RESUMEN
BACKGROUND: Anti-CD4 antibodies induce long-term graft survival by incompletely understood mechanisms, and CD4-ligation with HIV gp120-derivatives attenuates interleukin (IL)-2 receptor signaling. We examined the latter in the context of the CD4-modulating antibody 16H5. MATERIALS AND METHODS: We performed immunoblots to assess the IL-2-induced phosphorylation of signal transducer and activator of transcription (STAT)5 and Akt in the presence or absence of 16H5. Furthermore, we documented the effects of 16H5 on the induction of STAT5, activating protein (AP)-1, and myc by IL-2 in DNA-binding assays. 3H-thymidine incorporation of the human lymphoid cell line CMO, which exhibits constitutive activation of the STAT5 pathway and IL2-independent growth, was also measured during 16H5 treatment. RESULTS: In human T lymphocytes, 16H5 attenuated both the tyrosine phosphorylation of STAT5 by IL-2 and the IL-2-induced DNA-binding of this transcription factor. In contrast, 16H5 had no effect on the serine phosphorylation of Akt by IL-2 or on the IL-2-induced DNA-binding of myc. Signal transduction involving AP-1 was unaffected by 16H5 and IL-2. 16H5 also attenuated CMO cell proliferation. CONCLUSIONS: 16H5 targets the STAT5 signaling pathway to attenuate IL-2 receptor signal transduction in human T cells. This observation provides a molecular explanation for the immunomodulatory actions of anti-CD4 antibodies.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de la Leche , Transactivadores/fisiología , Anticuerpos/farmacología , Antígenos CD4/inmunología , División Celular/efectos de los fármacos , Línea Celular Transformada/efectos de los fármacos , Humanos , Interleucina-2/farmacología , Receptores de Interleucina-2/fisiología , Factor de Transcripción STAT5 , Transducción de SeñalAsunto(s)
Actinas/fisiología , Antígenos CD4/fisiología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Proteínas de la Leche , Receptores de Interleucina-2/fisiología , Transducción de Señal/fisiología , Linfocitos T/inmunología , Actinas/química , Línea Celular , Células Cultivadas , Citoesqueleto/fisiología , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Humanos , Receptores de Interleucina-2/genética , Factor de Transcripción STAT5 , Linfocitos T/fisiología , Transactivadores/metabolismoAsunto(s)
Antígenos CD4/fisiología , Proteínas de Unión al ADN/metabolismo , Interleucina-2/farmacología , Proteínas de la Leche , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal , Linfocitos T/fisiología , Transactivadores/metabolismo , Animales , Apoptosis , Humanos , Interferón gamma/deficiencia , Interferón gamma/genética , Interferón gamma/fisiología , Interleucina-2/fisiología , Janus Quinasa 3 , Ligandos , Ratones , Ratones Noqueados , Factor de Transcripción STAT5 , Linfocitos T/inmunologíaRESUMEN
Blocking the CD28-B7 T lymphocyte costimulatory pathway with the recombinant protein CTLA4Ig induces long term allograft survival in rodents. It has been suggested that this results from selective activation of the Th2 immune pathway. To test this hypothesis, we compared vascularized cardiac allograft survival in wild-type (IL-4 +/+) and homozygous IL-4 gene-knockout (IL-4 -/-) mice. We report in this study that long term survival (>100 days) of fully allogeneic grafts can be induced readily in IL-4 -/- recipients treated with a short course of CTLA4Ig. We also demonstrate that IL-4 -/- mice are deficient in Th2-type cytokine expression following in vitro or in vivo allostimulation. These results suggest that IL-4 production and subsequent generation of a Th2-type immune response are not obligatory for CTLA4Ig-induced long term acceptance of vascularized allografts.
Asunto(s)
Antígeno B7-1/inmunología , Antígenos CD28/inmunología , Refuerzo Inmunológico de Injertos , Supervivencia de Injerto/inmunología , Trasplante de Corazón/inmunología , Inmunoconjugados , Inmunosupresores/farmacología , Interleucina-4/deficiencia , Activación de Linfocitos/inmunología , Abatacept , Enfermedad Aguda , Animales , Antígenos CD , Antígenos de Diferenciación/uso terapéutico , Antígeno CTLA-4 , Rechazo de Injerto/prevención & control , Supervivencia de Injerto/genética , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Interleucina-4/biosíntesis , Interleucina-4/genética , Isoantígenos/fisiología , Activación de Linfocitos/genética , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/biosíntesis , Proteínas Recombinantes de Fusión/uso terapéutico , Bazo/patología , Células Th2/patologíaRESUMEN
It is generally assumed that IFNgamma plays a central role in acute allograft rejection. To test this hypothesis, we transplanted fully allogeneic (MHC class I and II incompatible) C3H/HeJ (H2k) murine hearts to IFNgamma-/- (IFNgamma gene-knockout) and IFNgamma+/+ BALB/c (H2d) mice. The phenotype of IFNgamma-/- mice was confirmed by demonstrating absent IFNgamma protein production by Con A stimulated IFNgamma-/- splenocytes. Both IFNgamma-/- and IFNgamma+/+ strains rejected transplanted hearts acutely: graft survival (mean +/- SD) was 5.2+/-0.4 and 6.0+/-0.0 days, respectively. Histologic examination revealed similar patterns of acute cellular rejection in both mouse groups. IFNgamma mRNA was present in hearts rejected by IFNgamma+/+ mice but was absent in those rejected by IFNgamma-/- mice. IL-2, IL-4, IL-10, and TNFalpha mRNA expression, on the other hand, was similar in grafts rejected by either strain. We also observed that hapten-induced delayed-type hypersensitivity (DTH) response was significantly reduced but not absent in IFNgamma-/- mice. Our results demonstrate that IFNgamma is not required for acute cellular rejection of fully allogeneic murine hearts. We propose that non-DTH mechanisms of allograft destruction could be enhanced in the absence of IFNgamma and thus lead to robust acute rejection.
Asunto(s)
Trasplante de Corazón/inmunología , Hipersensibilidad Tardía/etiología , Interferón gamma/farmacología , Enfermedad Aguda , Adyuvantes Inmunológicos/farmacología , Animales , Citocinas/genética , Rechazo de Injerto/genética , Rechazo de Injerto/prevención & control , Haptenos/efectos adversos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , ARN Mensajero/análisisAsunto(s)
Anticuerpos Monoclonales/toxicidad , Anticuerpos Monoclonales/uso terapéutico , Complejo CD3/inmunología , Supervivencia de Injerto/inmunología , Trasplante de Corazón/inmunología , Terapia de Inmunosupresión/métodos , Interleucina-10/uso terapéutico , Animales , Humanos , Inmunosupresores/uso terapéutico , Inmunosupresores/toxicidad , Interleucina-10/farmacología , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/farmacología , Factores de Tiempo , Trasplante Homólogo/inmunologíaRESUMEN
The rejection of the transplanted allograft is dependent on T cell activation, which requires T cell receptor engagement by antigen and costimulatory signals delivered by T cell surface molecules such as CD28. CTLA4-Ig is a fusion protein that has previously been shown to block the CD28-mediated costimulatory signal and inhibit immune responses in vitro and in vivo. In this report we show that treatment of the C3H/He recipient of a BALB/c vascularized cardiac allograft with a 12-day course of CTLA4-Ig produced indefinite graft survival (> 100 days) in the majority of recipients. In addition, these recipients demonstrated donor-specific transplantation tolerance when tested with donor-specific (BALB/c) and third-party (C57BL/10) skin grafts. These results demonstrate that CTLA4-Ig can induce transplantation tolerance in the adult murine cardiac allograft model.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos de Diferenciación/farmacología , Supervivencia de Injerto/efectos de los fármacos , Trasplante de Corazón/inmunología , Inmunoconjugados , Abatacept , Animales , Antígenos CD , Antígeno CTLA-4 , Humanos , Cadenas Pesadas de Inmunoglobulina/farmacología , Terapia de Inmunosupresión/métodos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Factores de Tiempo , Trasplante Homólogo/inmunologíaRESUMEN
Dendritic cells (DC) play a critical role in the initiation of T cell-mediated immune responses, and express costimulatory molecules that are required for optimal activation of unprimed T cells. Studies on the regulation of the costimulatory molecules on DC have produced evidence from several systems that GM-CSF can up-regulate expression of CTLA4 counter receptor (CTLA4-CR) (but not intercellular adhesion molecule 1 (ICAM-1) and heat stable Ag (HsAg)) on DC. This is demonstrated on splenic DC, Langerhans cells, kidney DC in culture, and in a skin-explant culture system, in which the increased expression of CTLA4-CR on Langerhans cells (LC) occurs concomitantly with their migration out of skin. Interestingly, despite the ability of both GM-CSF and IFN-gamma to increase CTLA4-CR and maintain similar levels of ICAM-1, HsAg, and MHC molecule expression, the functional consequences of these cytokines on splenic DC are distinctly different. GM-CSF enhances the ability of DC to stimulate both T cell proliferation and cytokine release, whereas IFN-gamma causes no increase in immunostimulatory function. Further analysis of the CTLA4-CR on these cell populations by using the GL-1 and IG10 mAbs has shown that GM-CSF-cultured DC express high levels of both B7-1 and B7-2, whereas IFN-gamma-cultured DC express increased levels of only B7-2. These results suggest that optimal stimulation of unprimed T cells to proliferate and release cytokines may require participation of both of these CTLA4 counter receptors, and confirm the importance of GM-CSF for the maturation of DC into potent stimulators of T cell activation.
Asunto(s)
Antígenos CD , Antígeno B7-1/análisis , Células Dendríticas/fisiología , Inmunoconjugados , Glicoproteínas de Membrana , Abatacept , Animales , Antígenos de Diferenciación/fisiología , Antígeno B7-2 , Secuencia de Bases , Antígeno CTLA-4 , Línea Celular , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Interferón gamma/farmacología , Células de Langerhans/fisiología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Datos de Secuencia MolecularAsunto(s)
Rechazo de Injerto/patología , Trasplante de Corazón/patología , Animales , Enfermedad Crónica , Citocinas/metabolismo , Rechazo de Injerto/inmunología , Trasplante de Corazón/inmunología , Ratones , Ratones Endogámicos , Ratas , Ratas Endogámicas Lew , Ratas Endogámicas WF , Trasplante HomólogoAsunto(s)
Tolerancia Inmunológica , Linfocitos T Reguladores/inmunología , Linfocitos T/inmunología , Inmunología del Trasplante , Animales , Citocinas/biosíntesis , Citocinas/genética , Trasplante de Corazón/inmunología , Hipersensibilidad Tardía , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Linfocitos T Citotóxicos/inmunologíaAsunto(s)
Fallo Renal Crónico/etnología , Trasplante de Riñón/estadística & datos numéricos , Sistema del Grupo Sanguíneo ABO/análisis , Centros Médicos Académicos , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Población Negra , Niño , Preescolar , Femenino , Georgia/epidemiología , Supervivencia de Injerto , Humanos , Lactante , Fallo Renal Crónico/cirugía , Trasplante de Riñón/tendencias , Masculino , Persona de Mediana Edad , Trasplante Homólogo , Población BlancaRESUMEN
Whereas dendritic cells (DC) are known to be potent activators of T cells both in vitro and in vivo, the critical costimulatory molecules expressed on DC are not well characterized. Using immunocytochemical and molecular techniques we find that splenic DC express B7/BB1, the counter-receptor for CD28. Moreover, expression of B7/BB1 is upregulated on epidermal Langerhans cells (LC) during their functional maturation into potent T cell stimulators. In blocking experiments, we find that participation of B7/BB1 is required for optimal proliferation of unprimed, allogeneic T cells in DC-driven, primary mixed leukocyte reactions. These data demonstrate that the regulated expression of B7/BB1 on DC may be important in the initiation of a primary T cell response.
Asunto(s)
Linfocitos B/inmunología , Células Dendríticas/inmunología , Receptores de Superficie Celular/biosíntesis , Linfocitos T/inmunología , Animales , Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Secuencia de Bases , Antígenos CD28 , Células Dendríticas/citología , Citometría de Flujo , Activación de Linfocitos , Prueba de Cultivo Mixto de Linfocitos , Masculino , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Superficie Celular/genéticaRESUMEN
Activated CD4+ Th2 cells release cytokines (IL-4,-10) that block activation & cytokine (IL-2/IFN-gamma) release by proinflammatory T (CD4+,CD8+) effector cells. To test the hypothesis that peripheral tolerance to alloantigen is linked to differential activation of CD4+ Th2 cells we measured cytokine transcripts in heart grafts (C57BL/10----C3H/HeJ) and spleens of mice rendered "tolerant" by donor-specific blood transfusion, anti-CD4 mAb pretreatment, and cyclosporine administration. The expression of IL-2/IFN-gamma transcripts was reduced greater than 90% in grafts from tolerant recipients. IL-4/IL-10 transcripts were generally enhanced and persisted in graft and recipient spleen. Accordingly adoptive transfer studies were performed to determine whether Th2-like effectors, which express Fc receptors (FcR), mediate suppression in this model. Unfractionated mononuclear cells (MC) (5 x 10(6), isolated from spleens of heart graft recipients made tolerant by DST, prolonged the survival of test grafts greater than 90 days in irradiated (680 rads) recipients reconstituted with a sufficient number of MC from spleens of naive C3H to precipitate rejection of the test graft in 18.2 days (MST, n = 5). Conversely adoptive transfer of inocula depleted of FcR+ cells on immune complex columns or with anti-FcR mAb 24G2 caused test grafts to be rejected in 8-11 days. These results suggest that peripheral tolerance to alloantigen may be linked to differential activation of Th2 cells that induce anergy by suppression. The possibility that Th2-like effectors mediate peripheral tolerance to self is discussed.
Asunto(s)
Linfocitos B/inmunología , Trasplante de Corazón/inmunología , Animales , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/inmunología , Ciclosporina/administración & dosificación , Citocinas/fisiología , Hipersensibilidad Tardía/inmunología , Tolerancia Inmunológica , Inmunoterapia Adoptiva , Interferón gamma/farmacología , Interleucina-10/análisis , Interleucina-2/análisis , Interleucina-4/análisis , Masculino , Ratones , Reacción en Cadena de la Polimerasa , Receptores Fc/análisis , Bazo/química , Bazo/fisiología , Linfocitos T Colaboradores-Inductores/ultraestructura , Trasplante Homólogo/fisiologíaRESUMEN
Previous studies in this laboratory have documented tumor necrosis factor alpha (TNF) release by macrophage laden glomeruli in the accelerated autologous form of nephrotoxic serum nephritis (AA-NTSN). We now report that the administration of anti-TNF antiserum to rats with the AA-NTSN reduces albuminuria in a dose related manner (day 8 postinduction) and limits glomerular necrosis (P less than 0.05) without affecting the endogenous creatinine clearance (CCr). Protease inhibitors block cytolytic activity of TNF in vitro and reduce glomerular necrosis in experimental nephritis in vivo. The combined administration of anti-TNF antiserum and an amidine-type protease inhibitor (BABIM) to rats with the AA-NTSN caused a greater diminution of albuminuria and histopathology than observed in rats treated with either agent alone, and also prevented the fall in CCr otherwise observed in this model system. Since, in our studies, BABIM did not inhibit cytolytic TNF activity in vitro, we conclude that the effects of combined administration of these two agents are mediated by independent mechanisms. Our results highlight the pathogenic significance of local TNF release in immune renal disease accompanied by prominent glomerular macrophage accumulation.
Asunto(s)
Sueros Inmunes/inmunología , Enfermedades del Sistema Inmune/patología , Glomérulos Renales/patología , Nefritis/patología , Inhibidores de Proteasas/farmacología , Factor de Necrosis Tumoral alfa/inmunología , Albuminuria/orina , Animales , Anticuerpos/inmunología , Membrana Basal/inmunología , Creatinina/metabolismo , Sueros Inmunes/fisiología , Inmunoglobulina G/análisis , Riñón/patología , Glomérulos Renales/inmunología , Masculino , Ratones , Nefritis/inmunología , Nefritis/metabolismo , Ratas , Ratas Endogámicas Lew , OvinosRESUMEN
Although mononuclear phagocytes were once considered the sole source of tumor necrosis factor alpha (TNF), it is now understood that other cell types, including T and B lymphocytes, may be stimulated to produce this factor. Herein we describe the release of a cytotoxic activity in vitro by isolated rat glomeruli and cultured glomerular mesangial cells. Immunoperoxidase staining with monoclonal antibodies against Ia and leukocyte common antigen-documented cultured mesangial cell populations were free of mononuclear phagocytes. Cytotoxic activities generated by isolated glomeruli and mesangial cell cultures eluted on gel chromatography in two peaks corresponding to molecular weights of 17 and 40 kDa and were fully inhibitable by anti-recombinant murine TNF antiserum. These data strongly suggest that intrinsic glomerular mesangial cells contribute to TNF release by intact glomeruli. Intraglomerular generation of TNF and perhaps other cell types may bear upon the pathogenesis of immune glomerular injury.
Asunto(s)
Glomérulos Renales/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Cultivadas , Cromatografía en Gel , Citotoxicidad Inmunológica , Citotoxinas/análisis , Técnicas In Vitro , Masculino , Peso Molecular , Miosinas/biosíntesis , Ratas , Ratas Endogámicas LewRESUMEN
Cytotoxic activity expressed by renal glomeruli of LEW rats with the accelerated autologous form of nephrotoxic serum nephritis (AA-NTSN) was assessed using the lymphotoxin (LT) sensitive line aL929. Glomeruli isolated on day 3 following nephritis induction were much more cytotoxic for aL929 in coculture than glomeruli of unmodified LEW (p less than 0.0025). Such a cytolysis was due to a soluble factor eluted on gel chromatography in a single peak corresponding to molecular weight (MW) of 40-45 kDa. The cytotoxic activity, harvested in 24-hour culture supernatants of nephritic glomeruli (NGS) and the chromatographic peak were totally inhibitable by antiserum neutralizing recombinant murine tumor necrosis factor alpha (rMuTNF). An abundant glomerular macrophage accumulation on day 3 and reduction of cytotoxicity (42.3%, p less than 0.01) upon irradiation limiting the mononuclear phagocyte infiltrate suggest that macrophages are at least partly responsible for TNF production by nephritic glomeruli. This study provides the initial documentation on generation of TNF in glomerular inflammatory injury induced by immune reaction.
Asunto(s)
Glomérulos Renales/lesiones , Nefritis/etiología , Factor de Necrosis Tumoral alfa/metabolismo , Albuminuria/etiología , Animales , Células Cultivadas , Citotoxicidad Inmunológica , Glomérulos Renales/inmunología , Glomérulos Renales/metabolismo , Macrófagos/inmunología , Masculino , Nefritis/inmunología , Ratas , Ratas Endogámicas LewRESUMEN
The role of Platelet Activating Factor (PAF) in experimental immune glomerulonephritis was assessed by administering the specific PAF receptor antagonist CV-3988 to inbred LEW rats with the Accelerated Autologous Nephrotoxic Serum Nephritis (AA-NTSN). Intravenous administration of CV-3988 caused a marked and sustained reduction in albuminuria and renal histopathological changes. Conversely, although CV-3988 appeared to modulate the fall in glomerular filtration rate (GFR) in the AA-NTSN, this trend was not statistically significant. Renal glomeruli isolated on day 1 after nephritis induction spontaneously released 16.9 +/- 2.2 ng of PAF per 200 glomeruli, while in glomeruli of healthy rats this secretion was virtually undetectable. The administration of CV-3988 to rats with AA-NTSN did not affect the following: binding of intravenously injected sheep anti-rat glomerular basement membrane (GBM) antibody; levels of autologous antibody to sheep immunoglobulin G; the functional integrity of circulating neutrophils. We conclude that local PAF generation and release is intimately linked with the pathogenesis of glomerular injury in this form of immune disease.
Asunto(s)
Glomérulos Renales/lesiones , Nefritis/etiología , Glicoproteínas de Membrana Plaquetaria , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores Acoplados a Proteínas G , Albuminuria/prevención & control , Animales , Creatinina/sangre , Modelos Animales de Enfermedad , Glomérulos Renales/patología , Masculino , Nefritis/inmunología , Nefritis/prevención & control , Éteres Fosfolípidos/farmacología , Factor de Activación Plaquetaria/fisiología , Ratas , Ratas Endogámicas LewRESUMEN
We have previously documented the importance of tumor necrosis factor (TNF) alpha in the pathogenesis of nephrotoxic-serum nephritis in rats. In this study, we evaluated the possible relevance of the well-established cytocidal TNF activity to the mechanism of glomerular injury by assessing sensitivity of rat mesangial and epithelial cell populations to recombinant murine TNF alpha (rMuTNF). Radiolabelled confluent mesangial cell cultures that were incubated with rMuTNF released significantly more 3H-thymidine than control monolayers (maximum specific release was 11.4 +/- 4.9% at 1,000 pg/ml of rMuTNF). rMuTNF was, however, approximately 1,000-fold less cytolytic in mesangial cells than in the lymphotoxin-sensitive L929 cell line. Conversely, glomerular epithelial cells were not affected by exposure to rMuTNF under the same conditions. These results suggest that the cytolytic effect of TNF may contribute to glomerular injury in nephrotoxic-serum nephritis.
Asunto(s)
Mesangio Glomerular/efectos de los fármacos , Glomérulos Renales/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Epitelio/efectos de los fármacos , Masculino , Radioisótopos , Ratas , Ratas Endogámicas Lew , Proteínas Recombinantes/farmacología , Timidina/metabolismoAsunto(s)
Trasplante de Corazón , Tolerancia Inmunológica , Linfocinas/genética , Transcripción Genética , Animales , Circulación Coronaria , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Miocardio/metabolismo , Hibridación de Ácido Nucleico , ARN/genética , ARN/aislamiento & purificación , Trasplante Homólogo , Trasplante IsogénicoRESUMEN
Rat glomeruli were isolated utilizing differential sieving technique. Establishment and maintenance of epithelial and mesangial cell lines originating from glomeruli were based upon their individual growth requirements and simple cloning procedures. Despite repeated passages cells retained their specific morphologic features in cultures for approximately six months. Our attempts to grow rat glomerular endothelial cells with use of the platelet-derived growth factor (PDGF), endothelial mitogen (EM), thrombin and fibroblast-conditioned medium ended in failure. Although occasionally endothelial-like cells were seen in glomerular outgrowths, we did not succeed in cloning and propagating them in subsequent passages. It is concluded that growth in vitro of each type of rat kidney glomerular cells is dependent on specific conditions and stimulants. Moreover, endothelial cells of rat renal tufts proved refractory to cultivation using methods reported effective with regard to other mammalian species.