RESUMEN
Pancreatic cancer (PC) remains the predominant type of upper gastrointestinal tract cancer, associated with heightened morbidity and a survival rate below 12%. While immunotherapy has brought about transformative changes in the standards of care for most solid tumors, its application in PC is hindered by the ''cold tumor'' microenvironment, marked by the presence of immunosuppressive cells. Modest response rates in PC are attributed, in part to, the fibrotic stroma that obstructs the delivery of systemic immunotherapy. Furthermore, the occurrence of immune-related adverse events (iRAEs) often necessitates the use of sub-therapeutic doses or treatment discontinuation. In the pursuit of innovative approaches to enhance the effectiveness of immunotherapy for PC, implantable drug delivery devices and scaffolds emerge as promising strategies. These technologies offer the potential for sustained drug delivery directly to the tumor site, overcoming stromal barriers, immunosuppression, T cell exclusion, immunotherapy resistance, optimizing drug dosage, and mitigating systemic toxicity. This review offers a comprehensive exploration of pancreatic ductal adenocarcinoma (PDAC), the most common and aggressive form of PC, accompanied by a critical analysis of the challenges the microenvironment presents to the development of successful combinational immunotherapy approaches. Despite efforts, these approaches have thus far fallen short in enhancing treatment outcomes for PDAC. The review will subsequently delve into the imperative need for refining delivery strategies, providing an examination of past and ongoing studies in the field of localized immunotherapy for PDAC. Addressing these issues will lay the groundwork for the development of effective new therapies, thereby enhancing treatment response, patient survival, and overall quality of life for individuals diagnosed with PDAC.
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AIMS: Application of exogenous nucleotides can modulate wound healing via the activation of purinergic receptors. However, evidence for the release of endogenous nucleotides and the subsequent activation of purinergic receptors in this process has not been well defined. Therefore, the current study aimed to investigate wound-mediated nucleotide release and autocrine purinergic signalling during HaCaT keratinocyte wound closure following scratch injury. MAIN METHODS: An in vitro scratch wound apparatus was employed to study wound healing over 24-h in the presence of modulators of ATP release, P2 receptors and pathways downstream of P2 receptor activation. KEY FINDINGS: Adenosine 5'-triphosphate (ATP) was released from scratched cells. The ectonucleotidase apyrase and pharmacological inhibition of the nucleotide release hemichannel, pannexin-1, decreased wound closure over time. The non-selective P2Y receptor antagonist suramin and the selective P2Y2 receptor antagonist AR-C118925XX, but not other P2 antagonists, decreased wound closure. AR-C118925XX decreased wound closure in a concentration-dependent fashion. However, exogenous P2Y2 receptor agonists, ATP or uridine 5'-triphosphate, did not enhance wound closure. PCR and immunoblotting confirmed P2Y2 receptor expression in HaCaT cells. U73122, a phospholipase C antagonist, and 2-aminoethoxydiphenylborate, an inositol 1,4,5-trisphosphate receptor-sensitive Ca2+-release channel antagonist, decreased wound closure consistent with P2Y2 receptor activation. Absence of extracellular or intracellular Ca2+ or inhibition of intracellular Ca2+-release also impaired wound closure. SIGNIFICANCE: These data describe a novel autocrine signalling mechanism in which wound-mediated release of endogenous ATP in response to mechanical scratching of HaCaT cells activates P2Y2 receptors to facilitate wound closure.
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Adenosina Trifosfato/metabolismo , Comunicación Autocrina , Receptores Purinérgicos P2Y2/metabolismo , Cicatrización de Heridas , Células HEK293 , Humanos , Antagonistas del Receptor Purinérgico P2Y/farmacología , Suramina/farmacologíaRESUMEN
Missense variants are associated with various phenotypic traits and disorders in dogs. The canine P2RX7 gene, coding the ATP-gated P2X7 receptor ion channel, contains four known missense variants. The current study aimed to examine the presence of these variants in a random sample of pedigree and mixed-pedigree dogs. Exons 3, 8, 11 and 13 of the P2RX7 gene, encoding these four respective variants, in 65 dogs were assessed by Sanger sequencing and combined with existing sequencing data from another 69 dogs. The distribution of these variants was then evaluated in all 134 dogs combined and separately within individual breeds including 35 different pure breeds. The rs23314713 (p.Phe103Leu) and rs23315462 (p.Pro452Ser) variants were present in 47 and 40% of all dogs studied respectively, with the rs23314713 variant associated with brachycephalic breeds. Among pedigree dogs, the rs23314713 and rs23315462 variants were associated with brachycephalic and non-brachycephalic breeds respectively. The rs851148233 (p.Arg270Cys) and rs850760787 (p.Arg365Gln) variants were present only in dogs of Cocker Spaniel and Labrador Retriever pedigrees respectively. No other missense variants were found in exons 3, 8, 11 and 13 of the P2RX7 gene within the dogs. In conclusion, the rs23314713 and rs23315462 missense variants of the P2RX7 gene are present in a large proportion of dogs, with the rs23314713 variant associated with a number of brachycephalic breeds. However, the association of this variant with dogs of bulldog ancestry, not brachycephaly per se, cannot be excluded.
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Craneosinostosis/veterinaria , Enfermedades de los Perros/genética , Perros/genética , Mutación Missense , Receptores Purinérgicos P2X7/genética , Animales , Cruzamiento , Craneosinostosis/genética , LinajeRESUMEN
Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative method for blood cancers and other blood disorders, but is limited by the development of graft-versus-host disease (GVHD). GVHD results in inflammatory damage to the host liver, gastrointestinal tract and skin, resulting in high rates of morbidity and mortality in HSCT recipients. Activation of the A2A receptor has been previously demonstrated to reduce disease in allogeneic mouse models of GVHD. This study aimed to investigate the effect of A2A activation on disease development in a humanised mouse model of GVHD. Immunodeficient non-obese diabetic-severe combined immunodeficiency-interleukin (IL)-2 receptor γnull (NSG) mice injected with human (h) peripheral blood mononuclear cells (hPBMCs), were treated with either the A2A agonist CGS 21680 or control vehicle. Contrary to the beneficial effect of A2A activation in allogeneic mouse models, CGS 21680 increased weight loss, and failed to reduce the clinical score or increase survival in this humanised mouse model of GVHD. Moreover, CGS 21680 reduced T regulatory cells and increased serum human IL-6 concentrations. Conversely, CGS 21680 reduced serum human tumour necrosis factor (TNF)-α concentrations and leukocyte infiltration into the liver, indicating that A2A activation can, in part, reduce molecular and histological GVHD in this model. Notably, CGS 21680 also prevented healthy weight gain in NSG mice not engrafted with hPBMCs suggesting that this compound may be suppressing appetite or metabolism. Therefore, the potential benefits of A2A activation in reducing GVHD in HSCT recipients may be limited and confounded by adverse impacts on weight, decreased T regulatory cell frequency and increased IL-6 production.
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Agonistas del Receptor de Adenosina A2/uso terapéutico , Adenosina/análogos & derivados , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Fenetilaminas/uso terapéutico , Adenosina/efectos adversos , Adenosina/uso terapéutico , Agonistas del Receptor de Adenosina A2/efectos adversos , Animales , Peso Corporal/efectos de los fármacos , Citocinas/inmunología , Modelos Animales de Enfermedad , Femenino , Enfermedad Injerto contra Huésped/inmunología , Humanos , Ratones , Fenetilaminas/efectos adversos , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
Graft-versus-host disease (GVHD) is a life-threatening consequence of allogeneic haematopoietic stem cell transplantation, a curative therapy for haematological malignancies. The ATP-gated P2X7 receptor channel is implicated in the development of GVHD. P2X7 activity on human leukocytes can be influenced by gain-of-function (GOF) and loss-of-function (LOF) single nucleotide polymorphisms (SNPs) in the P2RX7 gene. In this study, the P2RX7 gene was sequenced in 25 human donors and the P2X7 activity on subsets of peripheral blood T cells, natural killer (NK) cells and monocytes was measured using an ATP-induced dye uptake assay. GOF and LOF SNPs representing 10 of the 17 known P2RX7 haplotypes were identified, and correlated with P2X7 activity on all leukocyte subsets investigated. Notably, invariant (i) NK T cells displayed the highest P2X7 activity amongst all cell types studied. To determine if donor P2X7 activity influenced the development of GVHD, immunodeficient NOD-SCID-IL2Rγnull (NSG) mice were injected with human peripheral blood mononuclear cells isolated from donors of either GOF (hP2X7GOF mice) or LOF (hP2X7LOF mice) P2RX7 genotype. Both hP2X7GOF and hP2X7LOF mice demonstrated similar human leukocyte engraftment, and showed comparable weight loss, GVHD clinical score and overall survival. Donor P2X7 activity did not affect human leukocyte infiltration or GVHD-mediated tissue damage, or the relative expression of human P2X7 or human interferon-γ (hIFNγ) in tissues. Finally, hP2X7GOF and hP2X7LOF mice demonstrated similar concentrations of serum hIFNγ. This study demonstrates that P2X7 activity correlates with donor P2RX7 genotype on human leukocyte subsets important in GVHD development, but does not affect GVHD development in a humanised mouse model of this disease.
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Enfermedad Injerto contra Huésped/genética , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Animales , Genotipo , Humanos , Leucocitos Mononucleares/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Polimorfismo de Nucleótido SimpleRESUMEN
Allogeneic haematopoietic stem cell transplantation (HSCT) is a frequent curative therapy for numerous haematological malignancies. However, HSCT is limited by the occurrence of graft-versus-host disease (GVHD), with current therapies restricted to general immunosuppression. Activation of the P2X7 receptor by extracellular adenosine triphosphate (ATP) causes inflammation and tissue damage in GVHD. Short-term pharmacological blockade of P2X7 has been shown to reduce clinical disease and/or reduce inflammatory markers in allogeneic and humanized mouse models of GVHD. The current study demonstrates that long-term P2X7 blockade by intra-peritoneal injection of Brilliant Blue G (BBG) thrice weekly for up to 10â¯weeks did not impact human (h) peripheral blood mononuclear cell (PBMC) engraftment, predominantly T cells, in blood at 3â¯weeks post-hPBMC injection or in spleens at end-point in humanized mice. Histological analysis demonstrated long-term BBG treatment reduced leukocyte infiltration in the livers of humanized mice. Immunohistochemical analysis demonstrated that BBG treatment reduced liver apoptosis. Long-term BBG treatment did not alter clinical disease, mRNA expression of pro-inflammatory markers in tissues or serum human interferon (IFN)-γ concentrations. Therefore, this study demonstrates that P2X7 activation plays a role in GVHD pathogenesis in the livers of humanized mice, supporting a role for this receptor in GVHD development in HSCT recipients.
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Enfermedad Injerto contra Huésped/tratamiento farmacológico , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Hepatitis/prevención & control , Antagonistas del Receptor Purinérgico P2X/uso terapéutico , Receptores Purinérgicos P2X7/fisiología , Colorantes de Rosanilina/uso terapéutico , Animales , Citocinas/sangre , Modelos Animales de Enfermedad , Femenino , Leucocitos/efectos de los fármacos , Leucocitos/fisiología , Hígado/efectos de los fármacos , Hígado/inmunología , Hígado/patología , RatonesRESUMEN
Graft-versus-host disease (GVHD) remains a major problem after allogeneic haematopoietic stem cell transplantation, a curative therapy for haematological malignancies. Previous studies have demonstrated a role for the adenosine triphosphate (ATP)-gated P2X7 receptor channel in allogeneic mouse models of GVHD. In this study, injection of human peripheral blood mononuclear cells (PBMCs) into immunodeficient non-obese diabetic-severe combined immunodeficiency-interleukin (NOD-SCID-IL)-2Rγnull (NSG) mice established a humanized mouse model of GVHD. This model was used to study the effect of P2X7 blockade in this disease. From five weeks post-PBMC injection, humanized mice exhibited clinical signs and histopathology characteristic of GVHD. The P2X7 antagonist, Brilliant Blue G (BBG), blocked ATP-induced cation uptake into both murine and human cells in vitro. Injection of BBG (50 mg/kg) into NSG mice did not affect engraftment of human leucocytes (predominantly T cells), or the clinical score and survival of mice. In contrast, BBG injection reduced circulating human interferon (IFN)-γ significantly, which was produced by human CD4+ and CD8+ T cells. BBG also reduced human T cell infiltration and apoptosis in target organs of GVHD. In conclusion, the P2X7 antagonist BBG reduced circulating IFN-γ in a humanized mouse model of GVHD supporting a potential role for P2X7 to alter the pathology of this disease in humans.
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Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Trasplante de Células Madre Hematopoyéticas , Interferón gamma/sangre , Antagonistas del Receptor Purinérgico P2X/uso terapéutico , Colorantes de Rosanilina/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Enfermedad Injerto contra Huésped/inmunología , Humanos , Subunidad gamma Común de Receptores de Interleucina/genética , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Receptores Purinérgicos P2X7/metabolismo , Trasplante HomólogoRESUMEN
The P2X7 receptor is a trimeric ATP-gated cation channel important in health and disease. We have observed that the specific phospholipase D (PLD)1 antagonist, CAY10593 impairs P2X7-induced shedding of the 'low affinity' IgE receptor, CD23. The current study investigated the mode of action of this compound on P2X7 activation. Measurements of ATP-induced ethidium(+) uptake revealed that CAY10593 impaired P2X7-induced pore formation in human RPMI 8226 B cells, P2X7-transfected HEK-293 cells and peripheral blood mononuclear cells. Concentration response curves demonstrated that CAY10593 impaired P2X7-induced pore formation in RPMI 8226 cells more potently than the PLD2 antagonist CAY10594 and the non-specific PLD antagonist halopemide. Electrophysiology measurements demonstrated that CAY10593 also inhibited P2X7-induced inward currents. Notably, RT-PCR demonstrated that PLD1 was absent in RPMI 8226 cells, while choline-Cl medium or 1-butanol, which block PLD stimulation and signalling respectively did not impair P2X7 activation in these cells. This data indicates that CAY10593 impairs human P2X7 independently of PLD1 stimulation and highlights the importance of ensuring that compounds used in signalling studies downstream of P2X7 activation do not affect the receptor itself.
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Neoplasias Experimentales/metabolismo , Fosfolipasa D/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Transducción de Señal/efectos de los fármacos , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Humanos , Antagonistas del Receptor Purinérgico P2X/farmacologíaRESUMEN
The human P2X7 receptor is a two-transmembrane ionotropic receptor which has a ubiquitous distribution and is most highly expressed on immune cells. In macrophages and similar myeloid cells primed by lipopolysaccharide (LPS), activation of P2X7 by extracellular ATP opens a cation channel/pore allowing massive K+ efflux associated with processing and secretion of pro-inflammatory cytokines interleukin (IL)-1ß and IL-18. A variety of other downstream effects follows P2X7 activation over several minutes including shedding of certain surface molecules, membrane blebbing, microvesicle/exosome release and apoptosis of the cell. High concentrations of ATP (>100 µM) are required to activate P2X7 but it remains unclear where these levels exist, other than in inflammatory foci or confined spaces such as in bone. A variety of potent selective antagonists of P2X7 activation have recently become available, allowing clinical trials to be undertaken in inflammatory and immune-mediated disorders. Proteomic studies have shown that P2X7 exists as a large multiprotein complex which includes non-muscle myosin heavy chain and other elements of the cytoskeleton. In the absence of its ATP ligand and serum, P2X7 has an alternate function in the recognition and phagocytosis of non-opsonized foreign particles, including bacteria and apoptotic cells. The P2RX7 gene has many polymorphic variants and isoforms which increase or decrease function of the receptor. Genetic association studies have linked loss-of-function polymorphisms with reactivation of latent tuberculosis as well as symptomatic infection with certain other obligate intracellular pathogens. The many roles involving P2X7 suggest that this receptor is essential to fundamental aspects of the innate immune response.
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Inmunidad Innata , Receptores Purinérgicos P2X7/inmunología , Receptores Purinérgicos P2X7/metabolismo , Apoptosis , Humanos , Interleucina-1/inmunología , Interleucina-1/metabolismo , Fagocitosis , Proteómica , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7/genéticaRESUMEN
Mycobacterium tuberculosis is one of the most successful human pathogens, surviv ing in latent foci of infection in one third of humanity, yet causing lung necrosis in sufficient individuals to ensure its transmission. Each stage of the host response to M. tuberculosis is under genetic control, including the initial encounter with mycobacteria by macrophages, epithelial cells and dendritic cells in the lung, induction of the inductive T cell response, and killing by activated macrophages within granulomas. Although environmental factors are important determinants of progression to disease, there is a genetic component underlying susceptibility to tuberculosis (TB), the basis of which may vary in different populations. Recent studies using a variety of methods have defined a number of susceptibility alleles for the development of active TB. Many of these influence macrophage responses to mycobacteria. We have studied the influence of loss of function polymorphisms in the human P2X7 gene on the capacity of macrophages to kill M. tuberculosis. Activation of the P2X7 receptor, an ATP-gated Ca2+ channel, leads to the formation of pores, the activation of phospholipase D, and the induction of apoptosis with death of the infecting mycobacteria. Macrophages from subjects who are heterozygote, homozygote or compound heterozygote for these polymorphisms fail to undergo apoptosis and show partial or complete inhibition of mycobacterial killing. One of these non-functioning polymorphisms was significantly associated with increased susceptibility to TB disease, particularly extrapulmonary disease, in two unrelated cohorts of TB patients. Insights into the genetic regulation of susceptibility to human TB may identify novel methods for controlling latent M. tuberculosis and reducing the burden of tuberculosis.
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Predisposición Genética a la Enfermedad/genética , Mycobacterium tuberculosis/inmunología , Polimorfismo Genético , Tuberculosis/genética , Tuberculosis/inmunología , Humanos , Macrófagos/inmunología , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/inmunología , Receptores Purinérgicos P2X7RESUMEN
Interleukin (IL)-18 is an important proinflammatory cytokine processed and released from cells of the monocyte lineage by activation of the P2X(7) receptor by extracellular adenosine 5'-triphosphate (ATP). We examined if a loss-of-function polymorphism of the human P2X(7) receptor (glutamic acid-496 to alanine) impairs this process. Using a whole blood-based assay, ATP-induced release of IL-18 from homozygous subjects after 120 min incubation with ATP was 42% of that from wild-type subjects. Moreover, the level of ATP-induced IL-18 release from lipopolysaccharide (LPS)-primed monocytes of homozygous subjects after 30 and 60 min incubation with ATP was 21 and 44%, respectively, of that from wild-type monocytes. Nigericin, a K(+) ionophore, induced a similar release of IL-18 from monocytes of either genotype. ATP-induced ethidium(+) uptake in LPS-primed, monocytes of homozygous subjects was only 11% of that in wild-type monocytes, while P2X(7) surface expression on LPS-primed, homozygous monocytes was 44% of that on wild-type monocytes.
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Adenosina Trifosfato/farmacología , Interleucina-18/metabolismo , Monocitos/metabolismo , Polimorfismo Genético/genética , Receptores Purinérgicos P2/genética , Líquido Extracelular/efectos de los fármacos , Líquido Extracelular/metabolismo , Humanos , Interleucina-18/genética , Monocitos/efectos de los fármacos , Receptores Purinérgicos P2X7RESUMEN
P2X(7) receptor/channels mediate ATP-induced apoptosis in a range of cells including lymphocytes. HEK293 cells were transfected with wild-type human P2X(7) receptor or site-directed mutant constructs (K193A, K311A and E496A) known to be non-functional from measurements of barium/ethidium influx in the presence of ATP or 2',3'-O-(4-benzoylbenzoyl)-ATP. An antibody was designed against an epitope from a loop adjacent to the extracellular ATP site. The epitope was unavailable in cells expressing normal functional surface receptors. Non-functional surface receptors as well as intracellular receptors selectively bound the antibody. So did B-lymphocytes from chronic lymphocytic leukemia patients expressing non-functional (E496A) mutant receptor.
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Linfocitos B/metabolismo , Riñón/metabolismo , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/farmacología , Línea Celular , Etidio/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Riñón/citología , Riñón/embriología , Mutagénesis Sitio-Dirigida , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7RESUMEN
B lymphocytes are known to synthesise the P2X7 subtype of the P2X purinergic receptor family; however, the identification of the other six P2X subtypes on these cells has been limited by the absence of specific antibodies. In this study, we used a panel of anti-P2X polyclonal antibodies and confocal microscopy to examine the presence of each P2X receptor on human B lymphocytes. We observed that P2X1, P2X2, P2X4 and P2X7 subtypes, but not P2X3, P2X5 and P2X6 subtypes, are present on B lymphocytes.
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Linfocitos B/metabolismo , Leucemia Linfocítica Crónica de Células B/sangre , Receptores Purinérgicos P2/análisis , Receptores Purinérgicos/análisis , Adenosina Trifosfato/metabolismo , Anticuerpos/metabolismo , Linfocitos B/inmunología , Reacciones Cruzadas , Epítopos , Humanos , Inmunohistoquímica , Activación del Canal Iónico/fisiología , Microscopía Confocal , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/inmunología , Receptores Purinérgicos P2X , Relación Estructura-ActividadRESUMEN
In this study we compared the effects of subinflammatory and inflammatory doses of solar-simulated ultraviolet (UV) radiation on enhancement of skin tumor growth, sensitization to haptens and cellular changes within the epidermis of C3H/HeN mice. Tumors transplanted into mice 3 days after exposure to inflammatory, but not subinflammatory, doses of UV radiation had a higher growth rate than those tumors inoculated into unirradiated control mice. Both doses of UV radiation suppressed the induction of contact hypersensitivity and induced tolerance when hapten was painted onto the skin 3 days after irradiation. Skin exposed to the higher, but not the lower, dose of UV radiation contained significantly increased numbers of CD11b+, CD45+ MHC class II- and CD45+ MHC class II(hi) inflammatory cells 3 days post-irradiation. The immunosuppression correlated with a reduction in Langerhans cells and dendritic epidermal T cells. Collectively, this suggests that suppression to contact sensitizers is due to the UV radiation effects on Langerhans cells and dendritic epidermal T cells. While these effects may also suppress the induction of anti-tumor immunity, at higher doses of UV radiation inflammatory cells may enhance tumor growth by a non-immunological mechanism.
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Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/metabolismo , Rayos Ultravioleta , Animales , Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/metabolismo , Dermatitis por Contacto , Citometría de Flujo , Inflamación , Antígenos Comunes de Leucocito/biosíntesis , Antígeno de Macrófago-1/biosíntesis , Complejo Mayor de Histocompatibilidad , Ratones , Ratones Endogámicos C3H , Trasplante de Neoplasias , Sistema Solar , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
There is little evidence that cutaneous dendritic cells (DC), including epidermal Langerhans cells (LC), can induce immunity to UV radiation (UVR)-induced skin tumours. Here, it is shown that cells within skin can induce protective antitumour immunity against a UVR-induced fibrosarcoma. Transplantation of the skin overlying subcutaneous tumours onto naïve recipients could induce protective antitumour immunity, probably because the grafting stimulated the tumour Ag-loaded DC to migrate to local lymph nodes. This suggests that cutaneous APC can present tumour Ag to induce protective antitumour immunity. Previously, it has been shown that immunization of mice with MHC class II+ epidermal cells (EC) pulsed with tumour extracts could induce delayed-type hypersensitivity against tumour cells. Here, this same immunization protocol could induce protective immunity against a minimum tumorigenic dose of UVR-induced fibrosarcoma cells, but not higher doses. Epidermal cells obtained from semiallogeneic donors and pulsed with tumour extract could also induce protective immunity. However, presentation of BSA Ag from the culture medium was found to contribute to this result using semiallogeneic EC. The results suggest that LC overlying skin tumours may be able to induce protective immunity to UVR-induced tumours if stimulated to migrate from the skin.
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Células Presentadoras de Antígenos/inmunología , Células Dendríticas/inmunología , Neoplasias Inducidas por Radiación/inmunología , Neoplasias Cutáneas/inmunología , Piel/citología , Animales , Células Presentadoras de Antígenos/fisiología , Células Dendríticas/fisiología , Fibrosarcoma/inmunología , Humanos , Hipersensibilidad Tardía , Inmunidad Celular , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Albúmina Sérica Bovina/inmunología , Piel/inmunología , Trasplante de Piel , Rayos UltravioletaRESUMEN
The P2X(7) receptor is a ligand-gated cation-selective channel that mediates ATP-induced apoptosis of cells of the immune system. We and others have shown that P2X(7) is nonfunctional both in lymphocytes and monocytes from some subjects. To study a possible genetic basis we sequenced DNA coding for the carboxyl-terminal tail of P2X(7). In 9 of 45 normal subjects a heterozygous nucleotide substitution (1513A-->C) was found, whereas 1 subject carried the homozygous substitution that codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X(7) on lymphocytes was not affected by this E496A polymorphism, demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the E496A homozygote subject expressed nonfunctional receptor, whereas heterozygotes showed P2X(7) function that was half that of germline P2X(7). Results of transfection experiments showed that the mutant P2X(7) receptor was nonfunctional when expressed at low receptor density but regained function at a high receptor density. This density dependence of mutant P2X(7) function was also seen on differentiation of fresh monocytes to macrophages with interferon-gamma, which up-regulated mutant P2X(7) and partially restored its function. P2X(7)-mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X(7) compared with germline (8.6 versus 35.2%). The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X(7) receptor.
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Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Alanina , Células Cultivadas , Ácido Glutámico , Humanos , Leucocitos Mononucleares/metabolismo , Mutación Puntual , Polimorfismo Genético , Receptores Purinérgicos P2X7 , Transducción de Señal/genética , Relación Estructura-ActividadRESUMEN
UV radiation causes a number of cellular changes within the skin which play a role in tumor outgrowth, including immunosuppression and production of growth-enhancing cytokines. Both of these enable tumors to grow but their relative importance in carcinogenesis is poorly defined. In this study, C3H/HeN mice were exposed to a single inflammatory dose of 410 mJ/cm(2) UVB radiation (plus 100 mJ/cm(2) UVA radiation) followed by the inoculation of a regressor squamous cell carcinoma into or the painting of oxazolone onto the treated skin. Tumors transplanted 2 or 3 but not 4 days after irradiation had a significantly higher growth rate than tumors inoculated into unirradiated control mice. In contrast, mice failed to respond to hapten when it was applied 2, 3 or 4 days after irradiation. Cytofluorimetric analysis demonstrated that the number of F4/80(+) Langerhans cells was not significantly reduced until 4 days after irradiation, while the number of dendritic epidermal T cells was significantly lower at all time points observed after UV-irradiation. Furthermore, a large cellular infiltration of CD11b(+), Gr-1(+), CD45(+) MHC class II(+) and CD45(+) MHC class II(-) cells into the epidermis was observed 2 and 3 days after irradiation, which corresponded with the enhanced tumor growth. To a lesser extent tumor growth was also associated with CD45(+) MHC class II(hi) cells, possibly the previously described UV-induced macrophage. In contrast, suppression of contact hypersensitivity corresponded with the reduction in dendritic epidermal T cells but not with other cell changes. The results suggest that, in this model, where immunosuppression did not appear to be responsible for enhanced tumor growth, inflammatory infiltrates may contribute to the promotion of skin tumor growth within UV-irradiated skin.
Asunto(s)
Carcinoma de Células Escamosas/patología , Movimiento Celular/efectos de la radiación , Células Epidérmicas , Epidermis/efectos de la radiación , Células de Langerhans/fisiología , Neoplasias Cutáneas/patología , Linfocitos T/fisiología , Rayos Ultravioleta/efectos adversos , Adyuvantes Inmunológicos/toxicidad , Animales , Carcinoma de Células Escamosas/etiología , División Celular/fisiología , Dermatitis por Contacto/etiología , Dermatitis por Contacto/inmunología , Dermatitis por Contacto/patología , Epidermis/inmunología , Femenino , Células de Langerhans/citología , Células de Langerhans/inmunología , Ratones , Ratones Endogámicos C3H , Trasplante de Neoplasias , Oxazolona/inmunología , Neoplasias Cutáneas/etiología , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Both genetic and environmental factors are thought to contribute to specific IgE responses, however, the relative contribution of each in the responses to individual ryegrass pollen allergens is largely unknown even though some responses to allergens have been linked to certain HLA complexes. OBJECTIVE: Using a large group of monozygotic and dizygotic twins, this study was designed to investigate the IgE binding profiles of individual ryegrass pollen (Lolium perenne) components to assess the relative contribution of genetic and environmental factors in determining IgE responses to specific allergens. METHODS: Ryegrass pollen proteins were separated by electrophoresis and immunoblotted with sera from 191 pairs of twins where at least one sibling had a SPT > 2 mm to perennial ryegrass. Concordance levels for individual ryegrass pollen components were compared between monozygotic and dizygotic twins in a subset group where both twins had SPT > 3 mm to perennial ryegrass. RESULTS: Immunoblotting revealed 23 individual IgE-binding components from ryegrass pollen. Although there was a significantly greater proportion of monozygotic twins with SPT wheals greater than 3 mm when compared with the dizygotic twins, the mean case-wise concordance for the immunoblot components was similar for both groups of twins. The mean case-wise concordance when at least four pairs of sera were involved was 44% for the MZ twins (n=11 components) and 45% for the DZ twins (n=12 components). We found no significant differences in concordance levels for any of the 23 individual components including allergens previously associated with HLA. CONCLUSION: Evidence for genetic control of allergen-specific IgE responses in a large population sample of twins to individual ryegrass allergens is limited, indicating that the IgE responses to specific ryegrass pollen allergens are determined largely by environmental factors.
Asunto(s)
Alérgenos/inmunología , Inmunoglobulina E/inmunología , Lolium/inmunología , Polen/inmunología , Gemelos Dicigóticos , Gemelos Monocigóticos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Australia , Sitios de Unión de Anticuerpos , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Immunoblotting , Inmunoglobulina E/sangre , Masculino , Persona de Mediana Edad , Proteínas de Plantas/inmunologíaRESUMEN
BACKGROUND: Although some studies have shown genetic control of specific IgE responses to purified grass allergens, studies with other allergens have not supported this. The extent of such control for house dust mite (HDM) (Dermatophagoides pteronyssinus) allergens is unclear. OBJECTIVE: We sought to determine the extent to which genetic factors control the specificity of IgE responses to individual HDM allergens by comparing the immunoblot patterns of IgE binding of serum from monozygotic and dizygotic members of a large cohort of Australian twins. METHODS: HDM proteins separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis were immunoblotted with sera from 317 twin pairs in which at least one twin had at least a weak HDM skin test response. Concordance levels for IgE binding to the individual HDM components were compared in the subset of 142 pairs of twins in which both twins were allergic to HDMs (skin prick test wheal diameter, > 3 mm). RESULTS: Over all 36 blotted bands, the mean case-wise concordance was 41% for monozygotic twins and 17% for dizygotic twins. Of the components detected, only those of molecular weights 23 kd and 16 kd were significantly different between the groups (p < 0.01). Differences observed between the monozygotic and dizygotic twins could be partly explained by overall IgE hyperresponsiveness. CONCLUSION: Evidence for genetic control of IgE responses to 36 IgE-binding HDM components from a large sample of twins showed significant differences in concordance for two components and nonsignificant differences for several others. In the monozygotic twins, concordance never exceeded 67% for any band, and most monozygotic individuals recognized components their co-twin did not. Genetic control of overall atopy in monozygotic twins is far stronger than that controlling specific sensitization to HDM allergens.