Late Time Points Research Articles

Late onset pancytopenia post chimeric antigen receptor T cell therapy triggered by a COVID-19 vaccination: a case report

Cytopenia is a common side effect after chimeric antigen receptor T cell (CAR-T) therapy but is unusual at late timepoints when it is most commonly due to bone marrow (BM) failure, myelodysplasia (MDS) or other secondary malignancies. In this case report, we describe a patient who developed severe aplastic anaemia 18 months after CD19 CAR-T therapy, and shortly after COVID-19 vaccination. This is the first description of severe aplastic anaemia triggered by COVID-19 vaccination as a cause of post-CAR-T cytopenia. The patient described was treated with a combination of eltrombopag and ciclosporin due to frailty and had a rapid and complete clinical response to this therapy, providing an effective option for the treatment of immune-related cytopenia post-CAR-T in an increasingly elderly and frail population.

CNSC-04. UNDERSTANDING PEDIATRIC BRAIN TUMOR CHEMOTHERAPY INDUCED LONG-TERM DEFICITS IN HIGH-ORDER NEUROCOGNITIVE AND SENSORIMOTOR FUNCTION

Abstract Because of major advancements in cancer treatment especially chemotherapy, 85% of children with cancer are predicted to survive for 5 years or more. However, chemotherapy can cause long-term health problems when childhood cancer survivors enter adulthood (known as late effects). The neurocognitive features of these late effects (chemobrain) include trouble in concentrating, memorizing, and decreased processing speed, and results in lower IQ and academic achievement, poor hand-eye coordination, and slower development over time. Despite such a negative impact on the quality of life of cancer-surviving children, the mechanism is poorly understood and there is no FDA-approved drug to prevent or cure these side effects. To gain a better understanding of the mechanism underlying chemotherapy-induced cognitive impairments, we developed a pre-clinical model using carboplatin in juvenile mice to study long-term neurological defects. Carboplatin is commonly used chemotherapy drug in pediatric brain tumor patients. We have tested the capacity of different doses of carboplatin and its long-term effect on cognitive and sensorimotor function and performed single nuclear RNA sequencing (snRNAseq) of the hippocampus. Our preliminary data demonstrated dose-dependent deficits in executive and sensorimotor function 7-9 weeks after carboplatin administration in the mice of both sexes. snRNAseq of the hippocampus showed changes in the glial cell number as well as genes associated with oxidative phosphorylation and neurodegeneration at early and late time points. Bioenergetics using Seahorse in the brain synaptosomes revealed mitochondrial dysfunction in the form of decreased maximum respiratory capacity long after carboplatin treatment. This study established a preclinical model that can be leveraged to mechanistically understand the long-term neurocognitive side effects of brain tumor chemotherapy during childhood. Future studies will aim to identify potential targets to prevent and/or treat the neurotoxic effects of cancer treatment in pediatric patients.

IMMU-60. MYELOID POPULATIONS MODULATE GD2 CAR T CELL ACTIVITY IN DIFFUSE MIDLINE GLIOMA

Abstract H3K27M-mutated diffuse midline gliomas (DMGs) are universally lethal cancers in children and young adults. Our team previously demonstrated efficacy of GD2-targeting chimeric antigen receptor T cells (GD2-CAR T-cells) in preclinical models of DMG of the pons (also called diffuse intrinsic pontine glioma (DIPG) and DMG of the spinal cord, and opened a Phase I clinical trial (NCT04196413) treating patients with first intravenous (IV) followed by repeated infusions of intracerebroventricular (ICV) GD2-CAR T-cells. We employed high-dimensional analyses to define immune states contributing to CAR-T activity in patients. Single cell RNA-sequencing (scRNAseq) was conducted on 555,406 single cells from 115 cerebrospinal fluid (CSF) samples of 13 patients before and after CAR-T treatment. This is the largest CSF CAR-T dataset in central nervous system (CNS) tumors and provides insights into the immune biology surrounding CAR-T treatment for CNS malignancies. Patient CSF samples were dominated by T cell and myeloid populations. After CAR-T infusion, patient CSF exhibited an increased fraction of regulatory T cells and myeloid populations from baseline. Myeloid cells in early timepoints after ICV administration demonstrated a unique pro-inflammatory signature, while CSF samples from IV and late ICV timepoints exhibited a suppressive signature. To further explore the immune biology of these myeloid contributors, we developed a patient-derived xenograft model of DMG relapse following low-dose ICV GD2 CAR-T treatment. Using a pharmacological CSF1R-inhibitor, we demonstrate that depletion of microglia/myeloid cells at a specific window following CAR-T administration enhances durability of tumor control. Together, these data display the power of in-depth correlative analyses to identify distinct immune populations that drive durability of response. Key findings from these data will allow for optimization of CAR-T therapies for H3K27M+ DMG patients, providing hope to shift the paradigm of this fatal disease.

EPCO-53. CHARACTERIZING THE FUNCTIONAL IMPACT OF GERMLINE-INHERITED RISK ALLELES IN GLIOMA EVOLUTION

Abstract Efforts to understand etiology of glioma led researchers to investigate associated genetic factors. Genome Wide Association Studies (GWAS) conducted to that end, uncovered an association of various single nucleotide polymorphisms (SNP) among hallmark oncogenes and tumor suppressors as risk variants in glioma incidence. Here, we perform in vitro modeling of four risk alleles/SNPs((rs723527 (EGFR), rs55705857 (MYC), rs7572263 (IDH1), rs78378222 (TP53)) mapped to non-coding regulatory regions of the genome, to assess their causal contribution to glioma evolution. To model the aforementioned risk alleles in an in-vitro system, we used a CRISPR-knock in strategy in human induced pluripotent stem cells (hiPSCs) and verified by Sanger sequencing. To study the effects of the risk alleles on brain cell types, we generated a cerebral organoid model from the edited and control lines and subjected them to downstream morphological and transcriptomic analyses. While we did not see morphological changes in the histological sections relative to controls at the early 14-day timepoint, we observed increased differentiation in both control and risk cohorts, along with an absence of neural rosettes, at the late 90-day timepoint. Bulk RNA-seq of the organoids at 14-days resulted in positive enrichment of oncogenic signaling pathways and negative enrichment of DNA repair pathways in the risk allele cohorts. Furthermore, single cell RNA seq of 14 and 90-day samples revealed a sharp decrease in neural stem cell clusters in both IDH and TP53 cohorts, while control organoids demonstrated robust production of immature neurons at 90 days. Overall, we observed a significant increase in differentiation into mature cell clusters in the control, relative to the risk allele organoids. Ongoing efforts are aimed at understanding this apparent differentiation block in the IDH and TP53 compartments, relative to control. Further analysis will lead to addressing key understudied areas of glioma etiology and associated mechanisms of risk incidence.

Targeting AXL cellular networks in kidney fibrosis

IntroductionThe incidence of chronic kidney disease (CKD) is increasing, in parallel with risk factors including obesity and diabetes mellitus. AXL plays a central role in CKD, providing a rationale to evaluate clinical AXL targeting agents.MethodsTo determine the efficacy and underlying molecular mechanisms of AXL inhibition in CKD, we employed a murine unilateral ureteral obstruction (UUO) model preventively treated with a selective AXL kinase inhibitor (bemcentinib) during disease progression. We isolated kidneys at an early (3 days) or late (15 days) timepoint and profiled the cell populations using mass cytometry.ResultsPreventive treatment with bemcentinib significantly attenuated fibrosis in the UUO model. The anti-fibrotic effect correlated with a decrease in mesangial cells and inhibition of innate immune cell infiltration, while the proportion of epithelial cells increased. We mapped AXL expression to a unique network of cells in the kidney: mesangial cells, pericytes, macrophages and dendritic cells.DiscussionWe propose that AXL targeting affects an important cellular interaction network underlying fibrotic progression. These results support the clinical application of AXL targeting agents to treat CKD.

Immunologic Profiling of CSF in Subarachnoid Neurocysticercosis Reveals Specific Interleukin-10-Producing Cell Populations During Treatment.

Subarachnoid neurocysticercosis (SANCC) is the most severe form of Taenia solium CNS infection and accounts for the majority of neurocysticercosis-associated mortality. Inflammation is important in the treatment of SANCC because overactivity can lead to serious complications, but excessive suppression may be counterproductive toward parasite eradication. A relative abundance of CSF IL-10 to IL-12 has been associated with increased treatment duration for patients with SANCC, suggesting that IL-10 plays an important role in this disease process. To better understand SANCC immunology and the major sources of IL-10 during anthelmintic treatment, we took an unbiased and comprehensive approach to phenotype the immune cell populations in the CSF and peripheral blood of patients with SANCC. Eight samples of CSF cells collected from 5 patients with SANCC during treatment were evaluated using single-cell RNA sequencing. Matched CSF and peripheral blood mononuclear cells from 4 patients were assessed using flow cytometry. Staining for extracellular and intracellular markers allowed for the characterization of IL-10-producing T cells. The CSF during SANCC contains a diversity of immune cell populations including multiple myeloid and lymphoid populations. Although there were changes in the composition of CSF cells during treatment, the largest population at both early and late time points was CD4+ T cells. Within this population, we identified 3 sources of IL-10 unique to SANCC CSF compared with controls: natural regulatory T cells (nTregs), induced regulatory T cells (iTregs), and Th17 cells. The abundance and phenotype of these IL-10-producing populations differed between CSF and blood in patients with SANCC, but iTregs were the single most productive population in the CSF. During treatment, these IL-10 producers persisted in consistent proportions despite decreases in parasite antigen over time. This profile of immune cell populations in the CSF provides a comprehensive blueprint of the local and systemic immunology associated with SANCC. The identification of IL-10-producing cells in the CSF and peripheral blood deepens our understanding of the immunosuppressive phenotype that deters SANCC treatment success. Finally, the discovery that these IL-10 producers persist throughout treatment highlights the endurance of these populations in the CNS.

Persistent angina following revascularization for chronic coronary syndromes: a systematic review and meta-analysis of randomized controlled trials

Abstract Introduction Recent randomized clinical trials (RCTs) have demonstrated that revascularization plus optimal medical therapy for patients with chronic coronary syndromes (CCS) results in greater reduction in angina than medical therapy alone. However, the frequency of persistent angina following revascularization for CCS is not well understood. Purpose To quantify the frequency of persistent angina following revascularization in contemporary RCTs of revascularization plus medical therapy vs. medical therapy alone among patients with CCS. Methods A literature search through January of 2024 was conducted in PubMed and Cochrane Central Register of Controlled Trials for contemporary RCTs reporting angina frequency following randomization to revascularization plus medical therapy vs. medical therapy alone. The primary outcome of interest was the frequency of persistent angina in the revascularization arms of these studies at early (3-12 months) and late (3-5 years) time points. A random effects model was used to combine results from individual studies. Results From a total of 5287 citations, the search yielded 7 RCTs that met inclusion criteria. These studies randomized 5719 patients to an invasive or revascularization strategy. Revascularization was exclusively by percutaneous coronary intervention (PCI) in 4 studies and by coronary artery bypass grafting or PCI in 3 studies. At the early time point, the combined frequency of persistent angina was 39.6% (95% CI: 30.6-49.4). For the two blinded, sham-controlled studies, the rate of persistent angina was significantly greater at 67.2% (95%CI: 54.9-77.4) compared to 30.5% (95% CI: 23.1-39) in the unblinded studies (P<0.001). In the 5 studies with 3-5 years of follow-up, the frequency of persistent angina was 33.3% (95% CI: 19.2-51.2). Conclusion Persistent angina is common after revascularization for CCS and suggests alternative causes and treatments of residual angina should be considered.

POSTOPERATIVE VISUAL fiELD DEfiCITS ARE NOT ASSOCIATED WITH REDUCTION IN QUALITY OF LIFE IN GLIOBLASTOMA PATIENTS

Abstract AIMS Preservation of functional integrity is a core goal of glioblastoma surgery. Visual field deficits can significantly impact quality of life and patient function. Both tumour and surgical effects may affect visual function in these patients. The objective of this Study was to evaluate the effect of visual field deficits on HRQoL in the preoperative and early postoperative period (prior to chemoradiotherapy). METHOD 48 patients with glioblastoma received preoperative, early postoperative (withon 1 week) and delayed postoperative testing (6 weeks) for visual function usinga combination of the OCS-Bridge neurocognitive screening tool and formal visual field testing. Patients also completed the EORTC QLQ C30 with BN-20 and VFQ-25 visual function questionaires at the same timepoints. Statistical tests were performed using R Statistical Software (v4.1.2; R Core Team 2021). RESULTS The proportion of patients with visual field deficits at preoperative, early postoperative and late early postoperative timepoints were 20 (41.7%), 28 (58.3%) and 24 (50%) respectively. Most patients without a preoperative deficit did not have an early 17/20 85% or late postoperative deficit 19/24 79.1%. The proportion of patients with deficits at early and late postoperative timepoints was significantly higher in patients with preoperative deficits compared to patients without preoperative deficits, (Chi square p = 0.004) and (p = 0.008) respectively. There was no significant differences between HRQoL measures between timepoints or between patients with deficits at any timepoint compared to patients without deficits. CONCLUSION Postoperative visual field deficits are related to preoperative tumour-effects rather than surgery. Patients can be counselled that while they do not recover following surgery, it is not associated with significant reductions in HRQoL. Patients with preoperative visual field deficits may be considered for resection without significant worry about the preservation of visual field function since these deficits do not recover postoperatively.

Multiomics profiling of mouse polycystic kidney disease progression at a single-cell resolution

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and causes significant morbidity, ultimately leading to kidney failure. PKD pathogenesis is characterized by complex and dynamic alterations in multiple cell types during disease progression, hampering a deeper understanding of disease mechanism and the development of therapeutic approaches. Here, we generate a single-nucleus multimodal atlas of an orthologous mouse PKD model at early, mid, and late timepoints, consisting of 125,434 single-nucleus transcriptomic and epigenetic multiomes. We catalog differentially expressed genes and activated epigenetic regions in each cell type during PKD progression, characterizing cell-type-specific responses to Pkd1 deletion. We describe heterogeneous, atypical collecting duct cells as well as proximal tubular cells that constitute cyst epithelia in PKD. The transcriptional regulation of the cyst lining cell marker GPRC5A is conserved between mouse and human PKD cystic epithelia, suggesting shared gene regulatory pathways. Our single-nucleus multiomic analysis of mouse PKD provides a foundation to understand the earliest changes molecular deregulation in a mouse model of PKD at a single-cell resolution.

Environmental stress and salivary cortisol levels in preterm infants.

Preterm infants are exposed to numerous environmental stressors during their Neonatal Intensive Care Unit (NICU) stay, particularly during the first week after birth. The aim of this study is to assess whether salivary cortisol levels are correlated with Neonatal Infant Stressor Scale (NISS) scores in preterm infants during the first week of life. We also quantified the changes in both NISS scores and cortisol levels in the first week, and whether cortisol levels are associated with gestational age. Preterm infants (n = 38, birth weight <1250 g and/or gestational age <29 weeks) were included. Saliva samples were collected on day 0-3 (early) and day 4-7 (late), and cortisol concentrations were measured by immunoassay. NISS scores were assessed retrospectively for the six hours preceding each saliva collection. NISS scores were not significantly correlated with salivary cortisol levels at either time point. However, infants born at <28 weeks gestation had higher median cortisol levels than infants born at >28 weeks (p = 0.0068), and there was also a significant inverse relationship between NISS score and gestational age (p = 0.04). There was no significant difference between the early and late time points for either NISS scores or cortisol levels. Cortisol levels are elevated in infants <28 weeks gestation but do not correlate with NISS scores. NISS scores are inversely related to gestational age, likely reflecting increased exposure to interventions and invasive procedures for the smallest infants.

Copper-61 is an advantageous alternative to gallium-68 for PET imaging of somatostatin receptor-expressing tumors: a head-to-head comparative preclinical study.

Gallium-68 positron emission tomography (68Ga-PET) with the two registered somatostatin analogs, [68Ga]Ga-DOTA-Tyr3-octreotide ([68Ga]Ga-DOTA-TOC) and [68Ga]Ga-DOTA-Tyr3-octreotate ([68Ga]Ga-DOTA-TATE), where DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, is routinely used for imaging of somatostatin receptor (SST)-expressing tumors. We investigated copper-61 (61Cu) as an alternative radiometal for PET imaging of SST-expressing tumors. Compared to gallium-68, copper-61 (t1/2 = 3.33 h, E β + max = 1.22 MeV) can be produced on a large scale, enables late time point imaging, and has the therapeutic twin copper-67. Herein, DOTA-TOC and 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA)-TOC were labeled with copper-61 and compared with the clinically used [68Ga]Ga-DOTA-TOC. [61Cu]CuCl2 was produced from an irradiated natural nickel target. DOTA-TOC and NODAGA-TOC were labeled with [61Cu]CuCl2 in ammonium acetate buffer so to achieve a reaction pH of 5-6 and a temperature of 95°C for DOTA-TOC or room temperature for NODAGA-TOC. The radioligands were evaluated head-to-head in vitro using human embryonic kidney (HEK)-SST2 cells (affinity, binding sites, cellular uptake, and efflux) and in vivo using HEK-SST2 xenografts [PET/computed tomography (CT) imaging, biodistribution, and pharmacokinetics] and compared with [68Ga]Ga-DOTA-TOC, which was prepared using a standard procedure. Dosimetry estimates were made for [61Cu]Cu-NODAGA-TOC. [61Cu]Cu-DOTA-TOC and [61Cu]Cu-NODAGA-TOC were prepared at an apparent molar activity of 25 MBq/nmol with radiochemical purities of ≥96% and ≥98%, respectively. In vitro, both presented a sub-nanomolar affinity for SST2 (IC50 = 0.23 and 0.34 nM, respectively). They were almost entirely internalized upon binding to SST2-expressing cells and had similar efflux rates at 37°C. In vivo, [61Cu]Cu-DOTA-TOC and [61Cu]Cu-NODAGA-TOC showed the same accumulation in SST2-expressing tumors. However, PET/CT images and biodistribution analyses clearly showed an unfavorable biodistribution for [61Cu]Cu-DOTA-TOC, characterized by accumulation in the liver and the abdomen. [61Cu]Cu-NODAGA-TOC displayed favorable biodistribution, comparable with [68Ga]Ga-DOTA-TOC at 1 h post-injection (p.i.). Notwithstanding, [61Cu]Cu-NODAGA-TOC showed advantages at 4 h p.i., due to the tumor retention and improved tumor-to-non-tumor ratios. The effective dose (2.41 × 10-3 mSv/MBq) of [61Cu]Cu-NODAGA-TOC, but also the dose to the other organs and the kidneys (9.65 × 10-2 mGy/MBq), suggested a favorable safety profile. Somatostatin receptor 61Cu-PET imaging not only matches the performance of 68Ga-PET at 1 h p.i. but has advantages in late-time imaging at 4 h p.i., as it provides improved tumor-to-non-tumor ratios. [61Cu]Cu-NODAGA-TOC is superior to [61Cu]Cu-DOTA-TOC in vivo. The use of the chelator NODAGA allows quantitative labeling with copper-61 at room temperature and enables the straightforward use of a kit formulation for simple manufacturing in medical centers.

Immunoregulatory Macrophages Modify Local Pulmonary Immunity and Ameliorate Hypoxic Pulmonary Hypertension.

Macrophages play a significant role in the onset and progression of vascular disease in pulmonary hypertension, and cell-based immunotherapies aimed at treating vascular remodeling are lacking. To evaluate the effect of pulmonary administration of macrophages modified to have an anti-inflammatory/proresolving phenotype in attenuating early pulmonary inflammation and progression of experimentally induced pulmonary hypertension. Mouse bone marrow-derived macrophages were polarized in vitro to a regulatory (M2reg) phenotype. M2reg profile and anti-inflammatory capacity were assessed in vitro upon lipopolysaccharide/IFNγ (interferon-γ) restimulation, before their administration to 8- to 12-week-old mice. M2reg protective effect was evaluated at early (2-4 days) and late (4 weeks) time points during hypoxia (8.5% O2) exposure. Levels of inflammatory markers were quantified in alveolar macrophages and whole lung, while pulmonary hypertension development was ascertained by right ventricular systolic pressure (RVSP) and right ventricular hypertrophy measurements. Bronchoalveolar lavage from M2reg-transplanted hypoxic mice was collected and its inflammatory potential evaluated on naive bone marrow-derived macrophages. M2reg macrophages expressing Tgfβ, Il10, and Cd206 demonstrated a stable anti-inflammatory phenotype in vitro, by downregulating the induction of proinflammatory cytokines and surface molecules (Cd86, Il6, and Tnfα) upon a subsequent proinflammatory stimulus. A single dose of M2reg attenuated hypoxic monocytic recruitment and perivascular inflammation. Early hypoxic lung and alveolar macrophage inflammation leading to pulmonary hypertension development was significantly reduced, and, importantly, M2reg attenuated right ventricular hypertrophy, right ventricular systolic pressure, and vascular remodeling at 4 weeks post-treatment. Adoptive transfer of M2reg halts the recruitment of monocytes and modifies the hypoxic lung microenvironment, potentially changing the immunoreactivity of recruited macrophages and restoring normal immune functionality of the lung. These findings provide new mechanistic insights into the diverse role of macrophage phenotype on lung vascular homeostasis that can be explored as novel therapeutic targets.

Mathematical modeling suggests heterogeneous replication of Mycobacterium tuberculosis in rabbits.

How quickly Mycobacterium tuberculosis ( Mtb ) replicates and dies in lungs of infected individuals is likely to determine the outcome of the infection - either control/clearance of the bacteria by the host immune response or progression to active disease, tuberculosis ( TB ). And yet, only a few studies, primarily in mice, rigorously estimated the rates of Mtb replication and death during an in vivo infection. We infected rabbits with a novel clinical isolate of Mtb carrying an unstable, "replication clock" plasmid and followed the dynamics of bacteria over time. Interestingly, previous methods developed to estimate Mtb replication and death rates using similar data for Mtb infection of mice failed to describe our novel data on Mtb dynamics in rabbits; we showed that heterogeneous dynamics of Mtb in semi-independent subpopulations in lungs of Mtb-infected rabbits may be one explanation of this failure of the method. Our results highlight potential differences in Mtb dynamics in different mammalian hosts and suggest ways to evaluate heterogeneity of Mtb replication and death rates in vivo.

Metabolism and disposition of zamicastat in rats

The metabolism and disposition of zamicastat, a reversible dopamine β-hydroxylase (DβH) inhibitor, developed for treatment of Pulmonary Arterial Hypertension (PAH), were investigated in rats after oral and intravenous administration of [14C]-zamicastat. Zamicastat was rapidly absorbed and widely distributed to peripheral tissues, with total radioactivity almost completely recovered 168 h post-dose. Its main route of excretion was via faeces, whilst urine and expired air had minor roles. Maximum plasma concentration of zamicastat-related radioactivity occurred in the first hours, remaining quantifiable up to 144 h. The unchanged zamicastat plasma peak was 2 h post-dose and declined to low levels over 24 h. Zamicastat metabolism occurs largely during the first 8 h with only one metabolite identified in the latest time-point (96 h), the isocyanic acid/thiocyanic acid (tautomeric forms). Zamicastat metabolic pathway involved multiple reactions comprising desulphurisation, oxidative desulphurisation, N-debenzylation followed by further oxidation or N-acetylation, and the unexpected multistep metabolic pathway leading to isocyanic acid/thiocyanic acid.

Context-specific anti-inflammatory roles of type III interferon signaling in the lung in nonviral injuries.

Type III interferons (λ1, λ2, and λ3) are potent antiviral cytokines in the lung. However, their roles in nonviral lung injuries are less well understood. This study investigates the activation of type III interferon signaling in three distinct models of lung injuries caused by diverse stimuli: the bacterial pathogen Pseudomonas aeruginosa, bacterial endotoxin LPS, and the chemotherapeutic agent bleomycin. Our data show that, despite inducing a potent inflammatory response, Pseudomonas and LPS did not increase IFNλ secretion. In contrast, bleomycin instillation increased secretion of IFNλ in the airways at both early and late time points. Consistent with limited secretion, type III interferon signaling had a minimal role in the host response to both Pseudomonas and LPS, as measured by pathogen burden, inflammatory response, and lung injury. Conversely, a deficiency in type III interferon signaling led to increased inflammatory signaling and elevated acute lung injury in the bleomycin model on day 3. This elevated early injury resulted in impaired recovery in IFNLR1 knockout mice, as evidenced by their recovery from bleomycin-induced weight loss. Taken together, these data suggest a context-specific activation of type III interferon signaling, where it plays an anti-inflammatory role in the lung.

Novel Fluorescence-Based Methods to Determine Infarct and Scar Size in Murine Models of Reperfused Myocardial Infarction.

Determination of infarct and scar size following myocardial infarction (MI) is commonly used to evaluate the efficacy of potential cardioprotective treatments in animal models. However, histological methods to determine morphological features in the infarcted heart have barely improved since implementation while still consuming large parts of the tissue and offering little options for parallel analyses. We aim to develop a new fluorescence technology for determining infarct area and area at risk that is comparable to 2,3,5-triphenyltetrazolium chloride (TTC) staining but allows for multiple analyses on the same heart tissue. For early and late time points following MI, we compared classical histochemical approaches with fluorescence staining methods. Reperfused MI was induced in male mice, the hearts were extracted 24 h, 7-, 21-, or 28-days later and fluorescently stained by combining Hoechst and phalloidin. This approach allowed for clear visualization of the infarct area, the area at ischemic risk and the remote area not affected by MI. The combined fluorescence staining correlated with the classic TTC/Evans Blue staining 24 h after MI (r = 0.8334). In later phases (>7 d) post-MI, wheat germ agglutinin (WGA) is equally accurate as classical Sirius Red (r = 0.9752), Masson's (r = 0.9920) and Gomori's Trichrome (r = 0.8082) staining for determination of scar size. Additionally, feasibility to co-localize fluorescence-stained immune cells in specific regions of the infarcted myocardium was demonstrated with this protocol. In conclusion, this new procedure for determination of post-MI infarct size is not inferior to classical TTC staining, yet provides substantial benefits, including the option for unbiased software-assisted analysis while sparing ample residual tissue for additional analyses. Overall, this enhances the data quality and reduces the required animal numbers consistent with the 3R concept of animal experimentation.

Acute kidney injury results in long-term alterations of kidney lymphatics in mice.

The long-term effects of a single episode of acute kidney injury (AKI) induced by bilateral ischemia-reperfusion injury (BIRI) on kidney lymphatic dynamics are not known. The purpose of this study was to determine if alterations in kidney lymphatics are sustained in the long term and how they relate to inflammation and injury. Mice underwent BIRI as a model of AKI and were followed up to 9 mo. Although kidney function markers normalized following initial injury, histological analysis revealed sustained tissue damage and inflammation for up to 9 mo. Transcriptional analysis showed both acute and late-stage lymphangiogenesis, supported by increased expression of lymphatic markers, with unique signatures at each phase. Expression of Ccl21a was distinctly upregulated during late-stage lymphangiogenesis. Three-dimensional tissue cytometry confirmed increased lymphatic vessel abundance, particularly in the renal cortex, at early and late timepoints postinjury. In addition, the study identified the formation of tertiary lymphoid structures composed of CCR7+ lymphocytes and observed changes in immune cell composition over time, suggesting a complex and dynamic response to AKI involving tissue remodeling and immune cell involvement. This study provides new insights into the role of lymphatics in the progression of AKI to chronic kidney disease.NEW & NOTEWORTHY Here, we perform the first, comprehensive study of long-term lymphatic dynamics following a single acute kidney injury (AKI) event. Using improved three-dimensional image analysis and an expanded panel of transcriptional markers, we identify multiple stages of lymphatic responses with distinct transcriptional signatures, associations with the immune microenvironment, and collagen deposition. This research advances kidney lymphatic biology, emphasizing the significance of longitudinal studies in understanding AKI and the transition to chronic kidney disease.

Early and late responses to Fusarium Head blight in durum wheat: Focus on phenylpropanoid biosynthetic pathway

Durum wheat is among the cereal crops most susceptible to Fusarium Head Blight (FHB), a fungal disease that can lead to significant yield losses. Despite this, only limited research efforts have been directed towards understanding FHB resistance in durum wheat. Wheat grains naturally contain phenolic compounds, and anthocyanins are particularly present in the so-called pigmented wheat genotypes, such as purple pericarp ones. In this study the effects of the biotic stress caused by Fusarium graminearum infection on phenylpropanoid biosynthetic pathway in durum wheat spikes were explored, considering three genotypes with different susceptibility (including a purple pericarp genotype), and two time points (an early stage time point: 2 days post infection, and a late stage time point: 21 days post infection). At early infection stage, the F. graminearum infection triggered upregulation of all the considered genes involved in the phenylpropanoid pathway in the resistant genotype, while, in the purple pericarp genotype, the infection caused an increase in quercetin accumulation in the soluble fraction of spike extract. At late infection stage, the infection caused (in all the genotypes) a degradation of secondary cell wall and the release of the hydroxycinnamic acids esterified with arabinoxylans (ferulic acid and p-coumaric acid) and lignin-derived monomers (vanillic acid). Furthermore, chalcone synthase gene (CHS) and the transcription factor Ppm1 (Purple pericarp MYB 1) were boosted in the pigmented genotype due to infection at late infection stage. These findings contribute to the understanding of host-pathogen interactions for future breeding programs focused on improving FHB resistance in durum wheat varieties, with a particular focus on pigmented genotypes.

Abstract B005: Characterizing the role of fibroadipogenic progenitors in pancreatic cancer cachexia: the role of miR-27a-3p

Abstract Introduction: Approximately 80% of patients with pancreatic cancer suffer from cachexia- a debilitating condition characterized by involuntary loss of muscle and fat. While muscle wasting has mostly been investigated from a whole tissue level, the impact of how different cell types contribute to muscle wasting remains elusive. The current study focuses on characterizing the role of muscle resident fibroadipogenic progenitors (FAPs) in pancreatic cancer cachexia. Dysregulation of FAPs produces sustained fibrogenic and/or adipogenic signals, which may lead to fibrofatty infiltration- a condition often observed in patients with cachexia. Using an experimental model of pancreatic cancer cachexia, we aim to understand (i) the overall contribution of FAPs to muscle wasting, (ii) the role of microRNAs (miRNAs- a class of small RNAs considered as global modulators of gene expression) in FAPs dysregulation and (iii) if modulating miRNAs in cancer improves muscle micro and macroenvironment. Methods: A male pancreatic cancer cell line was utilized to investigate the cachexia progression over time where day 14 (D14), 21 (D21), and day 26 (D26) were considered early, intermediate, and late time points, respectively. Mice were orthotopically implanted with 2 million pancreatic cancer cells (referred to as the KPC) and control mice received sham surgery (referred to as the control). Single nucleus RNA seq (snRNA seq) from quadriceps muscle, RNA seq, and miRNA profiling from sorted FAPs were performed. miR-27a-3p was knocked down in pancreatic cancer cells using a lentiviral system. Results: snRNA seq identified 13 clusters in quadriceps muscle including FAPs. Further, 4 subpopulations of FAPs with predominantly distinct transcriptional profiles were identified indicating that FAPs may be functionally heterogeneous. Further, muscle wasting genes such as Bmp4 and Osmr were present predominantly in FAPs suggesting that dysregulation of FAPs may contribute to muscle wasting. Time-resolved RNA seq analysis from sorted FAPs showed that alterations in adipogenesis (observed on day 14) preceded fibrosis (observed on d21), suggesting that dysregulation of FAPs is an early event in the muscle wasting trajectory, potentially contributing to fibrofatty infiltration. miR-27a-3p was chosen as a candidate molecule that was downregulated in FAPs and upregulated in the tumor when compared to controls. Inhibiting miR-27a-3p on FAPs increased adipogenesis and treatment with mimic reduced adipogenesis. Knocking down miR-27a in cancer prevented muscle wasting in vivo, and reduced FAPs dysregulation, suggesting that miR-27a-3p plays a dual role in tumor and muscle microenvironment. Conclusions: The study shows for the first time that dysregulation of FAPs significantly contributes to muscle wasting, potentially mediated through miR-27a-3p which when modulated in tumor reduces tumor progression and improves the muscle micro and macroenvironment. Citation Format: Ashok Narasimhan, Chun Wai Cheung, Nasim Kajabadi, Bruce Lin, Lin Wei Tung, Chihkai Chang, Fabio MV Rossi. Characterizing the role of fibroadipogenic progenitors in pancreatic cancer cachexia: the role of miR-27a-3p [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B005.

Different Antigen-Specific CD4+ and CD8+ T-Cell Response against HCMV Proteins in Pregnant Women with Primary Infection and in Control Subjects with Remote Infection.

Background/Objectives: Human cytomegalovirus (HCMV) is the most frequent cause of congenital infections. The HCMV-specific T-cell response in primary infection may help define reliable correlates of immune protection in pregnancy. In this study, the antigen-specific T-cell response against different HCMV proteins (IE-1, pp65, gB, gHgLpUL128L) was investigated in pregnant women with primary infection and in control subjects with remote infection to identify possible components of a vaccine. Methods: Blood samples from 35 pregnant women with HCMV primary infection and 30 HCMV-seropositive healthy adult subjects with remote infection were tested. The antigen-specific T-cell response was measured using cytokine intracellular staining after stimulation with IE-1, pp65, gB and gHgLpUL128L peptides pool. Results: The pp65-specific CD4+ T-cell response was higher in pregnant women with HCMV primary infection at the late time point and in control subjects with remote infection, while the pregnant women at the early time point showed a higher gB-specific CD8+ T-cell response. Regarding the CD4+ and CD8+ T-cell phenotypes, we observed that HCMV-specific CD4+ and CD8+ T cells expressing CD45RA+ remained constant in pregnant women with primary infection at the early and late time points and in subjects with remote infection, while HCMV-specific CD4+ and CD8+ T cells expressing IL-7R+ or producing IL-2 were higher in control subjects with remote infection than in pregnant women with HCMV primary infection. Conclusions: The T-cell response was higher against gB in the early phase of infection and against pp65 in the late phase. Therefore, these proteins should be taken into consideration as candidates for a vaccine.