Novel MAGIC composite scores using both clinical symptoms and biomarkers best predict treatment outcomes of acute GVHD.

Acute graft-vs-host disease (GVHD) grading systems that use only clinical symptoms at treatment initiation such as Minnesota risk identify standard and high risk categories but lack a low risk category suitable to minimize immunosuppressive strategies. We developed a new grading system that includes a low risk stratum based on clinical symptoms alone and determined whether the incorporation of biomarkers would improve the model's prognostic accuracy. We randomly divided 1863 patients in the Mount Sinai Acute GVHD International Consortium (MAGIC) who were treated for GVHD into training and validation cohorts. Patients in the training cohort were divided into 14 groups based on similarity of clinical symptoms and similar NRM; we used a classification and regression tree (CART) algorithm to create three Manhattan risk groups that produced a significantly higher area under the receiver operating characteristic curve (AUC) for 6-month NRM than the Minnesota risk classification (0.69 vs. 0.64, P=0.009) in the validation cohort. We integrated serum GVHD biomarker scores with Manhattan risk using patients with available serum samples and again used a CART algorithm to establish three MAGIC composite scores that significantly improved prediction of NRM compared to Manhattan risk (AUC, 0.76 vs. 0.70, P=0.010). Each increase in MAGIC composite score also corresponded to a significant decrease in day 28 treatment response (80% vs. 63% vs. 30%, P<0.001). We conclude that the MAGIC composite score more accurately predicts response to therapy and long term outcomes than systems based on clinical symptoms alone and may help guide clinical decisions and trial design.

Association between bilirubin and biomarkers of metabolic health and oxidative stress in the MARK-AGE cohort.

Recent studies have shown that elevated concentrations of unconjugated bilirubin (UCB) may be a protective host factor against the development of noncommunicable diseases (NCDs), whereas low levels of UCB are associated with the opposite effect. The results of this European study, in which 2,489 samples were tested for their UCB concentration using high-performance liquid chromatography (HPLC) and additional data from the MARK-AGE database were used for analysis, provide further evidence that elevated UCB concentrations are linked to a lower risk of developing NCDs and may act as a predictive marker of biological aging as individuals with elevated UCB concentrations showed favorable outcomes in metabolic health and oxidative-stress-related biomarkers. These findings underline the significance of studying individuals with moderate hyperbilirubinemia and investigate UCB routinely, also in the setting of aging, since this condition affects millions of people worldwide but has been underrepresented in clinical research and practice until now.

A validated HPLC-MS/MS method for the quantification of systemic mifepristone after subcutaneous application in mice.

Mifepristone (RU486, MIF) is a synthetic steroidal hormone with progesterone and glucocorticoid receptor antagonistic characteristics. MIF is commonly used for pharmalogical abortions, but also for the treatment of endometrial and endocrine disorders. The goal of the study was to establish and validate a targeted HPLC-MS/MS method for the quantification of MIF and one of its active metabolites metapristone (MET) in plasma after subcutaneous implantation of slow-release MIF pellets in female BALB/c mice. Additionally, we aimed to apply the analytical method to tissue of several organs to understand the tissue-specific distribution of both analytes after release into systemic circulation. Sample preparation comprised a simple liquid-liquid extraction with diethylether and required 100 µl of plasma or homogenates of approximately 50 mg of tissue. The presented HPLC-MS/MS method showed high sensitivity with baseline separation of MIF, MET, and the internal standard levonorgestrel within a run time of only 8.0 minutes and comparable limits of quantification for plasma and tissue homogenates ranging from 40 pg ml-1 to 105 pg ml-1 for MIF and MET. The presented study is suitable for murine plasma and tissues and can be easily applied to human samples.

Ruxolitinib for the treatment of acute graft-versus-host disease: a retrospective analysis.

Steroid-refractory acute graft-versus-host disease (aGvHD) is a serious complication after allogeneic hematopoietic stem cell transplantation, associated with significant mortality. Ruxolitinib was the first drug approved for aGvHD, based on results of the REACH2 trial; however, real-world data are limited. We retrospectively analyzed the safety and efficacy of ruxolitinib for treatment of aGvHD at our center from March 2016 to August 2022 and assessed biomarkers of risk. We identified 49 patients receiving ruxolitinib as second- (33/49), third- (11/49), fourth- (3/49), or fifth-line (2/49) treatment. Ruxolitinib was started on median day 11 (range, 7-21) after aGvHD onset; median duration of administration was 37 days (range, 20-86), with 10 patients continuing treatment at last follow-up. Median follow-up period was 501 days (range, 95-905). In the primary analysis at the 1-month assessment, overall response rate was 65%, and failure-free survival was 78%. Infectious complications ≥ CTCAE Grade III were observed in 10/49 patients within 1-month followup. Patients responding to ruxolitinib therapy required fewer steroids and exhibited lower levels of the serum biomarkers regenerating islet-derived protein 3-alpha, suppression of tumorigenicity 2, and the Mount Sinai Acute GVHD International Consortium algorithm probability. A univariate regression model revealed steroid-dependent aGvHD as a significant predictor of better response to ruxolitinib. Within 6-months follow-up, four patients experienced recurrence of underlying malignancy, and eight died due to treatment-related mortality. Overall, ruxolitinib was welltolerated and showed response in heavily pretreated patients, with results comparable to those of the REACH2 trial. Biomarkers may be useful predictors of response to ruxolitinib.

Efficacy and safety of ruxolitinib in the treatment of chronic graft-versus-host disease: a retrospective analysis.

Steroid-refractory chronic graft-versus-host disease (cGvHD) is associated with significant morbidity and mortality, with ruxolitinib being the first drug approved for its treatment. We retrospectively analyzed the safety and efficacy of ruxolitinib for treatment of cGvHD at our center between 07/2015 and 12/2022 and identified 48 patients receiving ruxolitinib as second (18/48) or advanced (30/48) treatment line. Ruxolitinib was started on median day 340 (range 119-595) after cGvHD onset; median duration of administration was 176 (range, 79-294) days with 16/48 patients continuing treatment at last follow-up. National Institutes of Health organ grading and the intensity of immunosuppression were assessed at the start of ruxolitinib treatment and repeated after 1, 3, 6, and 12 months. Response assessment was terminated at the start of any additional new immunosuppressant treatment. The median time of follow-up was 582 (range, 104-1161) days. At the primary analysis after six months on ruxolitinib treatment, the overall response rate was 33%, and failure-free survival was 58%. Infectious adverse events ≥ CTCAE grade III were observed in 10/48 patients. The response rate was not associated with the severity of cGvHD, number of previous treatment lines, or number of additional agents combined with ruxolitinib applying a univariate regression model. At the time of the 12-month follow-up, four patients experienced recurrence of the underlying malignancy and two patients had experienced non-relapse-related mortality. Overall, ruxolitinib was relatively well-tolerated and showed outcomes comparable to the REACH3 trial in a heavily pretreated patient population.

Informal and formal long-term care utilization and unmet needs in Europe: examining socioeconomic disparities and the role of social policies for older adults.

The rising number of older adults with limitations in their daily activities has major implications for the demands placed on long-term care (LTC) systems across Europe. Recognizing that demand can be both constrained and encouraged by individual and country-specific factors, this study explains the uptake of home-based long-term care in 18 European countries with LTC policies and pension generosity along with individual factors such as socioeconomic status. Using data from the Survey of Health, Ageing and Retirement in Europe conducted in 2019, we apply a two-part multilevel model to assess if disparities in use of LTC are driven by disparities in needs or disparities in use of care when in need. While individual characteristics largely affect the use of care through its association with disparities in need, country-level characteristics are important for the use of care when in need. In particular, the better health of wealthier and more educated individuals makes them less likely to use any type of home-based personal care. At the country level, results show that the absence of a means-tested benefit scheme and the availability of cash-for-care benefits (as opposed to in-kind) are strongly associated with the use of formal care, whether it is mixed (with informal care) or exclusive. LTC policies are, however, shown to be insufficient to significantly reduce unmet needs for personal care. Conversely, generous pensions are significantly associated with lower unmet needs, underscoring the importance of considering the likely adverse effects of future pension reforms.

Glucose Metabolism as a Potential Therapeutic Target in Cytarabine-Resistant Acute Myeloid Leukemia.

Altered glycolytic metabolism has been associated with chemoresistance in acute myeloid leukemia (AML). However, there are still aspects that need clarification, as well as how to explore these metabolic alterations in therapy. In the present study, we aimed to elucidate the role of glucose metabolism in the acquired resistance of AML cells to cytarabine (Ara-C) and to explore it as a therapeutic target. Resistance was induced by stepwise exposure of AML cells to increasing concentrations of Ara-C. Ara-C-resistant cells were characterized for their growth capacity, genetic alterations, metabolic profile, and sensitivity to different metabolic inhibitors. Ara-C-resistant AML cell lines, KG-1 Ara-R, and MOLM13 Ara-R presented different metabolic profiles. KG-1 Ara-R cells exhibited a more pronounced glycolytic phenotype than parental cells, with a weaker acute response to 3-bromopyruvate (3-BP) but higher sensitivity after 48 h. KG-1 Ara-R cells also display increased respiration rates and are more sensitive to phenformin than parental cells. On the other hand, MOLM13 Ara-R cells display a glucose metabolism profile similar to parental cells, as well as sensitivity to glycolytic inhibitors. These results indicate that acquired resistance to Ara-C in AML may involve metabolic adaptations, which can be explored therapeutically in the AML patient setting who developed resistance to therapy.

Third-party fecal microbiota transplantation for high-risk treatment-naïve acute GVHD of the lower GI tract.

ABSTRACT: Disruption of the intestinal microbiome is observed with acute graft-versus-host disease (GVHD) of the lower gastrointestinal (LGI) tract, and fecal microbiota transplantation (FMT) has successfully cured steroid-refractory cases. In this open-label, single-arm, pilot study, third-party, single-donor FMT was administered in combination with systemic corticosteroids to participants with high-risk acute LGI GVHD, with a focus on treatment-naïve cases. Participants were scheduled to receive 1 induction dose (15 capsules per day for 2 consecutive days), followed by 3 weekly maintenance doses, consisting of 15 capsules per dose. The primary end point of the study was feasibility, which would be achieved if ≥80% of participants able to swallow ≥40 of the 75 scheduled capsules. Ten participants (9 treatment-naïve; 1 steroid-refractory) were enrolled and treated. The study met the primary end point, with 9 of 10 participants completing all eligible doses. Organ-specific LGI complete response rate at day 28 was 70%. Initial clinical response was observed within 1 week for all responders, and clinical responses were durable without recurrent LGI GVHD in complete responders. Exploratory analyses suggest that alpha diversity increased after FMT. Although recipient microbiome composition never achieved a high degree of donor similarity, expansion of donor-derived species and increases in tryptophan metabolites and short-chain fatty acids were observed within the first 7 days after FMT. Investigation into the use of microbiome-targeted interventions earlier in the treatment paradigm for acute LGI GVHD is warranted. This trial was registered at www.ClinicalTrials.gov as #NCT04139577.

Postprandial Micronutrient Variability and Bioavailability: An Interventional Meal Study in Young vs. Old Participants.

This study explores age- and time-dependent variations in postprandial micronutrient absorption after a micronutrient-rich intervention meal within the Biomiel (bioavailability of micronutrients in elderly) study. Comprising 43 healthy participants, the study compares young (n = 21; mean age 26.90 years) and old (n = 22; mean age 66.77 years) men and women, analyzing baseline concentrations and six-hour postprandial dynamics of iron (Fe), copper (Cu), zinc (Zn), selenium (Se), iodine (I), free zinc (fZn), vitamin C, retinol, lycopene, ß-carotene, α-tocopherol, and γ-tocopherol, along with 25(OH) vitamin D (quantified only at baseline). Methodologically, quantifications in serum or plasma were performed at baseline and also at 90, 180, 270, and 360 min postprandially. Results reveal higher baseline serum Zn and plasma lycopene concentrations in the young group, whereas Cu, Se, Cu/Zn ratio, 25(OH) vitamin D, α-tocopherol, and γ-tocopherol were higher in old participants. Postprandial variability of Zn, vitamin C, and lycopene showed a strong time-dependency. Age-related differences in postprandial metabolism were observed for Se, Cu, and I. Nevertheless, most of the variance was explained by individuality. Despite some limitations, this study provides insights into postprandial micronutrient metabolism (in serum/plasma), emphasizing the need for further research for a comprehensive understanding of this complex field. Our discoveries offer valuable insights for designing targeted interventions to address and mitigate micronutrient deficiencies in older adults, fostering optimal health and well-being across the lifespan.

Bacteria and bacteriophage consortia are associated with protective intestinal metabolites in patients receiving stem cell transplantation.

The microbiome is a predictor of clinical outcome in patients receiving allogeneic hematopoietic stem cell transplantation (allo-SCT). Microbiota-derived metabolites can modulate these outcomes. How bacteria, fungi and viruses contribute to the production of intestinal metabolites is still unclear. We combined amplicon sequencing, viral metagenomics and targeted metabolomics from stool samples of patients receiving allo-SCT (n = 78) and uncovered a microbiome signature of Lachnospiraceae and Oscillospiraceae and their associated bacteriophages, correlating with the production of immunomodulatory metabolites (IMMs). Moreover, we established the IMM risk index (IMM-RI), which was associated with improved survival and reduced relapse. A high abundance of short-chain fatty acid-biosynthesis pathways, specifically butyric acid via butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT, which catalyzes EC 2.8.3.8) was detected in IMM-RI low-risk patients, and virome genome assembly identified two bacteriophages encoding BCoAT as an auxiliary metabolic gene. In conclusion, our study identifies a microbiome signature associated with protective IMMs and provides a rationale for considering metabolite-producing consortia and metabolite formulations as microbiome-based therapies.

Energy and macronutrient restriction regulate bile acid homeostasis.

As we reported previously, caloric restriction (CR) results in an increased concentration of bile acids (BA) in the intestinal mucosa. We now investigated the background of this phenotype, trying to identify nutrition-related factors modulating BA levels. Male mice were submitted to various types of restrictive diets and BA levels and expression of associated factors were measured. We found that BA concentration is increased in the liver of CR mice, which corresponds to reduced expression of the Shp gene and elevated mRNA levels of Cyp27a1, Bal, and Ntcp, as well as CYP7A1 protein and gene expression. Correlation between decreased concentration of BAs in the feces, increased BAs levels in plasma, and elevated gene expression of BAs transporters in the ileum mucosa suggests enhanced BA uptake in the intestine of CR mice. Corresponding to CR upregulation of liver and ileum mucosa, BA concentration was found in animals submitted to other types of prolonged energy-restricting dietary protocols, including intermittent fasting and fasting-mimicking diet. While over-night fasting had negligible impact on BAs levels. Manipulation of macronutrient levels partly affected BA balance. Low-carbohydrate and ketogenic diet increased BAs in the liver but not in the intestine. Carbohydrate restriction stimulates BA synthesis in the liver, but energy restriction is required for the increase in BA levels in the intestine and its uptake.

Temporal dynamics of muscle mitochondrial uncoupling-induced integrated stress response and ferroptosis defense.

Mitochondria play multifaceted roles in cellular function, and impairments across domains of mitochondrial biology are known to promote cellular integrated stress response (ISR) pathways as well as systemic metabolic adaptations. However, the temporal dynamics of specific mitochondrial ISR related to physiological variations in tissue-specific energy demands remains unknown. Here, we conducted a comprehensive 24-hour muscle and plasma profiling of male and female mice with ectopic mitochondrial respiratory uncoupling in skeletal muscle (mUcp1-transgenic, TG). TG mice are characterized by increased muscle ISR, elevated oxidative stress defense, and increased secretion of FGF21 and GDF15 as ISR-induced myokines. We observed a temporal signature of both cell-autonomous and systemic ISR in the context of endocrine myokine signaling and cellular redox balance, but not of ferroptotic signature which was also increased in TG muscle. We show a progressive increase of muscle ISR on transcriptional level during the active phase (night time), with a subsequent peak in circulating FGF21 and GDF15 in the early resting phase. Moreover, we found highest levels of muscle oxidative defense (GPX and NQO1 activity) between the late active to early resting phase, which could aim to counteract excessive iron-dependent lipid peroxidation and ferroptosis in muscle of TG mice. These findings highlight the temporal dynamics of cell-autonomous and endocrine ISR signaling under skeletal muscle mitochondrial uncoupling, emphasizing the importance of considering such dissociation in translational strategies and sample collection for diagnostic biomarker analysis.

Incidence and Outcome of Atypical Manifestations of Chronic Graft-versus-Host Disease: Results From a Retrospective Single-Center Analysis.

Chronic graft-versus-host disease (cGVHD) is the leading cause of late nonrelapse mortality (NRM) after allogeneic hematopoietic stem cell transplantation (alloHSCT) and defined by 8 diagnostic target organs. Recently, provisional criteria for atypical manifestations of cGVHD that include manifestations in nonclassic organs as well as atypical manifestations in National Institutes of Health (NIH)-defined organs, were proposed by a NIH task force. Little is known about the incidence, risk factors, and impact on survival of atypical cGVHD, however. The aim of the present study was to analyze these parameters in a sequential patient population. We retrospectively screened 623 patients who underwent alloHSCT at the University Medical Center Regensburg between January 2008 and December 2020 for atypical cGVHD manifestations, applying the provisional NIH taskforce criteria. A total of 102 patients (16.4%) met the criteria, representing 25% of all cGVHD cases, and 14 patients (2.2%) had only atypical cGVHD. The most frequent manifestations were immune-mediated cytopenias (24.5%), renal cGVHD (13.7%) and (poly)serositis (13.7%). Multivariate analysis identified prior acute GVHD (odds ratio [OR], 2.28 and 2.93) and infusion of donor lymphocytes (OR, 1.77 for both) as risk factors for classic cGVHD and atypical cGVHD, whereas total body irradiation was an independent risk factor for atypical cGVHD manifestations only (OR, 1.76). Compared to patients without cGVHD, those with atypical and NIH-defined cGVHD showed similarly better overall survival (P = .034 and < .001) and low relapse-related mortality (P < .001 for both). NRM was significantly increased by atypical GVHD, but not by NIH-defined cGVHD (P = .019 and .10), which was driven only by a few atypical organ manifestations (eg, renal, restrictive lung disease, peripheral neuropathy), whereas others did not contribute to NRM (eg, thyroid gland, musculoskeletal, pancreas). In summary, atypical cGVHD is more common than previously estimated and has both similarities with and differences from NIH-defined cGVHD. In particular, the increased NRM and a subset of patients with only atypical cGVHD point to the urgent need to capture these manifestations in cGVHD cohorts, including analysis of treatment outcomes.

Triple Therapy with Metformin, Ketogenic Diet, and Metronomic Cyclophosphamide Reduced Tumor Growth in MYCN-Amplified Neuroblastoma Xenografts.

Neuroblastoma (NB) is a childhood cancer in which amplification of the MYCN gene is the most acknowledged marker of poor prognosis. MYCN-amplified NB cells rely on both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) for energy production. Previously, we demonstrated that a ketogenic diet (KD) combined with metronomic cyclophosphamide (CP) delayed tumor growth in MYCN-amplified NB xenografts. The anti-diabetic drug metformin (MET) also targets complex I of the OXPHOS system. Therefore, MET-induced disruptions of mitochondrial respiration may enhance the anti-tumor effect of CP when combined with a KD. In this study, we found that MET decreased cell proliferation and mitochondrial respiration in MYCN-amplified NB cell lines, while the combination of KD, MET, and low-dose CP (triple therapy) also reduced tumor growth and improved survival in vivo in MYCN-amplified NB xenografts. Gene ontology enrichment analysis revealed that this triple therapy had the greatest effect on the transcription of genes involved in fatty acid ß-oxidation, which was supported by the increased protein expression of CPT1A, a key mitochondrial fatty acid transporter. We suspect that alterations to ß-oxidation alongside the inhibition of complex I may hamper mitochondrial energy production, thus explaining these augmented anti-tumor effects, suggesting that the combination of MET and KD is an effective adjuvant therapy to CP in MYCN-amplified NB xenografts.

AML consolidation therapy: timing matters.

PURPOSE: Infections due to severe neutropenia are the most common therapy-associated causes of mortality in patients with acute myeloid leukemia (AML). New strategies to lessen the severity and duration of neutropenia are needed. METHODS: Cytarabine is commonly used for AML consolidation therapy; we compared high- and intermediate-dose cytarabine administration on days 1, 2, and 3 (AC-123) versus days 1, 3, and 5 (AC-135) in consolidation therapy of AML. Recently, clinical trials demonstrated that high-dose AC-123 resulted in a shortened white blood cell (WBC) recovery time compared with high-dose AC-135. Our main hypothesis is that this is also the case for different cytarabine dosage, granulocyte colony-stimulating factor (G-CSF) administration, and cycle lengths. We analyzed 334 treatment schedules on virtual cohorts of digital twins. RESULTS: Comparison of 32,565 simulated consolidation cycles resulted in a reduction in the WBC recovery time for AC-123 in 99.6% of the considered cycles (median reduction 3.5 days) without an increase in the number of leukemic blasts (lower value in 94.2% of all cycles), compared to AC-135. CONCLUSION: Our numerical study supports the use of AC-123 plus G-CSF as standard conventional AML consolidation therapy to reduce the risk for life-threatening infectious complications.

Multimodal immune cell phenotyping in GI biopsies reveals microbiome-related T cell modulations in human GvHD.

Acute graft-versus-host disease (aGvHD) is a significant complication after allogeneic hematopoietic stem cell transplantation (aHSCT), but major factors determining disease severity are not well defined yet. By combining multiplexed tissue imaging and single-cell RNA sequencing on gastrointestinal biopsies from aHSCT-treated individuals with fecal microbiome analysis, we link high microbiome diversity and the abundance of short-chain fatty acid-producing bacteria to the sustenance of suppressive regulatory T cells (Tregs). Furthermore, aGvHD severity strongly associates with the clonal expansion of mainly CD8 T cells, which we find distributed over anatomically distant regions of the gut, persistent over time, and inversely correlated with the presence of suppressive Tregs. Overall, our study highlights the pathophysiological importance of expanded CD8 T cell clones in the progression of aGvHD toward more severe clinical manifestations and strongly supports the further development of microbiome interventions as GvHD treatment via repopulation of the gut Treg niche to suppress inflammation.

Metabolic protein kinase signalling in neuroblastoma.

BACKGROUND: Neuroblastoma is a paediatric malignancy of incredibly complex aetiology. Oncogenic protein kinase signalling in neuroblastoma has conventionally focussed on transduction through the well-characterised PI3K/Akt and MAPK pathways, in which the latter has been implicated in treatment resistance. The discovery of the receptor tyrosine kinase ALK as a target of genetic alterations in cases of familial and sporadic neuroblastoma, was a breakthrough in the understanding of the complex genetic heterogeneity of neuroblastoma. However, despite progress in the development of small-molecule inhibitors of ALK, treatment resistance frequently arises and appears to be a feature of the disease. Moreover, since the identification of ALK, several additional protein kinases, including the PIM and Aurora kinases, have emerged not only as drivers of the disease phenotype, but also as promising druggable targets. This is particularly the case for Aurora-A, given its intimate engagement with MYCN, a driver oncogene of aggressive neuroblastoma previously considered 'undruggable.' SCOPE OF REVIEW: Aided by significant advances in structural biology and a broader understanding of the mechanisms of protein kinase function and regulation, we comprehensively outline the role of protein kinase signalling, emphasising ALK, PIM and Aurora in neuroblastoma, their respective metabolic outputs, and broader implications for targeted therapies. MAJOR CONCLUSIONS: Despite massively divergent regulatory mechanisms, ALK, PIM and Aurora kinases all obtain significant roles in cellular glycolytic and mitochondrial metabolism and neuroblastoma progression, and in several instances are implicated in treatment resistance. While metabolism of neuroblastoma tends to display hallmarks of the glycolytic "Warburg effect," aggressive, in particular MYCN-amplified tumours, retain functional mitochondrial metabolism, allowing for survival and proliferation under nutrient stress. Future strategies employing specific kinase inhibitors as part of the treatment regimen should consider combinatorial attempts at interfering with tumour metabolism, either through metabolic pathway inhibitors, or by dietary means, with a view to abolish metabolic flexibility that endows cancerous cells with a survival advantage.