The interleukin-1 family is a group of important cytokines that play a key regulatory role in the immune and inflammatory response(including infectious and non-bacterial injuries).Nowadays,the interleukin-1 family mai...The interleukin-1 family is a group of important cytokines that play a key regulatory role in the immune and inflammatory response(including infectious and non-bacterial injuries).Nowadays,the interleukin-1 family mainly includes 11 cytokines and has multiple roles in the pathology and physiology of inflammation.Moreover,accumulating number of research show that the interleukin-1 family and its receptors are involved in the occurrence and development of cardiovascular diseases.Therefore,we show here the review involving hotspots of the interleukin-1 family in the process of inflammation and its target therapy in cardiovascular diseases,including atherosclerosis,myocardial infarction and heart failure.展开更多
Background: Cytokines are mediators of disease. Expression levels in the blood could be of clinical relevance. Objective: Aim of this study was to show if serum levels of IL-1β could be of any clinical relevance conc...Background: Cytokines are mediators of disease. Expression levels in the blood could be of clinical relevance. Objective: Aim of this study was to show if serum levels of IL-1β could be of any clinical relevance concerning dogs. IL-1β was measured in serum samples of healthy dogs to find a reference range for healthy individuals. Measurements of IL-1β should show if this substance was a possible marker for early stages of inflammation. Therefore, a possible relation between serum levels and grades of leukocytosis was analyzed. Methods: IL-1β concentrations in the blood were assessed by the use of a human enzyme linked immunosorbent assay (ELISA). 39 dogs with different inflammatory diseases were analyzed to figure out if there was a correlation between IL-1β serum levels and the number of leukocytes in peripheral blood. The control group consisted of 16 healthy dogs. Results: about half of the samples IL-1β were detected. Most of the patients showed no detectable amounts of IL-1β. The IL-1β levels measured in the serum were stable for at least nine weeks when stored at ?20?C. The patients tested positively on IL-1β had mostly lower-grade leukocytosis compared to those who had no IL-1β in serum. All the dogs which were suffering from disease but still had no traceable IL-1β, showed a leukocytosis as a common symptom. Conclusion: This study showed that IL-1β could become an interesting marker for the detection of early stages of inflammation when leukocytosis does not yet appear in peripheral blood. Nonetheless, the possible use in diagnosis is restricted. This is due to the fact that there are only low amounts of IL-1β to be detected in the serum, even concerning patients are suffering from disease.展开更多
The course and severity of periodontitis can be significantly affected by bacterial virulence as well as host immunity dysfunction. Periodontal tissue destruction has been proved to result from cascade of cytokines sy...The course and severity of periodontitis can be significantly affected by bacterial virulence as well as host immunity dysfunction. Periodontal tissue destruction has been proved to result from cascade of cytokines synthesized by reactive cells upon stimulation by pathogenic bacteria and lipopolysaccharides within their cell membranes. The clinical use of genetically programmed cells, producing substances blocking IL-1, based on recombinant IL-1 antagonist, as well as cytokines activating fibroblasts and osteoblasts to regenerate the destroyed periodontal tissue could prove alternative to the conventional treatment. Another cytokine of interest in respect to periodontitis ethiopathogenesis is soluble tumor necrosis factor receptor I (sTNF RI). Observation of soluble TNF receptors as physiologic inhibitors of TNF led to its administration in therapeutic process as well as in therapy selected cases of aggressive periodontitis.展开更多
BACKGROUND Obesity is a recognized risk factor for endometrial cancer (EmCa) and other cancer types. Leptin levels are significantly increased in obese individuals. Leptin-induced signaling crosstalk [Notch, Interleuk...BACKGROUND Obesity is a recognized risk factor for endometrial cancer (EmCa) and other cancer types. Leptin levels are significantly increased in obese individuals. Leptin-induced signaling crosstalk [Notch, Interleukin-1 (IL-1) and leptin outcome, NILCO] has been associated with breast cancer progression. This complex signaling crosstalk affects cancer cell proliferation, migration, invasion, angiogenesis, apoptosis and chemoresistance. NILCO expression was previously detected in human EmCa. However, it is unknown whether leptin regulates NILCO and alters EmCa’s response to chemotherapeutics. It is hypothesized that leptin induces NILCO and increases aggressiveness and chemoresistance in EmCa cells. AIM To determine whether leptin induces NILCO molecules in EmCa affecting cell proliferation, aggressiveness and chemoresistance. METHODS Leptin’s effects on the expression of NILCO molecules [mRNAs and proteins for Notch receptors (Notch1-4), ligands (JAG1 and DLL4) and downstream effectors (survivin, Hey2), and leptin (OB-R) and IL-1 (IL-1R tI) receptors] was examined in EmCa cells (type I: Ishikawa, and HEC-1A, and type II: An3Ca and KLE) using Real-time PCR and Western blot analysis, respectively. In addition, the effects of leptin on cell cycle, proliferation and cell invasion were determined using cytometric analysis (Cellometer Vision CBA system), MTT cell proliferation and Matrigel-based invasion assays, respectively. Inhibitors of leptin (nanoparticlebound leptin peptide receptor antagonist-2, IONP-LPrA2), IL-1 (anti-IL-1R tI antibody) and Notch (siRNA interference RNA) were used to investigate NILCO’s effects on cell proliferation and invasion. Leptin’s effects on Paclitaxel cytotoxicity in EmCa cells was determined by the CCK8 and Cellometer-based Annexin V assays. RESULTS For the first time it was shown that leptin is an inducer of Notch in EmCa. Experimental data suggest that leptin induced the expression of NILCO molecules, promoted proliferation and S- phase progression, and reduced Paclitaxel cytotoxicity on EmCa cells. Leptin’s effects were higher in type II EmCa cells. The progression of this more aggressive form of the disease is associated with obesity. Remarkably, the use of the leptin signaling antagonist, IONPLPrA2, re-sensitized EmCa cells to Paclitaxel. CONCLUSION Present data suggest the notion that leptin-induced NILCO could be a link between obesity and EmCa progression and chemoresistance. Most aggressive type II EmCa cells were higher sensitive to leptin, which appears to increase proliferation, cell cycle progression, aggressiveness, and chemoresistance to Paclitaxel. Therefore, leptin and NILCO could be novel therapeutic targets for type II EmCa, which does not have targeted therapy. Overall, IONP-LPrA2 has a potential as a novel adjuvant drug to enhance the effectiveness of type II EmCa chemotherapy.展开更多
AIM To evaluate the impact of cytochrome P450 2C19(CYP2C19) and interleukin-1β(IL-1β) polymorphisms on the efficacy of Helicobacter pylori(H. pylori) eradication by using rabeprazole-based hybrid therapy.METHODS A t...AIM To evaluate the impact of cytochrome P450 2C19(CYP2C19) and interleukin-1β(IL-1β) polymorphisms on the efficacy of Helicobacter pylori(H. pylori) eradication by using rabeprazole-based hybrid therapy.METHODS A total of 88 H. pylori-infected patients were recruited to receive 14-d of hybrid therapy from March 2013 to May 2014. Three patients were excluded from analysis because of incomplete compliance. Either a follow-up endoscopy or 13 C-urea test was performed to determine the results of H. pylori eradication therapy. The genotypes of CYP2C19 and IL-1β were analyzed to investigate the impact on treatment effect. RESULTS The total eradication rate of H. pylori was 92.94%(79/85). According to the CYP2C19 genotypes, the rates of H. pylori eradication were 89.19% in extensive metabolizers(EM) and 95.83% in non-EM. The H.pylori eradication rates regarding the IL-1β genotypes were 92.59% in the normal acid secretion group and 93.10% in the low acid secretion group. After multivariable logistic regression analysis, both the genotypes of CYP2C19 and IL-1β had no significant influences on the eradication rates of H. pylori.CONCLUSION The CYP2C19 and IL-1β polymorphisms are not significantly independent factors of H.pylori eradication using rabeprazole-based hybrid therapy.展开更多
Targeting multiple immune mechanisms may overcome therapy resistance and further improve cancer immunotherapy for humans.Here,we describe the application of virus-like vesicles(VLV)for delivery of three immunomodulato...Targeting multiple immune mechanisms may overcome therapy resistance and further improve cancer immunotherapy for humans.Here,we describe the application of virus-like vesicles(VLV)for delivery of three immunomodulators alone and in combination,as a promising approach for cancer immunotherapy.VLV vectors were designed to deliver single chain interleukin(IL)-12,shorthairpin RNA(shRNA)targeting programmed death ligand 1(PD-L1),and a dominant-negative form of IL-17 receptor A(dn-IL17RA)as a single payload or as a combination payload.Intralesional delivery of the VLV vector expressing IL-12 alone,as well as the trivalent vector(designated CARG-2020)eradicated large established tumors.However,only CARG-2020 prevented tumor recurrence and provided long-term survival benefit to the tumor-bearing mice,indicating a benefit of the combined immunomodulation.The abscopal effects of CARG-2020 on the non-injected contralateral tumors,as well as protection from the tumor cell re-challenge,suggest immune-mediated mechanism of protection and establishment of immunological memory.Mechanistically,CARG-2020 potently activates Th1 immune mechanisms and inhibits expression of genes related to T cell exhaustion and cancer-promoting inflammation.The ability of CARG-2020 to prevent tumor recurrence and to provide survival benefit makes it a promising candidate for its development for human cancer immunotherapy.展开更多
Gene therapy constitutes a promising strategy for the treatment of osteoarthritis (OA). We assessed the use of electroporation (EP) of non-viral gene vectors, and compared its efficacy with that of adeno-associated vi...Gene therapy constitutes a promising strategy for the treatment of osteoarthritis (OA). We assessed the use of electroporation (EP) of non-viral gene vectors, and compared its efficacy with that of adeno-associated virus (AAV) vectors. EP- and AAV-mediated delivery of human interleukin-1 receptor antagonist (hIL-1Ra) was localized performed in the joints of rats following induction of OA. mRNA levels for hIL-1Ra, IL-1β, TNF-α, MMP-13 and ADAMTS-4 in the cartilage and synovial tissues were analyzed. Structural analyses of the subchondral bone at the medial femoral condyle were performed by Micro-CT after treatment. Knee joint specimens were staining with hematoxylin and eosin and Saffron O. Induction of hIL-1Ra by both EP and AAV inhibited inflammatory-induced sub-chondral bone reconstruction, and effectively suppressed IL-1β activity, as evidenced by decreased expression of MMP-13 and ADAMTS-4. Histological analyses revealed significant protection of cartilage, proteoglycan by EP and AAV. hIL-1Ra expression was similar in both the EP and AAV groups. Notably, this gene is not easier degraded transduced by EP compared with AAV. Taken together, these results show that EP offers transfection efficiency comparable to that of AAV, with the potential for longer gene expression, making EP a promising candidate for efficient non-viral delivery of OA gene therapy.展开更多
Background:The objective of this study is to evaluate the implications of interleukin-1β(IL-1β)in photoreceptor degeneration using a model of blue light in rodents.Methods:CD-1 mice(12-16 weeks-old)were exposed to b...Background:The objective of this study is to evaluate the implications of interleukin-1β(IL-1β)in photoreceptor degeneration using a model of blue light in rodents.Methods:CD-1 mice(12-16 weeks-old)were exposed to blue LED light(6000 lux at 450 nm)for 1 hour and then sacrificed at day 3 post-illumination.Mice were intraperitoneally treated or not with a peptide antagonist of the IL-1βreceptor,named Rytvela(or 101.10)twice per day until sacrifice.Several markers related to the inflammatory process such as F4/80,NLRP3,Caspase-1,IL-1βand glial fibrillary acidic protein(GFAP)were evaluated by immunohistochemistry.Photoreceptor cell death was assessed by TUNEL assay and Caspase-3 immunofluorescence.Results:Immunofluorescence experiments revealed an infiltration of positive F4/80 cells(microglia and macrophages)into the subretinal space in mice exposed to blue light,which was significantly(P<0.01)abrogated with Rytvela treatment.Co-localization of NLRP3,Caspase-1,and IL-1βwith F4/80 positive cells was clearly detected in the subretinal space,suggesting that these inflammatory cells are the main source of IL-1β.Interestingly,GFAP immunoreactivity,a marker of stress in Müller cells,was augmented in retinas exposed to the blue light,and reduced with Rytvela administration.The TUNEL assay showed that Rytvela prevents photoreceptor apoptosis in the retina of mice exposed to blue light.Likewise,co-culture of retinal explants with LPS-ATP activated bone marrow-derived macrophages resulted in a high number of TUNEL positive photoreceptors,which was reduced by treatment with Rytvela.Conclusions:These results show that Rytvela attenuated the inflammatory response and prevented the death of photoreceptors in a model of dry AMD.Modulation of IL-1βsignaling would be a useful therapeutic avenue for dry AMD,for which no approved treatment currently exists.展开更多
基金Key Research and Development Project of Hainan Province(No.ZDYF2021SHFZ089)Rapid Innovation Foundation for Key Laboratories of the Ministry of Education(No.KLET-202019)。
文摘The interleukin-1 family is a group of important cytokines that play a key regulatory role in the immune and inflammatory response(including infectious and non-bacterial injuries).Nowadays,the interleukin-1 family mainly includes 11 cytokines and has multiple roles in the pathology and physiology of inflammation.Moreover,accumulating number of research show that the interleukin-1 family and its receptors are involved in the occurrence and development of cardiovascular diseases.Therefore,we show here the review involving hotspots of the interleukin-1 family in the process of inflammation and its target therapy in cardiovascular diseases,including atherosclerosis,myocardial infarction and heart failure.
文摘Background: Cytokines are mediators of disease. Expression levels in the blood could be of clinical relevance. Objective: Aim of this study was to show if serum levels of IL-1β could be of any clinical relevance concerning dogs. IL-1β was measured in serum samples of healthy dogs to find a reference range for healthy individuals. Measurements of IL-1β should show if this substance was a possible marker for early stages of inflammation. Therefore, a possible relation between serum levels and grades of leukocytosis was analyzed. Methods: IL-1β concentrations in the blood were assessed by the use of a human enzyme linked immunosorbent assay (ELISA). 39 dogs with different inflammatory diseases were analyzed to figure out if there was a correlation between IL-1β serum levels and the number of leukocytes in peripheral blood. The control group consisted of 16 healthy dogs. Results: about half of the samples IL-1β were detected. Most of the patients showed no detectable amounts of IL-1β. The IL-1β levels measured in the serum were stable for at least nine weeks when stored at ?20?C. The patients tested positively on IL-1β had mostly lower-grade leukocytosis compared to those who had no IL-1β in serum. All the dogs which were suffering from disease but still had no traceable IL-1β, showed a leukocytosis as a common symptom. Conclusion: This study showed that IL-1β could become an interesting marker for the detection of early stages of inflammation when leukocytosis does not yet appear in peripheral blood. Nonetheless, the possible use in diagnosis is restricted. This is due to the fact that there are only low amounts of IL-1β to be detected in the serum, even concerning patients are suffering from disease.
文摘The course and severity of periodontitis can be significantly affected by bacterial virulence as well as host immunity dysfunction. Periodontal tissue destruction has been proved to result from cascade of cytokines synthesized by reactive cells upon stimulation by pathogenic bacteria and lipopolysaccharides within their cell membranes. The clinical use of genetically programmed cells, producing substances blocking IL-1, based on recombinant IL-1 antagonist, as well as cytokines activating fibroblasts and osteoblasts to regenerate the destroyed periodontal tissue could prove alternative to the conventional treatment. Another cytokine of interest in respect to periodontitis ethiopathogenesis is soluble tumor necrosis factor receptor I (sTNF RI). Observation of soluble TNF receptors as physiologic inhibitors of TNF led to its administration in therapeutic process as well as in therapy selected cases of aggressive periodontitis.
基金Supported by the National Cancer Institute at the National Institutes of Health,No.S21 MD000101,No.5G12 MD0076021,No.G12RR026250-03,No.NIH RR030341C06,No.RR18386 to Morehouse School of Medicinethe National Institute of General Medical Sciences,National Institutes of Health,No.5T32HL103104-04 to DDB
文摘BACKGROUND Obesity is a recognized risk factor for endometrial cancer (EmCa) and other cancer types. Leptin levels are significantly increased in obese individuals. Leptin-induced signaling crosstalk [Notch, Interleukin-1 (IL-1) and leptin outcome, NILCO] has been associated with breast cancer progression. This complex signaling crosstalk affects cancer cell proliferation, migration, invasion, angiogenesis, apoptosis and chemoresistance. NILCO expression was previously detected in human EmCa. However, it is unknown whether leptin regulates NILCO and alters EmCa’s response to chemotherapeutics. It is hypothesized that leptin induces NILCO and increases aggressiveness and chemoresistance in EmCa cells. AIM To determine whether leptin induces NILCO molecules in EmCa affecting cell proliferation, aggressiveness and chemoresistance. METHODS Leptin’s effects on the expression of NILCO molecules [mRNAs and proteins for Notch receptors (Notch1-4), ligands (JAG1 and DLL4) and downstream effectors (survivin, Hey2), and leptin (OB-R) and IL-1 (IL-1R tI) receptors] was examined in EmCa cells (type I: Ishikawa, and HEC-1A, and type II: An3Ca and KLE) using Real-time PCR and Western blot analysis, respectively. In addition, the effects of leptin on cell cycle, proliferation and cell invasion were determined using cytometric analysis (Cellometer Vision CBA system), MTT cell proliferation and Matrigel-based invasion assays, respectively. Inhibitors of leptin (nanoparticlebound leptin peptide receptor antagonist-2, IONP-LPrA2), IL-1 (anti-IL-1R tI antibody) and Notch (siRNA interference RNA) were used to investigate NILCO’s effects on cell proliferation and invasion. Leptin’s effects on Paclitaxel cytotoxicity in EmCa cells was determined by the CCK8 and Cellometer-based Annexin V assays. RESULTS For the first time it was shown that leptin is an inducer of Notch in EmCa. Experimental data suggest that leptin induced the expression of NILCO molecules, promoted proliferation and S- phase progression, and reduced Paclitaxel cytotoxicity on EmCa cells. Leptin’s effects were higher in type II EmCa cells. The progression of this more aggressive form of the disease is associated with obesity. Remarkably, the use of the leptin signaling antagonist, IONPLPrA2, re-sensitized EmCa cells to Paclitaxel. CONCLUSION Present data suggest the notion that leptin-induced NILCO could be a link between obesity and EmCa progression and chemoresistance. Most aggressive type II EmCa cells were higher sensitive to leptin, which appears to increase proliferation, cell cycle progression, aggressiveness, and chemoresistance to Paclitaxel. Therefore, leptin and NILCO could be novel therapeutic targets for type II EmCa, which does not have targeted therapy. Overall, IONP-LPrA2 has a potential as a novel adjuvant drug to enhance the effectiveness of type II EmCa chemotherapy.
文摘AIM To evaluate the impact of cytochrome P450 2C19(CYP2C19) and interleukin-1β(IL-1β) polymorphisms on the efficacy of Helicobacter pylori(H. pylori) eradication by using rabeprazole-based hybrid therapy.METHODS A total of 88 H. pylori-infected patients were recruited to receive 14-d of hybrid therapy from March 2013 to May 2014. Three patients were excluded from analysis because of incomplete compliance. Either a follow-up endoscopy or 13 C-urea test was performed to determine the results of H. pylori eradication therapy. The genotypes of CYP2C19 and IL-1β were analyzed to investigate the impact on treatment effect. RESULTS The total eradication rate of H. pylori was 92.94%(79/85). According to the CYP2C19 genotypes, the rates of H. pylori eradication were 89.19% in extensive metabolizers(EM) and 95.83% in non-EM. The H.pylori eradication rates regarding the IL-1β genotypes were 92.59% in the normal acid secretion group and 93.10% in the low acid secretion group. After multivariable logistic regression analysis, both the genotypes of CYP2C19 and IL-1β had no significant influences on the eradication rates of H. pylori.CONCLUSION The CYP2C19 and IL-1β polymorphisms are not significantly independent factors of H.pylori eradication using rabeprazole-based hybrid therapy.
基金This work was supported by an American Association of Immunologists Careers in Immunology Fellowship to Ju Chen,University of Connecticut Innovation Fund to Valerian Nakaar and Kepeng WangNational Institute of Health/National Cancer Institute(NIH/NCI R01CA262430,USA)to Kepeng Wang+1 种基金This research was also funded by NIDDK(Nos.R43DK113858 and R44DK113858,USA)by NIAID(No.R43AI149798,USA)to Valerian Nakaar.The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.The funders had no role in study design,data collection and interpretation,or the decision to submit the work for publication.
文摘Targeting multiple immune mechanisms may overcome therapy resistance and further improve cancer immunotherapy for humans.Here,we describe the application of virus-like vesicles(VLV)for delivery of three immunomodulators alone and in combination,as a promising approach for cancer immunotherapy.VLV vectors were designed to deliver single chain interleukin(IL)-12,shorthairpin RNA(shRNA)targeting programmed death ligand 1(PD-L1),and a dominant-negative form of IL-17 receptor A(dn-IL17RA)as a single payload or as a combination payload.Intralesional delivery of the VLV vector expressing IL-12 alone,as well as the trivalent vector(designated CARG-2020)eradicated large established tumors.However,only CARG-2020 prevented tumor recurrence and provided long-term survival benefit to the tumor-bearing mice,indicating a benefit of the combined immunomodulation.The abscopal effects of CARG-2020 on the non-injected contralateral tumors,as well as protection from the tumor cell re-challenge,suggest immune-mediated mechanism of protection and establishment of immunological memory.Mechanistically,CARG-2020 potently activates Th1 immune mechanisms and inhibits expression of genes related to T cell exhaustion and cancer-promoting inflammation.The ability of CARG-2020 to prevent tumor recurrence and to provide survival benefit makes it a promising candidate for its development for human cancer immunotherapy.
文摘Gene therapy constitutes a promising strategy for the treatment of osteoarthritis (OA). We assessed the use of electroporation (EP) of non-viral gene vectors, and compared its efficacy with that of adeno-associated virus (AAV) vectors. EP- and AAV-mediated delivery of human interleukin-1 receptor antagonist (hIL-1Ra) was localized performed in the joints of rats following induction of OA. mRNA levels for hIL-1Ra, IL-1β, TNF-α, MMP-13 and ADAMTS-4 in the cartilage and synovial tissues were analyzed. Structural analyses of the subchondral bone at the medial femoral condyle were performed by Micro-CT after treatment. Knee joint specimens were staining with hematoxylin and eosin and Saffron O. Induction of hIL-1Ra by both EP and AAV inhibited inflammatory-induced sub-chondral bone reconstruction, and effectively suppressed IL-1β activity, as evidenced by decreased expression of MMP-13 and ADAMTS-4. Histological analyses revealed significant protection of cartilage, proteoglycan by EP and AAV. hIL-1Ra expression was similar in both the EP and AAV groups. Notably, this gene is not easier degraded transduced by EP compared with AAV. Taken together, these results show that EP offers transfection efficiency comparable to that of AAV, with the potential for longer gene expression, making EP a promising candidate for efficient non-viral delivery of OA gene therapy.
文摘Background:The objective of this study is to evaluate the implications of interleukin-1β(IL-1β)in photoreceptor degeneration using a model of blue light in rodents.Methods:CD-1 mice(12-16 weeks-old)were exposed to blue LED light(6000 lux at 450 nm)for 1 hour and then sacrificed at day 3 post-illumination.Mice were intraperitoneally treated or not with a peptide antagonist of the IL-1βreceptor,named Rytvela(or 101.10)twice per day until sacrifice.Several markers related to the inflammatory process such as F4/80,NLRP3,Caspase-1,IL-1βand glial fibrillary acidic protein(GFAP)were evaluated by immunohistochemistry.Photoreceptor cell death was assessed by TUNEL assay and Caspase-3 immunofluorescence.Results:Immunofluorescence experiments revealed an infiltration of positive F4/80 cells(microglia and macrophages)into the subretinal space in mice exposed to blue light,which was significantly(P<0.01)abrogated with Rytvela treatment.Co-localization of NLRP3,Caspase-1,and IL-1βwith F4/80 positive cells was clearly detected in the subretinal space,suggesting that these inflammatory cells are the main source of IL-1β.Interestingly,GFAP immunoreactivity,a marker of stress in Müller cells,was augmented in retinas exposed to the blue light,and reduced with Rytvela administration.The TUNEL assay showed that Rytvela prevents photoreceptor apoptosis in the retina of mice exposed to blue light.Likewise,co-culture of retinal explants with LPS-ATP activated bone marrow-derived macrophages resulted in a high number of TUNEL positive photoreceptors,which was reduced by treatment with Rytvela.Conclusions:These results show that Rytvela attenuated the inflammatory response and prevented the death of photoreceptors in a model of dry AMD.Modulation of IL-1βsignaling would be a useful therapeutic avenue for dry AMD,for which no approved treatment currently exists.
