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Antibody-mediated rejection (AMR) is among the most frequent causes for graft loss after kidney transplantation. While there are no approved therapies, several case reports with daratumumab and the very recent phase 2 trial of felzartamab in AMR have indicated the potential efficacy of therapeutic interventions targeting CD38. Donor-derived cell-free DNA (dd-cfDNA) is an emerging biomarker with injury-specific release and a short half-life, which could facilitate early diagnosis of AMR and monitoring of treatment response. We describe two cases of patients with chronic active AMR, who were treated with monthly daratumumab infusions, and in whom donor-derived cell-free DNA (dd-cfDNA) was measured longitudinally to monitor treatment response. In both patients, daratumumab treatment led to stabilization of kidney function parameters, a strong decline of dd-cfDNA below the previously established threshold for rejection, and partial or complete histologic resolution of AMR activity. Our case series suggests that dd-cfDNA may be a useful companion biomarker for longitudinal monitoring of anti-CD38 treatment in patients with AMR.
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Anticuerpos Monoclonales , Biomarcadores , Ácidos Nucleicos Libres de Células , Rechazo de Injerto , Trasplante de Riñón , Humanos , Ácidos Nucleicos Libres de Células/sangre , Anticuerpos Monoclonales/uso terapéutico , Rechazo de Injerto/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Biomarcadores/sangre , Femenino , Donantes de Tejidos , Adulto , ADP-Ribosil Ciclasa 1RESUMEN
Antibody-mediated rejection (AMR) is a major cause of graft failure limiting long-term graft survival after kidney transplantation. Current diagnostic strategy to detect AMR is suboptimal and requires further improvement. Previously suggested treatment regimens for AMR could not demonstrate efficacy, however novel therapeutic agents are currently under investigation. Donor-derived cell-free DNA (dd-cfDNA) is a novel non-invasive biomarker for allograft injury, that has been mainly studied in the context of rejection. Its short-half-life in circulation and injury-dependent release are its key advantages that contribute to its superior diagnostic accuracy, compared to traditional biomarkers. Moreover, previous studies showed that dd-cfDNA-release is well-linked to histological and molecular features of AMR, and thus able to reflect real-time injury. Further observations suggest that dd-cfDNA can be used as a suitable screening tool for early detection of AMR in patients with donor-specific-anti-HLA-antibodies (DSA), as well as for monitoring AMR activity after anti-rejection treatment. The weight of evidence suggests that the integration of dd-cfDNA in the graft surveillance of patients with AMR, or those suspicious of AMR (e.g., due to the presence of donor-specific anti-HLA-antibodies) has an added value and might have a positive impact on outcomes in this specific cohort.
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Biomarcadores , Ácidos Nucleicos Libres de Células , Rechazo de Injerto , Trasplante de Riñón , Donantes de Tejidos , Trasplante de Riñón/efectos adversos , Humanos , Rechazo de Injerto/inmunología , Rechazo de Injerto/diagnóstico , Ácidos Nucleicos Libres de Células/sangre , Biomarcadores/sangre , Antígenos HLA/inmunología , Isoanticuerpos/inmunología , Isoanticuerpos/sangre , Supervivencia de Injerto/inmunologíaRESUMEN
Enlarged perivascular spaces (EPVS) are common in cerebral small vessel disease (CSVD) and have been identified as a marker of dysfunctional brain clearance. However, it remains unknown if the enlargement occurs predominantly around arteries or veins. We combined in vivo ultra-high-resolution MRI and histopathology to investigate the spatial relationship of veins and arteries with EPVS within the basal ganglia (BG). Furthermore, we assessed the relationship between the EPVS and measures of blood-flow (blood-flow velocity, pulsatility index) in the small arteries of the BG. Twenty-four healthy controls, twelve non-CAA CSVD patients, and five probable CAA patients underwent a 3 tesla [T] and 7T MRI-scan, and EPVS, arteries, and veins within the BG were manually segmented. Furthermore, the scans were co-registered. Six autopsy-cases were also assessed. In the BG, EPVS were significantly closer to and overlapped more frequently with arteries than with veins. Histological analysis showed a higher proportion of BG EPVS surrounding arteries than veins. Finally, the pulsatility index of BG arteries correlated with EPVS volume. Our results are in line with previous works and establish a pathophysiological relationship between arteries and EPVS, contributing to elucidating perivascular clearance routes in the human brain.
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BACKGROUND: Antibody-mediated rejection is a leading cause of kidney-transplant failure. The targeting of CD38 to inhibit graft injury caused by alloantibodies and natural killer (NK) cells may be a therapeutic option. METHODS: In this phase 2, double-blind, randomized, placebo-controlled trial, we assigned patients with antibody-mediated rejection that had occurred at least 180 days after transplantation to receive nine infusions of the CD38 monoclonal antibody felzartamab (at a dose of 16 mg per kilogram of body weight) or placebo for 6 months, followed by a 6-month observation period. The primary outcome was the safety and side-effect profile of felzartamab. Key secondary outcomes were renal-biopsy results at 24 and 52 weeks, donor-specific antibody levels, peripheral NK-cell counts, and donor-derived cell-free DNA levels. RESULTS: A total of 22 patients underwent randomization (11 to receive felzartamab and 11 to receive placebo). The median time from transplantation until trial inclusion was 9 years. Mild or moderate infusion reactions occurred in 8 patients in the felzartamab group. Serious adverse events occurred in 1 patient in the felzartamab group and in 4 patients in the placebo group; graft loss occurred in 1 patient in the placebo group. At week 24, resolution of morphologic antibody-mediated rejection was more frequent with felzartamab (in 9 of 11 patients [82%]) than with placebo (in 2 of 10 patients [20%]), for a difference of 62 percentage points (95% confidence interval [CI], 19 to 100) and a risk ratio of 0.23 (95% confidence interval [CI], 0.06 to 0.83). The median microvascular inflammation score was lower in the felzartamab group than in the placebo group (0 vs. 2.5), for a mean difference of -1.95 (95% CI, -2.97 to -0.92). Also lower was a molecular score reflecting the probability of antibody-mediated rejection (0.17 vs. 0.77) and the level of donor-derived cell-free DNA (0.31% vs. 0.82%). At week 52, the recurrence of antibody-mediated rejection was reported in 3 of 9 patients who had a response to felzartamab, with an increase in molecular activity and biomarker levels toward baseline levels. CONCLUSIONS: Felzartamab had acceptable safety and side-effect profiles in patients with antibody-mediated rejection. (Funded by MorphoSys and Human Immunology Biosciences; ClinicalTrials.gov number, NCT05021484; and EUDRACT number, 2021-000545-40.).
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Rechazo de Injerto , Isoanticuerpos , Trasplante de Riñón , Células Asesinas Naturales , Humanos , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Método Doble Ciego , Femenino , Masculino , Persona de Mediana Edad , Trasplante de Riñón/efectos adversos , Células Asesinas Naturales/inmunología , Adulto , Isoanticuerpos/sangre , Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales Humanizados/efectos adversos , Riñón/patología , Riñón/inmunología , Anciano , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales/efectos adversosRESUMEN
OBJECTIVE: Distinguishing arginine vasopressin deficiency (AVP-D; central diabetes insipidus) from primary polydipsia (PP), commonly referred to as psychogenic polydipsia, is challenging. Psychopathologic findings, commonly used for PP diagnosis in clinical practice, are rarely evaluated in AVP-D patients, and no comparative data between the two conditions currently exist. DESIGN: Data from two studies involving 82 participants [39 AVP-D, 28 PP, and 15 healthy controls (HC)]. METHODS: Psychological evaluations were conducted using standardized questionnaires measuring anxiety [State-Trait Anxiety Inventory (STAI)], alexithymia [Toronto Alexithymia Scale (TAS-20)], depressive symptoms (Beck's Depression Inventory-II (BDI-II), and overall mental health [Short Form-36 Health Survey (SF-36)]. Higher STAI, TAS-20, and BDI-II scores suggest elevated anxiety, alexithymia, and depression, while higher SF-36 scores signify better overall mental health. RESULTS: Compared to HC, patients with AVP-D and PP showed higher levels of anxiety (HC 28 points [24-31] vs AVP-D 36 points [31-45]; vs PP 38 points [33-46], P < .01), alexithymia (HC 30 points [29-37] vs AVP-D 43 points [35-54]; vs PP 46 points [37-55], P < .01), and depression (HC 1 point [0-2] vs AVP-D 7 points [4-14]; vs PP 7 points [3-13], P < .01). Levels of anxiety, alexithymia, and depression showed no difference between both patient groups (P = .58, P = .90, P = .50, respectively). Compared to HC, patients with AVP-D and PP reported similarly reduced self-reported overall mental health scores (HC 84 [68-88] vs AVP-D 60 [52-80], P = .05; vs PP 60 [47-74], P < .01). CONCLUSION: This study reveals heightened anxiety, alexithymia, depression, and diminished overall mental health in patients with AVP-D and PP. The results emphasize the need for careful interpretation of psychopathological characteristics to differentiate between AVP-D and PP.
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Síntomas Afectivos , Ansiedad , Depresión , Diabetes Insípida Neurogénica , Humanos , Femenino , Masculino , Adulto , Depresión/psicología , Persona de Mediana Edad , Ansiedad/psicología , Diabetes Insípida Neurogénica/psicología , Arginina Vasopresina/deficiencia , Polidipsia Psicogénica/psicología , Polidipsia Psicogénica/complicaciones , Adulto Joven , Polidipsia/psicología , Estudios de Casos y ControlesRESUMEN
Sex differences affect the management of several diseases in both male and female patients. However, the influence of sex on neuroendocrine neoplasms (NENs) has been scarcely investigated. Thus, this study aimed to compare tumor characteristics, treatment decisions, and overall survival in patients with NENs, stratified by sex. The retrospective analysis of the SwissNET cohort covered NENs of gastroenteropancreatic, pulmonary, or unknown origin from July 2014 to September 2022. The analysis included 1985 patients (46% female and 54% male). No significant difference in tumor grading was found between male and female patients. However, male patients presented with higher staging at time of diagnosis and with more lymph node and bone metastases. Surgery was performed more often in female compared to male patients (73.4% vs 68.7%, P = 0.023). Male patients received peptide receptor nuclide therapy (PRRT) earlier than female patients (7.8 months vs 13.1 months from time of diagnosis, P = 0.003). The median overall survival was significantly shorter for male compared to female patients (male: 18 years, female: not reached, P < 0.001, hazard ratio (HR) 1.55 (1.19-2.01), P = 0.001). Multivariable analyses revealed advanced age (HR 1.02 (1.01-1.04)), cancer of unknown origin (HR 2.01 (1.09-3.70)), higher grading (G3: HR 6.74 (4.22-10.76)), having metastases at the time of diagnosis (HR 2.11 (1.47-3.02)), and surgical treatment (HR 0.67 (0.48-0.93)) as independent predictors for overall survival. In conclusion, male sex was associated with worse outcome in NEN patients, likely due to more advanced tumor stage at the time of diagnosis. Further investigations are required to understand the underlying mechanisms of these sex differences.
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Tumores Neuroendocrinos , Neoplasias Pancreáticas , Humanos , Masculino , Femenino , Estudios Retrospectivos , Tumores Neuroendocrinos/patología , Pronóstico , Clasificación del Tumor , Modelos de Riesgos Proporcionales , Neoplasias Pancreáticas/patologíaRESUMEN
Donor-derived cell-free DNA (dd-cfDNA) is used as a biomarker for detection of antibody-mediated rejection (ABMR) and other forms of graft injury. Another potential indication is guidance of immunosuppressive therapy when no therapeutic drug monitoring is available. In such situations, detection of patients with overt or subclinical graft injury is important to personalize immunosuppression. We prospectively measured dd-cfDNA in 22 kidney transplant recipients (KTR) over a period of 6 months after conversion to belatacept for clinical indication and assessed routine clinical parameters. Patient and graft survival was 100% after 6 months, and eGFR remained stable (28.7 vs. 31.1 mL/min/1.73 m2, p = 0.60). Out of 22 patients, 2 (9%) developed biopsy-proven rejection-one episode of low-grade TCMR IA and one episode of caABMR. While both episodes were detected by increase in creatinine, the caABMR episode led to increase in absolute dd-cfDNA (168 copies/mL) above the cut-off of 50 copies/mL, while the TCMR episode did show slightly increased relative dd-cfDNA (0.85%) despite normal absolute dd-cfDNA (22 copies/mL). Dd-cfDNA did not differ before and after conversion in a subgroup of 12 KTR with previous calcineurin inhibitor therapy and no rejection (12.5 vs. 25.3 copies/mL, p = 0.34). In this subgroup, 3/12 (25%) patients showed increase of absolute dd-cfDNA above the prespecified cut-off (50 copies/mL) despite improving eGFR. Increase in dd-cfDNA after conversion to belatacept is common and could point towards subclinical allograft injury. To detect subclinical TCMR changes without vascular lesions, additional biomarkers or urinary dd-cfDNA should complement plasma dd-cfDNA. Resolving CNI toxicity is unlikely to be detected by decreased dd-cfDNA levels. In summary, the sole determination of dd-cfDNA has limited utility in the guidance of patients after late conversion to belatacept. Further studies should focus on patients undergoing early conversion and include protocol biopsies at least for patients with increased dd-cfDNA.
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Oncogenic KRAS is the key driver oncogene for several of the most aggressive human cancers. One key feature of oncogenic KRAS expression is an early increase in cellular reactive oxygen species (ROS) which promotes cellular transformation if cells manage to escape cell death, mechanisms of which remain incompletely understood. Here, we identify that expression of oncogenic as compared to WT KRAS in isogenic cellular systems renders cells more resistant to ferroptosis, a recently described type of regulated necrosis. Mechanistically, we find that cells with mutant KRAS show a specific lack of ferroptosis-induced lipid peroxidation. Interestingly, KRAS-mutant cells upregulate expression of ferroptosis suppressor protein 1 (FSP1). Indeed, elevated levels of FSP1 in KRAS-mutant cells are responsible for mediating ferroptosis resistance and FSP1 is upregulated as a consequence of MAPK and NRF2 pathway activation downstream of KRAS. Strikingly, FSP1 activity promotes cellular transformation in soft agar and its overexpression is sufficient to promote spheroid growth in 3D in KRAS WT cells. Moreover, FSP1 expression and its activity in ferroptosis inhibition accelerates tumor onset of KRAS WT cells in the absence of oncogenic KRAS in vivo. Consequently, we find that pharmacological induction of ferroptosis in pancreatic organoids derived from the LsL-KRASG12D expressing mouse model is only effective in combination with FSP1 inhibition. Lastly, FSP1 is upregulated in non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) as compared to the respective normal tissue of origin and correlates with NRF2 expression in PDAC patient datasets. Based on these data, we propose that KRAS-mutant cells must navigate a ferroptosis checkpoint by upregulating FSP1 during tumor establishment. Consequently, ferroptosis-inducing therapy should be combined with FSP1 inhibitors for efficient therapy of KRAS-mutant cancers.
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Proteínas Reguladoras de la Apoptosis , Carcinogénesis , Ferroptosis , Animales , Humanos , Ratones , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma Ductal Pancreático/patología , Transformación Celular Neoplásica , Neoplasias Pulmonares/genética , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Neoplasias PancreáticasRESUMEN
BACKGROUND: The long-term outcomes of solid organ transplantation remain suboptimal. Therefore, appropriate biomarkers are needed in addition to immunosuppressive drugs and other traditional approaches for graft monitoring to achieve personalized immunosuppression and reduce premature graft loss. METHODS: Donor-derived cell-free DNA (dd-cfDNA) is a minimally invasive biomarker of cell death due to graft injury. It can be quantified using droplet digital polymerase chain reaction and next-generation sequencing. Fractional dd-cfDNA determination can be affected by changes in recipient cfDNA, such as those caused by leukopenia or infection, leading to false-positive or false-negative results, respectively. Absolute quantification of dd-cfDNA helps in overcoming this limitation. RESULTS: Overall, there is sufficient evidence of the clinical validity of dd-cfDNA. It detects rejection episodes early at an actionable stage and reflects the severity of graft injury without being rejection-specific. Owing to its high negative predictive value, dd-cfDNA is very useful for ruling out graft injury. Dd-cfDNA complements histological findings and can help in avoiding unnecessary biopsies. It indicates a response to rejection treatment and detects underimmunosuppression. CONCLUSIONS: Monitoring changes in dd-cfDNA over time may be helpful in adapting immunosuppression to prevent graft rejection. Moreover, serial dd-cfDNA determination may increase the effectiveness of transplant recipient surveillance and facilitate personalized immunosuppression when combined with other relevant clinical and diagnostic findings.
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Ácidos Nucleicos Libres de Células , Trasplante de Riñón , Trasplante de Órganos , Humanos , Biomarcadores , Terapia de Inmunosupresión , Donantes de Tejidos , Rechazo de Injerto/diagnóstico , Rechazo de Injerto/prevención & controlRESUMEN
There is a need to improve personalized immunosuppression in organ transplantation to reduce premature graft loss. Biomarkers are needed to better detect rejection, asymptomatic graft injury, and under-immunosuppression. Assessment of minimal necessary exposure to guide tapering and prevent immune activation is also important. There is robust clinical evidence from a large number of published studies supporting the role of dd-cfDNA for monitoring graft integrity and detection or exclusion of rejection. Dd-cfDNA indicates graft cell death without being rejection specific. It can be determined in plasma through droplet digital PCR using preselected SNPs or next generation sequencing. Changes in recipient cfDNA (e.g., by infection) can affect the results of dd-cfDNA fractional determination. This limitation can be overcome using absolute dd-cfDNA quantification. The combination of fractional and absolute determination including total cfDNA is recommended for meaningful interpretation of the results. The value proposition for the patient includes earlier transplant injury detection and intervention, less full blown rejection risk, an alternative to invasive biopsies, and personalized immunosuppression with potential for improved long-term outcome. Transplant physicians benefit from better immunosuppressive guidance and having an alternative when biopsies are refused or contraindicated. Further advantages are improved biopsy interpretation, less trial and error changes in immunosuppression, and less time dealing with complications. The laboratory medicine specialist can provide more effective services. Hospital management and insurance companies could benefit from more cost-effective surveillance of transplant recipients. Potential cost savings would result from fewer biopsies as a result of the tests' high negative predictive value, fewer re-transplantations, and less organ failure with return to dialysis. A pathway to implementation and metrics is suggested to measure the effectiveness of dd-cfDNA testing.
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Background: Both bromodomain and extra-terminal domain (BET) proteins and spleen tyrosine kinase (SYK) represent promising targets in diffuse large B-cell (DLBCL) and Burkitt's lymphoma (BL). We evaluated the anti-lymphoma activity of the isoform-specific bivalent BET inhibitor AZD5153 (AZD) and the pan-BET inhibitor I-BET151 (I-BET) as single agents and in combination with SYK inhibitor Entospletinib (Ento) in vitro. Methods: The effect of the single agents on cell proliferation and metabolic activity was evaluated in two DLBCL and two BL cell lines. Proliferation, metabolic activity, apoptosis, cell cycle and morphology were further investigated after a combined treatment of AZD or I-BET and Ento. RNAseq profiling of combined AZD+Ento treatment was performed in SU-DHL-4 cells. Results: Both BET inhibitors reduced cell proliferation and metabolic activity in a dose- and time-dependent manner. Combined BET and SYK inhibition enhanced the anti-proliferative effect and induced a G0/G1 cell cycle arrest. SU-DHL-4 demonstrated a pronounced modulation of gene expression by AZD, which was markedly increased by additional SYK inhibition. Functional enrichment analyses identified combination-specific GO terms related to DNA replication and cell division. Genes such as ADGRA2, MYB, TNFRSF11A, S100A10, PLEKHH3, DHRS2 and FOXP1-AS1 were identified as possible key regulators. Conclusion: Simultaneous inhibition of BET and SYK enhanced the anti-proliferative effects, and induced a combination-specific gene expression signature.
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The molecular heterogeneity of feline mammary carcinomas (FMCs) represents a prognostic and therapeutic challenge. RNA-Seq-based comparative transcriptomic profiling serves to identify recurrent and exclusive differentially expressed genes (DEGs) across sample types and molecular subtypes. Using mass-parallel RNA-Seq, we identified DEGs and performed comparative function-based analysis across 15 tumours (four basal-like triple-negative [TN], eight normal-like TN, and three luminal B fHER2 negative [LB fHER2-]), two cell lines (CL, TiHo-0906, and TiHo-1403) isolated from the primary tumours (LB fHER2-) of two cats included in this study, and 13 healthy mammary tissue controls. DEGs in tumours were predominantly upregulated; dysregulation of CLs transcriptome was more extensive, including mostly downregulated genes. Cell-cycle and metabolic-related DEGs were upregulated in both tumours and CLs, including therapeutically-targetable cell cycle regulators (e.g. CCNB1, CCNB2, CDK1, CDK4, GTSE1, MCM4, and MCM5), metabolic-related genes (e.g. FADS2 and SLC16A3), heat-shock proteins (e.g. HSPH1, HSP90B1, and HSPA5), genes controlling centrosome disjunction (e.g. RACGAP1 and NEK2), and collagen molecules (e.g. COL2A1). DEGs specifically upregulated in basal-like TN tumours were involved in antigen processing and presentation, in normal-like TN tumours encoded G protein-coupled receptors (GPCRs), and in LB fHER2- tumours were associated with lysosomes, phagosomes, and endosomes formation. Downregulated DEGs in CLs were associated with structural and signalling cell surface components. Hence, our results suggest that upregulation of genes enhancing proliferation and metabolism is a common feature among FMCs and derived CLs. In contrast, the dissimilarities observed in dysregulation of membrane components highlight CLs' disconnection with the tumour microenvironment. Furthermore, recurrent and exclusive DEGs associated with dysregulated pathways might be useful for the development of prognostically and therapeutically-relevant targeted panels.
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Carcinoma , Perfilación de la Expresión Génica , Animales , Biomarcadores , Gatos , Ciclo Celular/genética , Línea Celular , Proteínas de Choque Térmico/genética , Transcriptoma , Microambiente TumoralRESUMEN
Transcriptional bursting is a common expression mode for most genes where independent transcription of alleles leads to different ratios of allelic mRNA from cell to cell. Here we investigated burst-like transcription and its consequences in cardiac tissue from Hypertrophic Cardiomyopathy (HCM) patients with heterozygous mutations in the sarcomeric proteins cardiac myosin binding protein C (cMyBP-C, MYBPC3) and cardiac troponin I (cTnI, TNNI3). Using fluorescence in situ hybridization (RNA-FISH) we found that both, MYBPC3 and TNNI3 are transcribed burst-like. Along with that, we show unequal allelic ratios of TNNI3-mRNA among single cardiomyocytes and unequally distributed wildtype cMyBP-C protein across tissue sections from heterozygous HCM-patients. The mutations led to opposing functional alterations, namely increasing (cMyBP-Cc.927-2A>G) or decreasing (cTnIR145W) calcium sensitivity. Regardless, all patients revealed highly variable calcium-dependent force generation between individual cardiomyocytes, indicating contractile imbalance, which appears widespread in HCM-patients. Altogether, we provide strong evidence that burst-like transcription of sarcomeric genes can lead to an allelic mosaic among neighboring cardiomyocytes at mRNA and protein level. In HCM-patients, this presumably induces the observed contractile imbalance among individual cardiomyocytes and promotes HCM-development.
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Little is known about optimally applying chemotherapeutic agents in a specific temporal sequence to rapidly reduce the tumor load and to improve therapeutic efficacy. The clinical optimization of drug efficacy while reducing side effects is still restricted due to an incomplete understanding of the mode of action and related tumor relapse mechanisms on the molecular level. The molecular characterization of transcriptomic drug signatures can help to identify the affected pathways, downstream regulated genes and regulatory interactions related to tumor relapse in response to drug application. We tried to outline the dynamic regulatory reprogramming leading to tumor relapse in relapsed MLL-rearranged pro-B-cell acute lymphoblastic leukemia (B-ALL) cells in response to two first-line treatments: dexamethasone (Dexa) and cytarabine (AraC). We performed an integrative molecular analysis of whole transcriptome profiles of each treatment, specifically considering public knowledge of miRNA regulation via a network-based approach to unravel key driver genes and miRNAs that may control the relapse mechanisms accompanying each treatment. Our results gave hints to the crucial regulatory roles of genes leading to Dexa-resistance and related miRNAs linked to chemosensitivity. These genes and miRNAs should be further investigated in preclinical models to obtain more hints about relapse processes.
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Antineoplásicos , MicroARNs , Leucemia-Linfoma Linfoblástico de Células Precursoras , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular , Citarabina/farmacología , Citarabina/uso terapéutico , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , RecurrenciaRESUMEN
Personalized immunosuppression (IS) promises to improve the balance of necessary control of alloreactivity and dose-dependent adverse effects of long-term IS such as kidney insufficiency, infections, and malignancies. The majority of liver transplantation (LT) recipients exhibit graft injuries (graft inflammation and/or fibrosis) that are not eligible for an IS reduction according to current Banff criteria, even when liver enzymes are normal or only marginally elevated. This cross-sectional study evaluated the noninvasive prediction of such subclinical graft injuries in surveillance liver biopsies via donor-derived cell-free DNA (dd-cfDNA). Absolute and fractional dd-cfDNA increased stepwise from patients without histological signs of rejection (n = 26) over subclinical graft injury (n = 61), including subclinical T cell-mediated rejection to clinical overt T cell-mediated rejection (n = 21). Thus, fractional plasma dd-cfDNA was significantly elevated paired to surveillance biopsies with relevant subclinical graft injury according to 2016 Banff criteria compared with those with minimal or absent histological graft injury. In contrast, the presence of donor-specific anti-human leukocyte antigen antibodies was not associated with the amount of dd-cfDNA. The sensitivity and specificity of fractional dd-cfDNA to noninvasively predict relevant subclinical graft injury was rather limited with 73% and 52% at the cutoff value of 2.1% fractional dd-cfDNA. The positive predictive value of fractional dd-cfDNA above 2.1% was 76% to noninvasively predict subclinical graft injury, calculated on the prevalence of graft injury in our prospective surveillance biopsy program, whereas the negative predictive values was not predictive (47%). In conclusion, dd-cfDNA has a rather limited diagnostic fidelity in addition to other noninvasive markers for the assessment of subclinical graft injury in personalized IS approaches after LT in a cross-sectional setting.
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Ácidos Nucleicos Libres de Células , Trasplante de Hígado , Humanos , Rechazo de Injerto/diagnóstico , Rechazo de Injerto/epidemiología , Rechazo de Injerto/genética , Trasplante de Hígado/efectos adversos , Estudios Transversales , Estudios Prospectivos , Donantes de TejidosRESUMEN
Transplant recipients exhibit an impaired protective immunity after SARS-CoV-2 vaccination, potentially caused by mycophenolate (MPA) immunosuppression. Recent data from patients with autoimmune disorders suggest that temporary MPA hold might greatly improve booster vaccination outcomes. We applied a fourth dose of SARS-CoV-2 vaccine to 29 kidney transplant recipients during a temporary (5 weeks) MPA/azathioprine hold, who had not mounted a humoral immune response to previous vaccinations. Seroconversion until day 32 after vaccination was observed in 76% of patients, associated with acquisition of virus-neutralizing capacity. Interestingly, 21/25 (84%) calcineurin inhibitor-treated patients responded, but only 1/4 belatacept-treated patients responded. In line with humoral responses, counts and relative frequencies of spike receptor binding domain-specific (RBD-specific) B cells were markedly increased on day 7 after vaccination, with an increase in RBD-specific CD27++CD38+ plasmablasts. Whereas overall proportions of spike-reactive CD4+ T cells remained unaltered after the fourth dose, frequencies were positively correlated with specific IgG levels. Importantly, antigen-specific proliferating Ki67+ and in vivo-activated programmed cell death 1-positive T cells significantly increased after revaccination during MPA hold, whereas cytokine production and memory differentiation remained unaffected. In summary, antimetabolite hold augmented all arms of immunity during booster vaccination. These data suggest further studies of antimetabolite hold in kidney transplant recipients.
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Antimetabolitos , Vacunas contra la COVID-19 , COVID-19 , Trasplante de Riñón , Anticuerpos Antivirales , Antimetabolitos/uso terapéutico , COVID-19/prevención & control , Vacunas contra la COVID-19/inmunología , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunosupresores/uso terapéutico , SARS-CoV-2 , Receptores de Trasplantes , VacunaciónRESUMEN
BACKGROUND: Canine prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) are typically characterized by metastasis and chemoresistance. Cell lines are important model systems for developing new therapeutic strategies. However, as they adapt to culturing conditions and undergo clonal selection, they can diverge from the tissue from which they were originally derived. Therefore, a comprehensive characterization of cell lines and their original tissues is paramount. METHODS: This study compared the transcriptomes of nine canine cell lines derived from PAC, PAC metastasis and TCC to their respective original primary tumor or metastasis tissues. Special interests were laid on cell culture-related differences, epithelial to mesenchymal transition (EMT), the prostate and bladder cancer pathways, therapeutic targets in the PI3K-AKT signaling pathway and genes correlated with chemoresistance towards doxorubicin and carboplatin. RESULTS: Independent analyses for PAC, PAC metastasis and TCC revealed 1743, 3941 and 463 genes, respectively, differentially expressed in the cell lines relative to their original tissues (DEGs). While genes associated with tumor microenvironment were mostly downregulated in the cell lines, patient-specific EMT features were conserved. Furthermore, examination of the prostate and bladder cancer pathways revealed extensive concordance between cell lines and tissues. Interestingly, all cell lines preserved downstream PI3K-AKT signaling, but each featured a unique therapeutic target signature. Additionally, resistance towards doxorubicin was associated with G2/M cell cycle transition and cell membrane biosynthesis, while carboplatin resistance correlated with histone, m- and tRNA processing. CONCLUSION: Comparative whole-transcriptome profiling of cell lines and their original tissues identifies models with conserved therapeutic target expression. Moreover, it is useful for selecting suitable negative controls, i.e., cell lines lacking therapeutic target expression, increasing the transfer efficiency from in vitro to primary neoplasias for new therapeutic protocols. In summary, the dataset presented here constitutes a rich resource for canine prostate and bladder cancer research.
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BACKGROUND: Circulating graft-derived cell-free DNA (dd-cfDNA) is a new marker of cardiac allograft damage that is used for noninvasive rejection diagnostics. We performed dd-cfDNA (%) in heart transplant recipients during the first posttransplant year. METHODS: In 87 patients, serial dd-cfDNA determination at predefined time-points was performed in 770 single samples. dd-cfDNA fraction (%) was measured using an established universal droplet digital polymerase chain reaction method, providing same-day turn-around. Rejection was diagnosed according to clinical parameters and biopsies. RESULTS: Median dd-cfDNA (%) was high (5.36%) immediately after reperfusion and decreased to a median (interquartile range) of 0.10% (0.05%-0.24%) in clinically stable patients by postoperative day 10. Compared to dd-cfDNA (%) samples in clinically stable patients, values were higher (P < 0.001) in biopsy-proven rejection ISHLT 1R (0.42% [0.15%-0.53%]) and 2R rejection (0.84% [0.39%-0.97%]). Moreover, dd-cfDNA (%) was already significantly increased 9-30 days before biopsy-proven rejection (0.36% [0.20%-0.61%]). An as yet unknown finding was a slightly, but significantly (P < 0.0001) higher dd-cfDNA (%) value in samples of stable patients with pericardial effusions (PEs) (n = 94; 0.18% [0.07%-0.30%]) compared to samples of non-PE patients (n = 132; 0.07% [0.04%-0.17%]). Using a cutoff of 0.35%, sensitivity and specificity of dd-cfDNA for cardiac rejection were 0.76 and 0.83 (area under the curve [AUC] ROC-curve: 0.81 [95% confidence interval, 0.73-0.89]). Omitting PE samples from the control group yielded an AUC of 0.86 [95% confidence interval, 0.76-0.95]. Samples drawn <12 hours after endomyocardial biopsy showed high (0.40% [0.15%-1.21%]) dd-cfDNA values, also in ISHLT0R (0.36% [0.10%-0.60%]). CONCLUSIONS: dd-cfDNA plasma values were significantly associated with cardiac rejection. However, PE or improper sampling (eg, shortly after biopsy) should be considered as confounders for rejection diagnoses using dd-cfDNA.
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Ácidos Nucleicos Libres de Células , Aloinjertos , Biomarcadores , Estudios de Cohortes , Rechazo de Injerto , Humanos , Donantes de TejidosRESUMEN
Bruton's tyrosine kinase (BTK) and phosphoinositide 3-kinase (PI3K) in the B-cell receptor (BCR) signaling pathway are considered potential therapeutic targets for the treatment of B-cell lymphomas, among which, diffuse large B-cell lymphoma (DLBCL) is the most common type. Herein, we comparatively evaluated the single and combined application of the BTK inhibitor ibrutinib and the selective PI3Kγ inhibitor AS-605240 in the canine DLBCL cell line CLBL-1. For further comparison, key findings were additionally analyzed in canine B-cell leukemia GL-1 and human DLBCL cell line SU-DHL-4. While ibrutinib alone induced significant anti-proliferative effects on all cell lines in a dose-dependent manner, AS-605240 only induced anti-proliferative effects at high concentrations. Interestingly, ibrutinib and AS-605240 acted synergistically, reducing cell proliferation and increasing apoptosis/necrosis in all cell lines and inducing morphological changes in CLBL-1. Moreover, the combined application of ibrutinib and AS-605240 reduced relative phosphorylation and, in some instances, the levels of the BTK, AKT, GSK3ß, and ERK proteins. Comparative variant analysis of RNA-seq data among canine B- and T-lymphoid cell lines and primary B-cell lymphoma samples revealed potentially high-impact somatic variants in the genes that encode PI3K, which may explain why AS-605240 does not singly inhibit the proliferation of cell lines. The combination of ibrutinib and AS-605240 represents a promising approach that warrants further in vivo evaluation in dogs, potentially bearing significant value for the treatment of human DLBCL.