tumour immune response. While many different immunosuppressive mechanisms are likely to come into play, recent evidence suggests that extracellular adenosine acting at A2A receptors may have a major role in down-modulating the immune response as cancerous tissues contain elevated levels of adenosine and adenosine break-down products. While there is no doubt that all cells possess plasma membrane adenosine transporters that mediate adenosine uptake and may also allow its release, it is now clear that most of extracellularly-generated adenosine originates from the catabolism of extracellular ATP. METHODOLOGY/PRINCIPAL FINDINGS: Measurement of extracellular ATP is generally performed in cell supernatants by HPLC or soluble luciferin-luciferase assay, thus it generally turns out to be laborious and inaccurate. We have engineered a chimeric plasma membrane-targeted luciferase that allows in vivo real-time imaging of extracellular ATP. With this novel probe we have measured the ATP concentration within the tumour microenvironment of several experimentally-induced tumours. CONCLUSIONS/SIGNIFICANCE: Our results show that ATP in the tumour interstitium is in the hundreds micromolar range, while it is basically undetectable in healthy tissues. Here we show that a chimeric plasma membrane-targeted luciferase allows in vivo detection of high extracellular ATP concentration at tumour sites. On the contrary, tumour-free tissues show undetectable extracellular ATP levels. Extracellular ATP may be crucial for the tumour not only as a stimulus for growth but also as a source of an immunosuppressive agent such as adenosine. Our approach offers a new tool for the investigation of the biochemical composition of tumour milieu and for development of novel therapies based on the modulation of extracellular purine-based signalling.

Involvement of the purinergic P2X7 receptor in the formation of multinucleated giant cells.

Multinucleated giant cells (MGC), a hallmark of chronic inflammatory reactions, remain an enigma of cell biology. There is evidence implicating the purinergic P2X7 receptor in the fusion process leading to MGC. To investigate this, we used HEK 293 cells stably transfected with either 1) the full-length rat P2X7 receptor (P2X7 cells), 2) a rat P2X7 receptor lacking the C-terminal domain (P2X7TC), or 3) a mock vector, and rat alveolar macrophages (MA) expressing the native receptor. P2X7 cells cultured in serum-free medium formed increased numbers of MGC and displayed a higher fusion index compared with mock transfectants. Stimulation of P2X7 pore-forming activity in P2X7 cells by polymyxin B (PMB) further increased significantly the formation of MGC. Conversely, blockers of P2X-receptors including oxidized ATP, brilliant blue G, and pyridoxal phosphate-6-azophenyl-2'-4'-disulfonic acid inhibited significantly MGC formation in both unstimulated and PMB-stimulated P2X7-transfected cells. In contrast, cells transfected with the truncated P2X7TC were devoid of pore-forming activity, did not respond to PMB stimulation, and failed to form enhanced numbers of MGC, thus behaving as mock transfectants. As found for P2X7-transfected cells, PMB also potentiated dose-dependently the formation of multinucleated MA by rat alveolar MA. Pretreatment with oxidized ATP abrogated the PMB stimulatory effects. Together, these data demonstrate unequivocally the participation of P2X7 receptor in the process of MGC formation. Our study also provides evidence suggesting that stimulation of the P2X7 receptor pathway in MA may mediate increased formation of MGC during chronic inflammatory reactions.