Multiple Markers Research Articles

Fasting-Mimicking Diet Inhibits Autophagy and Synergizes with Chemotherapy to Promote T-Cell-Dependent Leukemia-Free Survival.

Fasting mimicking diets (FMDs) are effective in the treatment of many solid tumors in mouse models, but their effect on hematologic malignancies is poorly understood, particularly in combination with standard therapies. Here we show that cycles of a 3-day FMD given to high-fat-diet-fed mice once a week increased the efficacy of vincristine to improve survival from BCR-ABL B acute lymphoblastic leukemia (ALL). In mice fed a standard diet, FMD cycles in combination with vincristine promoted cancer-free survival. RNA seq and protein assays revealed a vincristine-dependent decrease in the expression of multiple autophagy markers, which was exacerbated by the fasting/FMD conditions. The autophagy inhibitor chloroquine could substitute for fasting/FMD to promote cancer-free survival in combination with vincristine. In vitro, targeted inhibition of autophagy genes ULK1 and ATG9a strongly potentiated vincristine's toxicity. Moreover, anti-CD8 antibodies reversed the effects of vincristine plus fasting/FMD in promoting leukemia-free survival in mice, indicating a central role of the immune system in this response. Thus, the inhibition of autophagy and enhancement of immune responses appear to be mediators of the fasting/FMD-dependent cancer-free survival in ALL mice.

Multiple Phenotypes of Chronic Coronary Syndromes Identified by ABCDE Stress Echocardiography

Regional wall motion abnormality is considered a sensitive and specific marker of ischemia during stress echocardiography (SE). However, ischemia is a multifaceted entity associated with either coronary artery disease (CAD) or angina with normal coronary arteries, a distinction difficult to make using a single sign. The aim of this study was to evaluate the diagnostic potential of the five-step ABCDE SE protocol for CAD detection. From the 2016-2022 Stress Echo 2030 study data bank, 3,229 patients were selected (mean age, 66±12years; 2,089 men [65%]) with known CAD (n=1,792) or angina with normal coronary arteries (n=1,437). All patients were studied using both the ABCDE SE protocol and coronary angiography, within 3months. In step A, regional wall motion abnormality is assessed; in step B, B-lines and diastolic function; in step C, left ventricular contractile reserve; in step D, coronary flow velocity reserve in the left anterior descending coronary artery; and in step E, heart rate reserve. SE response ranged from a score of 0 (all steps normal) to a score of 5 (all steps abnormal). For CAD, rates of abnormal results were 347 for step A (19.4%), 547 (30.5%) for step B, 720 (40.2%) for step C, 615 (34.3%) for step D, and 633 (35.3%) for step E. For angina with normal coronary arteries, rates of abnormal results were 81 (5.6%) for step A, 429 (29.9%) for step B, 432 (30.1%) for step C, 354 (24.6%) for step D, and 445 (31.0%) for step E. The dominant "solitary phenotype" was step B in 109 patients (9.1%). Stress-induced ischemia presents with a wide range of diagnostic phenotypes, highlighting its complex nature. Using a comprehensive approach such as the advanced ABCDE score, which combines multiple markers, proves to be more valuable than relying on a single marker in isolation.

Preoperative systemic inflammation response index: Clinicopathologic predictor of pathological complete response in HER2-positive breast cancer patients receiving neoadjuvant systemic therapy.

Multiple pretreatment systemic inflammatory markers (SIMs) have been reported as predictors of pathological complete response (pCR) after neoadjuvant systemic therapy (NST) in patients with breast cancer (BC). However, the most significant SIM remains to be conclusively identified, and variations among different molecular subtypes remain unknown. The objective of the study was to identify the most significant SIM in patients with human epidermal growth factor receptor 2 (HER2) positive BC, to construct a pCR-predictive nomogram combining it with other clinicopathologic factors, and to evaluate its prognostic value on survival. We retrospectively reviewed the findings for 240 patients with stage I-III HER2-positive BC who underwent NST and subsequent surgery at Kaohsiung and Taichung Veterans General Hospital from 2011 to 2021. Clinicopathologic factors were analyzed by stepwise logistic regression with backward selection. The data were used to construct a nomogram plot for determining the pCR probability. Kaplan-Meier curves and log-rank test were used to evaluate disease-free survival (DFS) and overall survival (OS). Among the pretreatment SIMs, only the systemic inflammation response index (SIRI) was significantly related to pCR, with an optimal cutoff value of 1.27 × 10 9 /L. Stepwise logistic analyses indicated that clinical N stage, HER2 immunohistochemistry score, hormone receptor status, targeted therapy regimen, and SIRI were independent predictors of pCR, with an area under the curve of 0.722. The Hosmer-Lemeshow test and calibration curve revealed that the predictive ability was a good fit to actual observations. A nomogram was constructed based on the logistic model. The external validation of the model also revealed satisfactory discrimination and calibration. Kaplan-Meier analysis showed that patients with SIRI <1.27 had longer DFS and OS. Pretreatment SIRI <1.27 is predictive of pCR, DFS, and OS in HER2-positive BC. Our nomogram could efficiently predict pCR and facilitate clinical decision-making before neoadjuvant treatment.

Ukumela impilo randomised trial: preliminary findings of height-adjustable sit-to-stand workstations on health outcomes of South African office workers

BackgroundThe prevalence of sedentary behaviour has concurrently risen with multiple cardiometabolic risk markers independent of physical activity levels. Office-based workers accumulate the highest levels of sitting time during occupational times. This study aims to investigate the short-term effects of using height-adjustable sit-to-stand workstations on cardiometabolic risk markers of office-based workers in South Africa.ResultsSixty-two office-based workers were randomized into intervention (n = 44), and the control group (n = 18). Small improvements were observed in BMI, blood pressure, and cholesterol levels in this cohort.ConclusionThis preliminary investigation confirms that short-term height-adjustable sit-stand interventions are effective in reducing workplace sitting time and selected health outcomes. South Africa has been attributed with the highest burden of obesity in Sub-Saharan Africa, as a result, there is a need to implement long-term workplace intervention to reverse these implications.Trial registrationPan African Clinical Trial Registry, PACTR201911656014962 on the 12th of November 2019.

Relationship of peripheral inflammation and discrimination with memory: a study of Black and Latinx middle‐aged adults

AbstractBackgroundMinoritized groups experience higher stress due to structural racism and discrimination. These exposures have been linked to lower cognitive test scores and inflammation is a potential biological pathway underlying this association. Yet studies with multiple inflammatory markers in diverse populations remain limited.MethodWe assessed 635 community‐dwelling adults (mean age = 54.5, SD = 11; 66% women; 67% Latinx, 33% non‐Latinx Black) from the Offspring Cohort. We characterized peripheral inflammation using principal component analysis on 33 serum inflammatory markers. Discrimination was ascertained by the Everyday and Major Discrimination Scales. Episodic memory was measured using the Selective Reminding Test and working memory by the Digit Span Test. Linear models stratified by racialized/ethnic group tested the cross‐sectional relationship between inflammation and memory adjusting for age, sex, and diabetes. Further, we examined if relationship between discrimination and memory (adjusted for age, gender, and education) was attenuated when inflammation was added. Due to smaller sample size, the magnitude of associations corresponding to change in cognitive score associated with &gt;1 year of chronological age, rather than statistical significance, was used to highlight important associations.ResultTwo inflammatory factors were derived, with component 1 including FGF2, Fractalkine, GM‐CSF, IFNg, IL13, MCP3, TNFa, and TNFb, and Component 2 including IFNa2, IL1b, IL1ra, IL5, IL9, and MIP1a. Among Spanish‐speaking participants racialized as Latinx, both inflammatory factors were associated with lower memory scores. Only the first factor was associated with lower memory for English‐speaking participants racialized as Latinx. For participants racialized as Black, only the second factor associated with lower memory. Among Spanish‐speaking Latinx, Everyday Discrimination was associated with lower working memory. Everyday Discrimination was associated with lower episodic memory among Black respondents; this association was attenuated by 20% with inclusion of the inflammatory factor 1 in the models. Several associations that were opposite to hypothesized relationships were observed.ConclusionElevated peripheral inflammation was associated with worse memory among individuals racialized as Black and Latinx. Our results indicate peripheral inflammation may contribute to disparities in cognition and may be one of the ways in which disadvantage due to structural racism and discrimination is embodied among minoritized groups in the United States.

A role of tau‐induced astrocyte dysfunction and senescence in AD

AbstractBackgroundUnder certain conditions and during aging, cellular stress and damage can trigger cellular senescence, an irreversible state of cell cycle arrest accompanied by the expression of proinflammatory mediators known collectively as the “senescent‐associated secretory phenotype” (SASP). In Alzheimer’s disease (AD), neuronal tau becomes hyperphosphorylated and misfolded, forming pathogenic soluble aggregates (tau oligomers) and destabilizing the microtubule cytoskeleton. Pathogenic soluble tau is released from neurons during neuronal activation and is transmitted in a prion‐like fashion to postsynaptic cells. In addition to neurons, tau is expressed in a variety of brain cell types including astrocytes. In the present study, we tested the central hypothesis that soluble pathogenic tau can be transmitted to astrocytes and that tau transmission to astrocytes induces cytoskeleton destabilization and cellular dysfunction, contributing to the progression of AD.MethodsTo test the molecular consequences of tau transmission, we treated primary human astrocytes with purified tau oligomers. We used advanced biochemical tools, immunocytochemistry, and live‐cell microscopy to measure cell and nuclear size, cell cycle arrest, and SASP activation. We also co cultured human astrocytes undergoing tau‐induced senescence with primary mouse or non‐human primate neurons to determine the functional impact of senescent astrocytes on neurons, then utilized immunocytochemistry and 3D image analyses to measure parameters of dendrite and spine morphology.ResultsWe found that pathogenic soluble tau is readily transmitted to primary human astrocytes, and that pathogenic tau transmission triggers phosphorylation of endogenous astrocyte tau, leading to the destabilization of the microtubule cytoskeleton. Further, we found that transmission of pathogenic tau to astrocytes potently upregulated multiple markers of cellular senescence, demonstrating for the first time tau‐induced astrocyte senescence. Density of dendritic spines and dendritic area were pronouncedly decreased in neurons co‐cultured with astrocytes undergoing tau‐induced senescence.ConclusionWe showed transmission of pathogenic tau to human astrocytes and that this event triggers astrocyte senescence in mouse models of AD tauopathy. Our data suggest that senescent astrocytes may be critical mediators of neuronal dysfunction in AD tauopathy.

Paramagnetic rim lesions and the central vein sign: Characterizing multiple sclerosis imaging markers.

Paramagnetic rims and the central vein sign (CVS) are proposed imaging markers of multiple sclerosis (MS) lesions. Using 7 tesla magnetic resonance imaging, we aimed to: (1) characterize the appearance of paramagnetic rim lesions (PRLs); (2) assess whether PRLs and the CVS are associated with higher levels of MS pathology; and (3) compare the characteristics between subjects with and without PRLs in early MS. Prospective study of 32 treatment-naïve subjects around the time of diagnosis who were assessed for the presence of PRLs and the CVS. Comparisons of lesion volume and macromolecular pool size ratio (PSR) index, a proxy of myelin integrity, between PRLs and non-PRLs, and CVS-positive and CVS-negative lesions were carried out. Differences in clinical/demographic characteristics between patients with PRLs and those without were tested. Fifteen subjects had ≥1 PRL for a total of 36 PRLs, of which two-thirds had a full rim. PRLs predicted a larger lesion size and decreased PSR signal. Lesion volume and presence of cervical spine lesions were significantly different between subjects with PRLs and those without, although neither remained significant after adjusting for multiple comparisons. One hundred and eighty-one lesions with CVS were identified with no differences between CVS-positive and CVS-negative lesions in volume (p = .27) and PSR values (p = .62). PRLs, but not CVS-positive lesions, are larger and have lower myelin integrity. Our findings indicate that PRLs are associated with higher levels of lesion-specific pathology prior to the start of disease-modifying therapy.

Design a dual-response two-photon fluorescent probe for simultaneous imaging of mitochondrial viscosity and peroxynitrite in a thrombosis model

BackgroundVenous thromboembolism is a sudden cardiovascular disease that can lead to death, and its pathologic development is closely related to vascular viscosity and inflammation. However, direct evidence from in vivo is really scarce. The key limitation is that the combined probes cannot detect multiple markers simultaneously, which may lead to unreliable results. Therefore, to develop a single probe that can simultaneously monitor the variations of viscosity in the vascular microenvironment as well as inflammation level during venous thrombosis. ResultsA dual-responsive two-photon fluorescent probe, Cou-ONOO, was designed and synthesized. Cou-ONOO provides a visualization tool for monitoring the viscosity of the vascular as well as the inflammatory marker ONOO‾ during thromboembolism via dual-channel simultaneous imaging. As a single probe that can recognize dual targets, Cou-ONOO effectively avoids the problems from unreliable results caused by complex synthesis and differences in intracellular localization, diffusion, and metabolism of different dyes as using combinatorial probes. Using Cou-ONOO, simultaneous imaging the variations of viscosity and ONOO‾at the cellular and tissue levels was successfully performed. In addition, Cou-ONOO also successfully visualized and tracked the viscosity of the vascular microenvironment and ONOO‾ during venous embolism in mice. SignificanceExperimental results show that both viscosity and inflammation are abnormally overexpressed in the microenvironment at the thrombus site during venous thrombosis. An intuitive visualization tool to elucidate the variations of viscosity as well as inflammation level in the vascular microenvironment during thrombosis was provided, which will facilitate a better clinical understanding of the pathological process of thrombosis.

Expression of a Novel Chimeric Inhibitory Receptor Attenuates CD19 CAR T Cells: A Promising Strategy for Toxicity Mitigation

Introduction Chimeric antigen receptor (CAR) T cells targeting CD19 are highly effective in patients with refractory/relapsed acute lymphoblastic leukemia and lymphoma but often trigger severe cytokine release syndrome (sCRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). For r/rALL patients, as many as 77% develop Grade 3-5 CRS and 40% Grade 3-5 ICANS. sCRS occurs when hyperactivated CAR T cells undergo exponential expansion accompanied by the release of excessive amounts of inflammatory cytokines. This leads to a systemic pro-inflammatory state that can cause hypotension, multi-organ failure and even death. Tocilizumab is the only FDA-approved drug for CRS and despite concurrent corticosteroids, often fails to control CRS. Attempts to improve safety have been proposed, but have significant limitations, such as permanent removal of CARs. New and innovative methods to mitigate toxicities are needed. The purpose of this study was to design and test the efficacy of a novel autoregulatory element in the form of a chimeric inhibitory receptor (CIR), that when engaged by a specific CRS-associated pro-inflammatory molecule would trigger inhibitory signaling within CAR T cells. We hypothesize that as cytokine levels increase following activation, these signals can be redirected through responsiveness of the CIR, leading to an attenuation of CAR activity. In this study, we have tested a CIR construct that couples the recognition of IFNγ to the signaling domain of the TIGIT inhibitory receptor. We co-expressed this CIR with a CD19.28ζ CAR to evaluate its ability to attenuate activation and cytokine production and to assess its cytotoxic activity. Methods An anti-IFNγ scFv followed by the transmembrane and intracellular domains of TIGIT were arranged in sequence with the truncated EGFR receptor followed by the CD19.28ζ FMC63 CAR with intervening T2A cleavage domains and cloned into a retroviral backbone. Transduction efficiency was assessed by staining with biotinylated CD19-Fc protein or biotinylated EGFR. Following expansion, memory and effector cell differentiation was evaluated by staining for CD45RO, CD45RA, CCR7, CD127, and CD95. Activation following stimulation with NALM-6 tumor cells was measured by staining for CD69, CD25, CD38, PD-1, and CD107a. Cytotoxicity was evaluated using bioluminescence. To mimic cell types present during CRS, we utilized a coculture assay that consists of CAR T cells, NALM-6 tumor cells, and either macrophages or dendritic cells (DC) differentiated from autologous CD14 + monocytes. Cocultures were incubated for 48 hr. and supernatants were collected for analyses by Luminex. Results CD19 CARs co-expressing a CIR composed of an anti-IFNγ scFv coupled to TIGIT inhibitory signaling expanded during the manufacturing process as well as T cells expressing only CD19 CAR and displayed normal levels of CAR expression. The presence of the CIR did not affect the differentiation profile of memory and effector subsets. CIR-expressing CD19 CARs exhibited robust tumor cell killing of NALM-6 targets equivalent to CD19 CARs. Next, CD19 CAR/CIR cells were pretreated with IFNγ for 24 hr. and then cultured with NALM-6 cells. Exposure to IFNγ resulted in a significant abrogation in cytotoxicity, which was eventually restored with time. Activation profile analysis of CARs equipped with CIR signaling after target stimulation revealed an overall diminution of CARs expressing multiple (4 or 5) activation markers relative to CD19 CARs. Importantly, CIR signaling resulted in significant reductions in the levels of effector, stimulatory, and inflammatory mediators including IFNγ, TNFα, GM-CSF, and IL-17. CARs expressing the CIR exhibited slower killing kinetics when challenged with sequential stimulations by fresh tumor cells. However, in vivo anti-tumor cell activity was not adversely affected as CAR/CIR T cells robustly eliminated disease in leukemia-bearing NSG mice. Lastly, using an in vitro CRS assay, we sought to determine if attenuated CAR T cells would be less potent in activating myeloid cells. We observed that monocyte-derived macrophage and DC activation by CD19 CAR/CIR T cells was reduced as evidenced by decreased secretion of proinflammatory cytokines and chemokines. Conclusion These results provide a compelling proof of concept of a novel self-regulating CAR T cell design with the potential to mitigate severe toxicities in patients.

Long-Term Follow-up of the AST Trial of Treatment-Free Remission in Chronic Myeloid Leukemia Patients

Background: Treatment-free remission (TFR) is a new goal for chronic myeloid leukemia (CML) patients who achieved a long deep molecular response (DMR) with better quality of life, reduced adverse events and high economic impact. The primary objective of the AST trial was to estimate molecular relapse rate at 24 month in CP-CML patients who discontinued TKI. Secondary objective to explore predictive markers for sustained TFR through clinical and immunological analysis ( innate inmune response). This update provides longer follow-up data and includes a new cohort of patients. Patients and Methods: AST-Argentina Stop Trial (NCT05926128) is a multicenter prospective study that included patients from 15 centers. Recruited patients ≥ 18 years, treated with TKI for at least 4 years, sustained MR 4.0 for ≥ 2 years and confirmed typical BCR-ABL1 b3a2 and/or b2a2 transcripts. Molecular studies were centralized in 2 internationally standardized laboratories and were performed monthly during the first 6 months, every 2 months until the first year, and every 3 months during the second year. TKI was restarted due to loss of major molecular response (MMR). Expression of multiple NK cell immunophenotypic markers(CD3-CD56+) was analyzed by flow cytometry at discontinuation time and 3 months later. The nonparametric Mann-Whitney test was used to compare the differences between relapsed and responding patients. Molecular relapse-free survival was estimated using the Kaplan-Meier method and optimal cut-off points were obtained using ROC curves. Comparisons between survival curves were made using the Log rank test method. The differences were considered statistically significant with p-values less than 0.05. Multivariate analysis was performed using the COX regression model. Results: A total of 81 patients divided into 2 cohorts were evaluated: 46 patients recruited between February 2018 and July 2020 (1st cohort) and 35 patients recruited between July 2022 and April 2023. Between these two periods, recruitment was stopped due to the COVID19 pandemic. This analysis focuses on the 1st cohort that has completed 24 months in the study. The median age was 57.5 years (24-86), median duration of treatment before discontinuation was 10.5 years (4.2-20). TKI treatment prior to discontinuation was as follows: Imatinib in 34 (73%) patients, Dasatinib 8 (17%), and Nilotinib 4 (9%). According to Sokal risk score: 22 (48%) were low risk, 14 (30%) intermediate, and 10 (22%) high risk. Seventeen patients (37%) lost MMR, leading to a 63% molecular relapse-free survival rate with a median follow-up of 47 months (41-52). All patients who lost MMR recovered it after restarting the same TKI, with a median time to response of 2.8 months (1-7). With a prolonged follow-up, 2 late relapses were observed (18 and 42 months) (Fig 1). For a univariate analysis, the continuous variables were dichotomized seeking to maximize the difference between the different cohorts. The differences were significant in the variables time in DMR (cut-off point: 51 months, p=0.0166)(HR=0.97 (0.94 - 0.99); p=0.013). Two of the NK cell markers showed significant differences between groups at month 3 after starting treatment, PD1 (Mann-Whitney p=0.035; Log-Rank test p=0.033) and NK-p44 (Mann-Whitney p=0.015; Log -Rank test p=0.020). Multivariate analysis using the Cox model that included variables as gender, age at diagnosis, time in DMR, time in TKI prior to discontinuation, Sokal score, and type of inhibitor, the clinical predictor of time in DMR was the only predictor, significantly associated with a higher rate of TFR, (HR = 0.97 (0.95 - 0.99); p=0.020). No patient experienced disease progression to advanced stages or death for any reason. Conclusion:This is the first multicenter study of TKI discontinuation in CML patients in Argentina showing that TKI can be safely discontinued in those who achieve and maintain a DMR before discontinuation. Molecular relapse-free survival rate was slightly higher than that reported in the international literature, and longer follow-up confirmed the sustained response. DMR before discontinuation showed to be a robust predictor of TFR. Although the sample size was increased, longer follow-up is needed to validate these findings in the second cohort. Potential advantages of TFR will not only impact in patients' quality of life but also show considerable reduction in the use of economic resources.

NK Cell Dysfunction in CLL Is Mediated through SHP-1 Signaling and Is Associated with Poor Prognosis

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, presenting with a highly variable clinical course, outcome and response to therapy. This variability has been linked to a complex tumor microenvironment and genetic/epigenetic modifications that lead to immunosuppression. Indeed, the clinical hallmark of this disease is the susceptibility to infections and secondary malignancies. While T cell dysfunction in CLL has been well described, conflicting information exists on NK cell changes in CLL and their role in CLL immunosurveillance. Here, we analyzed samples from 75 untreated (or completed treatment &amp;gt;2 years ago) CLL patients and 30 healthy controls. We discovered a substantial variation in NK cell function compared with healthy controls, which we further classified into hyperfunctional, normofunctional and dysfunctional based on their ability to degranulate, and produce IFN- γ in response to K562 targets ( Figure 1). NK cells from ‘dysfunctional’ CLL samples also displayed impaired cytotoxicity against K562 targets, weaker synapse formation and Syk/Zap70 signaling in response to antibody-dependent cellular cytotoxicity (ADCC) compared to those from ‘functional’ samples. The NK functional status was strongly associated with multiple prognostic markers, including cytogenetics, IgVH mutation status, ZAP-70 expression and disease stage, with patients with dysfunctional NK cells having significantly higher rates of secondary malignancies and viral infections ( Table 1). Using multispectral flow cytometry we discovered high phenotypic heterogeneity with unique NK cell clusters present in the dysfunctional samples. Those clusters express a pattern of activation-induced exhaustion, with upregulation of multiple checkpoint molecules such as PD-1, TIGIT, LAG-3, KLRG-1, TIM-3 but not evidence of terminal differentiation (CD57 negative). Those clusters also showed concomitant expression of activating molecules such as NKG2D and DNAM-1, suggesting reversibility of their dysfunctional status. We subsequently found significantly higher (p&amp;lt;0.0001) constitutive phosphorylation of SHP-1 protein, a phosphatase downstream of multiple inhibitory NK cell receptors, in NK cells from dysfunctional samples. Treatment with a SHP-1 inhibitor NSC-87877 (EMD Millipore) or use of siRNA targeting SHP-1 (ThermoFisher scientific), resulted in reversal of NK dysfunction with full restoration of their functional capacity. Taken together, our data suggest that NK cells functional status plays an important role in CLL disease progression and secondary complications. The novel mechanism of NK cell immune dysfunction in CLL described here has the potential to improve outcomes with novel immunotherapies such as chimeric antigen receptors, checkpoint inhibitors and bi-specific antibody therapy that have shown disappointing results in CLL to date when compared with other B cell malignancies.

18F-FDG PET/CT Imaging Biomarkers for CAR-T Cell Therapy Outcomes in Large-B-Cell Lymphoma

Introduction: CAR-T cell therapy has transformed the care of patients with large-B-cell lymphoma (LBCL), but disease relapse and toxicities remain clinical challenges, while effective quantitative biomarkers are lacking. Our aim was to determine the role of 18F-FDG PET/CT imaging features as biomarkers in LBCL treated with CD19 CAR-T cell therapy. Methods: 18F-FDG PET/CT scans performed before CAR-T for LBCL were analyzed retrospectively by a board certified radiologist. Metabolic tumor volume (MTV), SUVmax, total lesion glycolysis (TLG), presence of bulky disease, and 106 radiomic features per lesion were recorded using the PET/CT and PyRadiomics plugins for FIJI. Laboratory values including IL-6, IL-10, TNF-α, LDH, ferritin, fibrinogen, and D-dimer pre CAR-T infusion were recorded. Response to CAR-T cell therapy was defined according to Lugano criteria. Cytokine release syndrome (CRS) and neurotoxicity were graded according to ASTCT guidelines. Progression-free survival (PFS) and overall survival (OS) were measured from time of CAR-T cell infusion; PFS events were death, relapse, and disease progression. Association between PET features, markers of inflammation and outcomes were studied with Spearman rank correlations, Cox, and logistic regression models. Multivariable generalized estimating equation models were applied to evaluate differential expression of radiomic features between responders and non-responders, accounting for the dependency structure among ROIs from the same individual. Results: A total of 180 LBCL patients (median age 66 years) treated with autologous CD19 CAR-T (93 axi-cel, 52 tisa-cel, 35 liso-cel) were included. Two time points were considered: 1) pre-apheresis scans (n=161; median SUVmax 19, MTV 56 mL, TLG 427 SUV*mL), and 2) pre-infusion, i.e., latest disease evaluation before CAR-T infusion including post-apheresis/post-bridging, as well as pre-apheresis scans for patients without additional imaging afterwards (n=180; median SUVmax 15, MTV 44 mL, TLG 338 SUV*mL), with a total of 341 PET/CT scans evaluated. At lymphodepletion, MTV was significantly correlated with multiple elevated inflammatory laboratory markers; at latest assessment, all PET and inflammatory markers showed significant correlations. MTV at last assessment before infusion was associated with increased risk of grade ≥ 2 CRS (OR 1.08 [95%CI 1.01-1.20], P=0.031), but not ICANS, in a multivariable logistic regression model adjusted for age, pre-lymphodepletion LDH, CAR-T product, bridging and SUVmax. A higher SUVmax at latest assessment was associated with a lower likelihood of non-complete response (CR) (OR 0.58 [0.41-0.80], P&amp;lt;0.001), while MTV lacked such an association after multivariable model. Increased MTV both before apheresis and at last disease assessment before infusion, but not SUVmax, were associated with decreased PFS (HR 1.11 [1.05-1.17] and HR 1.04 [1.02-1.07], respectively), in a multivariable adjustment. A similar pattern was observed for OS. Notably, the combination of MTV and LDH enabled clear separation of high and low PFS risk groups (P&amp;lt;0.001, Figure 1). Finally, when considering over 100 granular pre-infusion radiomic features ( Figure 2), which are not routinely evaluated in CAR-T patients, multiple novel determinants of CR were discovered, including gray level dependence, run length matrix, and shape-based features (P&amp;lt;0.05). Conclusions: To the best of our knowledge, this represents the most extensive analysis of quantitative 18F-FDG PET/CT features within an LBCL cohort undergoing CAR-T treatment. Our findings indicate that standard PET features (MTV and SUVmax) predict both toxicity and efficacy of CAR-T cell therapy, while also showing correlations with systemic inflammation. Additionally, we present, for the first time, the relevance of granular radiomic features in assessing the probability of treatment success. If confirmed, PET features could serve as effective biomarkers in CAR-T patients and have the potential to guide interventions in high-risk populations.

Multimodality Platelet Evaluation By Mass Cytometry and Genetic Analysis in Patients with Bleeding Disorders

Introduction: Bleeding disorders are a diverse group of conditions characterized by abnormal bleeding tendencies. Platelet dysfunction is a common underlying cause and although traditional platelet aggregometry is important in the clinical work-up, platelet functional assays often provide limited diagnostic insights, prompting the need for more advanced techniques and approaches. This study aims to explore the utility of mass cytometry combined with genetic analysis in assessing platelet function in patients with otherwise unspecified bleeding tendencies. Methods: In a single-center prospective study, enrolled patients with clinical and laboratory suspicion of platelet-related bleeding disorders were subjected to multimodality evaluation. Mass cytometry (CyTOF) allows for simultaneous evaluation of multiple platelet markers on single cells, including those involved in platelet activation, aggregation, and adhesion, through a panel of heavy metal-conjugated antibodies. Whole genome sequencing with targeted analysis was utilized to identify potential genetic variants associated with quantitative and qualitative platelet disorders and correlated with CyTOF findings. Results: CyTOF revealed altered platelet markers associated with activation, aggregation, and adhesion in 40% (n=14) of total participants (n=35) with suspected platelet-related bleeding disorders ( Table 1). CyTOF showed significant concordance with platelet aggregometry in 74% of participants (data not shown). Alterations in specific CyTOF markers (e.g., PAC-1, CD40L) correlated with bleeding risk by univariate analysis of patients stratified to high vs low risk groups based on ISTH bleeding assessment tool (ISTH-BAT) ( Table 1). Multivariate analysis using a random forest machine learning algorithm identified variables most predictive of bleeding risk, including CD40L, PAC-1, P-selectin, and mean platelet volume. Genetic analysis identified one or more variants in genes implicated in qualitative and quantitative platelet disorders in 61% (n=19) of total patients analyzed ( Figure 1). Abnormal CyTOF findings were associated with greater proportion of variants detected (79% with abnormal CyTOF vs 53% with normal CyTOF) ( Figure 1). Direct associations between specific mass cytometry findings and genetic variants (i.e., GPIIb, GPIIIa, GPIb, GPIX, etc.) were discovered for several patients, providing important information in evaluating variant pathogenicity and diagnostic validation. Conclusion: This study highlights the potential of integrating mass cytometry and genetic analysis to assist in the diagnosis of otherwise unspecified bleeding tendencies, bleeding risk stratification, and patient management. Our findings enhance the understanding of platelet function in patients with bleeding disorders and provide valuable insights into the complex relationship between platelet phenotypes and genetic determinants. Further investigations with a larger patient cohort are warranted to validate and expand upon these initial correlations and explore their clinical implications in diagnosis and management of bleeding disorders.

Vil-Cre specific Slfn3KO mice exhibit sex-specific differences in lung, stomach, cecum, kidney, and proximal colon differentiation markers and Slfn family members expression levels

The Schlafen (Slfn) family proteins are critical regulators of cell proliferation, induction of immune responses, differentiation, self-restoration, and cell cycle progression. Rodent Slfn3 and human ortholog SLFN12 are critical in the regulation of intestinal epithelial differentiation. Following previous work utilizing Vil-Cre epithelial-specific Slfn3 knockout (VC-Slfn3KO) mice to evaluate Slfn3's role in small intestinal epithelial differentiation, we sought to characterize and distinguish the effects of Slfn3 loss on Slfn family member mRNA expression and differentiation markers for other epithelial cells in the lung, stomach, cecum, and proximal colon. Quantitative PCR analysis of Slfn1, 2, 4, 5, 8, and 9 and multiple differentiation markers were evaluated. We observed gender-specific effects with the loss of Slfn3 on the other Slfn family members and epithelial differentiation markers expression. Lung Slfn4 and 5 were increased only in male VC-Slfn3KO while lung Slfn2 and 8 were decreased only in female VC-Slfn3KO compared to controls. Slfn1, 2, 4, and 9 were increased in the gastric mucosa of male VC-Slfn3KO mice compared to controls. Slfn5 was reduced in female VC-Slfn3KO proximal colonic mucosa compared to controls. Lung AT1 cell differentiation marker Hopx mRNA expression was decreased and Ager was increased in VC-Slfn3KO male mice compared to controls. Lung AT2 differentiation markers and surfactant genes Sftpc and Sftpd were decreased in male VC-Slfn3KO mice. Stomach transcription factors, Lgr5 and Notch1 were increased in male VC-Slfn3KO. Tff1 secretory protein gene was decreased in female VC-Slfn3KO mice. Sucrase isomaltase was greatly increased in male VC-Slfn3KO mice in both cecal and proximal colonic mucosa, but glucose transporter Glut2 was decreased only in the cecum of female VC-Slfn3KO. The changes induced by VC-Slfn3KO in the expression of epithelial differentiation markers and other Schlafen proteins in various target tissues, indicate a complex regulation of gene expression that is sex-dependent.

Synergistic effect of nickel and temperature on gene expression, multiple stress markers, and depuration: an acute toxicity in fish.

Aquatic animals are prone to extinction due to metal pollution and global climate change. Even though the fish and their products are also unsafe for human consumption, their exports have been rejected due to inorganic and organic contaminants. Nickel (Ni) is a metal that induces toxicity and accumulates in the aquatic ecosystem, posing health threats to humans, animals, and fish. In light of the above, our present investigation aimed to determine the median lethal concentration (96h-LC50) of nickel alone and concurrent with high temperature (34°C) (Ni + T) using static non-renewable bioassay toxicity test in Pangasianodon hypophthalmus. The groups treated under exposure to Ni reared under control condition (25-28.9°C) and Ni + T exposure group reread under 34°C. In this study, chose the definitive dose of Ni and Ni + T as 17, 18, 19, and 20mg L-1 after the range finding test. The median lethal concentration of Ni and Ni + T was determined as 19.38 and 18.75mg L-1, respectively at 96h. Oxidative stress viz. catalase (CAT), superoxide dismutase (SOD), glutathione-s-transferase (GST), and glutathione peroxidase (GPx) in the liver, gill, and kidney were noticeably elevated with Ni and Ni + T during 96h. Whereas, the CAT, GPx, and SOD gene expressions were significantly upregulated with Ni and Ni + T. Trilox equivalent anti-oxidant capacity (TEAC), cupric reducing anti-oxidant capacity (CUPRIC), ferric reducing ability of plasma (FRAP), ethoxy resorufin-O-deethylase (EROD), and acetylcholine esterase (AChE) were reduced due to exposure to Ni and Ni + T. Cellular metabolic stress and lipid peroxidation were highly affected due to Ni and Ni + T exposure. The immunological status, as indicated by total protein, albumin, globulin, A:G ratio, and nitro blue tetrazolium chloride (NBT), was severely affected by the toxicity of Ni and Ni + T. Moreover, the gene expression of interleukin (IL), tumor necrosis factor (TNFα), toll-like receptor (TLR), and total immunoglobulin (Ig) was remarkably downregulated following exposure to Ni and Ni + T. HSP 70, iNOS expression, ATPase, Na + /K + -ATPase, cortisol, and blood glucose was significantly elevated with Ni and Ni + T in P. hypophthalmus. The bioaccumulation of Ni in fish tissues and experimental water was determined. The kidney and liver tissues were highly accumulated with Ni, whereas DNA damage was reported in gill tissue. Interestingly, depuration study revealed that at the 28th day, the Ni bioaccumulation was below the maximum residue limit (MRL) level. Therefore, the present study revealed that Ni and Ni + T led to dysfunctional gene and metabolic regulation affecting physiology and genotoxicity. The bioaccumulation and depuration results also indicate higher residual occurrence of Ni in water and aquatic organisms for longer periods.

External Validation of a Novel Multimarker GFR Estimating Equation.

Using multiple markers may improve accuracy in glomerular filtration rate (GFR) estimation. We sought to externally validate and compare the performance a novel multi-marker estimated GFR (panel eGFR) among Black and White persons using the Genetic Epidemiology Network of Arteriopathy cohort. We included 224 sex, race/ethnicity, and measured GFR (mGFR)-category-matched persons, with GFR measured using urinary clearance of iothalamate. We calculated panel eGFR using serum creatinine, valine, myo-inositol, cystatin C, age, and sex. We compared its reliability with current eGFR equations (2021 CKD-EPI creatinine [eGFR-Cr] and creatinine with cystatin C [eGFR-Cr-CysC]) using median bias, precision, and accuracy metrics. We evaluated each equation's performance in age, sex, and race subgroups. In the overall cohort, 49% were Black individuals and mean mGFR was 79 ml/min/1.73m2. Panel eGFR overestimated mGFR (bias: -2.4 ml/min/1.73m2; 95% confidence interval [CI]: -4.4, -0.7), eGFR-Cr-CysC underestimated mGFR (bias: 4.8 ml/min/1.73m2; 95% CI: 2.1, 6.7), while eGFR-Cr was unbiased (bias: 2.0 ml/min/1.73m2; 95% CI: -1.1, 4.6). All equations had comparable accuracy. Among Black males age <65 years, both eGFR-Cr (bias: 17.0 ml/min/1.73m2; 95% CI: 8.6, 23.5) and eGFR-Cr-CysC (bias: 14.5 ml/min/1.73m2; 95% CI: 6.0, 19.7) underestimated mGFR whereas panel eGFR was unbiased (bias: 1.7 ml/min/1.73m2; 95% CI: -3.4, 10.0). Metrics of accuracy for all eGFRs were acceptable in all subgroups except for panel eGFR in Black females <65 years (P30: 73.3%). Panel eGFR can be used to estimate mGFR and may have utility among Black males age <65 years where current CKD-EPI equations are biased.

Three-Dimensional Coordinate Calibration Models for Augmented Reality Applications in Indoor Industrial Environments

The calibration of three-dimensional (3D) coordinates in augmented reality systems is a complex activity. It involves the recognition of environmental characteristics and technology that models the 3D space of the device, determining its position and orientation. Single markers suffer from numerical instability, particularly when they are small within the camera image. In the industrial environment, it is common for augmented reality applications to cover large spaces, making it difficult to maintain attributes such as precision and accuracy. To address this issue, our study proposes a two-step calibration model that leverages multiple markers for accurate localization in a larger indoor environment. We developed the calibration model using Unity3D, Mixed Reality ToolKit, and Vuforia and evaluated it in terms of precision and accuracy in a proof of concept using the MS Hololens device. Our findings reveal that employing two markers significantly reduces angular discrepancies between points in the real and augmented environments. Moreover, our results underscore that registration accuracy improves as the number of calibration points increases. The results show improvements in determining the axes that define the 3D space, with a direct influence on the position of the points observed in the experiment.

Feasibility for non-invasive prenatal fetal blood group and platelet genotyping by massively parallel sequencing: A single test system for multiple atypical red cell, platelet and quality control markers.

Non-invasive prenatal tests (NIPT) to predict fetal red cell or platelet antigen status for alloimmunised women are provided for select antigens. This study reports on massively parallel sequencing (MPS) using a red cell and platelet probe panel targeting multiple nucleotide variants, plus individual identification single nucleotide polymorphisms (IISNPs). Maternal blood samples were provided from 33 alloimmunised cases, including seven with two red cell antibodies. Cell-free and genomic DNA was sequenced using targeted MPS and bioinformatically analysed using low-frequency variant detection. The resulting maternal genomic DNA allele frequency was subtracted from the cell-free DNA counterpart. Outcomes were matched against validated phenotyping/genotyping methods, where available. A 2.5% subtractive allele frequency threshold was set after comparing MPS predictions for K, RhC/c, RhE/e and Fya /Fyb against expected outcomes. This threshold was used for subsequent predictions, including HPA-15a, Jka /Jkb , Kpa /Kpb and Lua . MPS outcomes were 97.2% concordant with validated methods; one RhC case was discordantly negative and lacked IISNPs. IISNPs were informative for 30/33 cases as controls. NIPT MPS is feasible for fetal blood group genotyping and covers multiple blood groups and control targets in a single test. Noting caution for the Rh system, this has the potential to provide a personalised service for alloimmunised women.

Phylogenomic analyses shed light on the relationships of chiton superfamilies and shell-eye evolution.

Mollusca is the second-largest animal phylum with over 100,000 extant species representing eight classes. Across 1000 extant species in the class Polyplacophora, chitons have a relatively constrained morphology but with some notable deviations. Several genera possess "shell eyes", i.e., true eyes with a lens and retina that are embedded within the dorsal shells. The phylogeny of the major chiton clades is mostly well established, in a set of superfamily-level and higher level taxa supported by various approaches, including morphological studies, multiple gene markers, mitogenome-phylogeny, and phylotranscriptomic approaches. However, one critical lineage has remained unclear, namely Schizochiton which was controversially suggested as being the potential independent origin of chiton shell eyes. Here, with the draft genome sequencing of Schizochiton incisus (superfamily Schizochitonoidea) plus assemblies of transcriptome data from other polyplacophorans, we present phylogenetic reconstructions using both mitochondrial genomes and phylogenomic approaches with multiple methods. We found that phylogenetic trees from mitogenomic data are inconsistent, reflecting larger scale confounding factors in molluscan mitogenomes. However, a consistent and robust topology was generated with protein-coding genes using different models and methods. Our results support Schizochitonoidea as the sister group to other Chitonoidea in Chitonina, in agreement with the established classification. Combined with evidence from fossils, our phylogenetic results suggest that the earliest origin of shell eyes is in Schizochitonoidea, and that these structures were also gained secondarily in other genera in Chitonoidea. Our results have generated a holistic review of the internal relationship within Polyplacophora, and a better understanding of the evolution of Polyplacophora.

A SEVA-based, CRISPR-Cas3-assisted genome engineering approach for Pseudomonas with efficient vector curing

ABSTRACT The development of CRISPR-Cas-based engineering technologies has revolutionized the microbial biotechnology field. Over the years, the Class II Type II CRISPR-Cas9 system has become the gold standard for genome editing in many bacterial hosts. However, the Cas9 system does not allow efficient genomic integration in Pseudomonas putida , an emerging Synthetic Biology host, without the assistance of lambda-Red recombineering. In this work, we utilize the alternative Class I Type I-C CRISPR-Cas3 system from Pseudomonas aeruginosa as a highly efficient genome editing tool for P. putida and P. aeruginosa . This system consists of two vectors, one encoding the Cas genes, CRISPR array and targeting spacer, and a second Standard European Vector Architecture vector, containing the homologous repair template. Both vectors are Golden Gate compatible for rapid cloning and are available with multiple antibiotic markers, for application in various Gram-negative hosts and different designs. By employing this Cas3 system, we successfully integrated an 820-bp cassette in the genome of P. putida and performed several genomic deletions in P. aeruginosa within a week, with an efficiency of &gt;83% for both hosts. Moreover, by introducing a universal self-targeting spacer, the Cas3 system rapidly cures all helper vectors, including itself, from the host strain in a matter of days. As such, this system constitutes a valuable engineering tool for Pseudomonas , to complement the existing range of Cas9-based editing methods, and facilitates genomic engineering efforts of this important genus. IMPORTANCE The CRISPR-Cas3 editing system as presented here facilitates the creation of genomic alterations in Pseudomonas putida and Pseudomonas aeruginosa in a straightforward manner. By providing the Cas3 system as a vector set with Golden Gate compatibility and different antibiotic markers, as well as by employing the established Standard European Vector Architecture (SEVA) vector set to provide the homology repair template, this system is flexible and can readily be ported to a multitude of Gram-negative hosts. Besides genome editing, the Cas3 system can also be used as an effective and universal tool for vector curing. This is achieved by introducing a spacer that targets the origin-of-transfer, present on the majority of established (SEVA) vectors. Based on this, the Cas3 system efficiently removes up to three vectors in only a few days. As such, this curing approach may also benefit other genomic engineering methods or remove naturally occurring plasmids from bacteria.