Participation of preovulatory follicles in the activation of primordial follicles in mouse ovaries.

The mechanisms behind the selection and initial recruitment of primordial follicles (PmFs) from the non-growing PmF pool during each estrous cycle in females remain largely unknown. This study demonstrates that PmFs closest to the ovulatory follicle are preferentially activated in mouse ovaries under physiological conditions. PmFs located within 40 µm of the ovulatory follicles were more likely to be activated compared to those situated further away during the peri-ovulation period. Repeated superovulation treatments accelerated the depletion of the PmF reserve, whereas continuous suppression of ovulation delayed PmF reserve consumption. Spatial transcriptome sequencing of peri-ovulatory follicles revealed that ovulation primarily induces the degradation and remodeling of the extracellular matrix (ECM). This ECM degradation reduces mechanical stress around PmFs, thereby triggering their activation. Specifically, Cathepsin L (CTSL), a cysteine proteinase and lysosomal enzyme involved in ECM degradation, initiates the activation of PmFs adjacent to ovulatory follicles in a distance-dependent manner. These findings highlight the link between ovulation and selective PmF activation, and underscore the role of CTSL in this process under physiological conditions.

Remote actuation and on-demand activation of biomaterials pre-incorporated with physical cues for bone repair.

The high incidence of bone defect-related diseases caused by trauma, infection, and tumor resection has greatly stimulated research in the field of bone regeneration. Generally, bone healing is a long and complicated process wherein manipulating the biological activity of interventional scaffolds to support long-term bone regeneration is significant for treating bone-related diseases. It has been reported that some physical cues can act as growth factor substitutes to promote osteogenesis through continuous activation of endogenous signaling pathways. This review focuses on the latest progress in bone repair by remote actuation and on-demand activation of biomaterials pre-incorporated with physical cues (heat, electricity, and magnetism). As an alternative method to treat bone defects, physical cues show many advantages, including effectiveness, noninvasiveness, and remote manipulation. First, we introduce the impact of different physical cues on bone repair and potential internal regulatory mechanisms. Subsequently, biomaterials that mediate various physical cues in bone repair and their respective characteristics are summarized. Additionally, challenges are discussed, aiming to provide new insights and suggestions for developing intelligent biomaterials to treat bone defects and promote clinical translation.

68Ga-labeled fibroblast activation protein inhibitor (FAPI) PET/CT for locally advanced or recurrent pancreatic cancer staging and restaging after chemoradiotherapy.

Purpose: 68Ga-labeled fibroblast activation protein inhibitor (FAPI) is a novel PET tracer with great potential for staging pancreatic cancer. Data on locally advanced or recurrent disease is sparse, especially on tracer uptake before and after high dose chemoradiotherapy (CRT). The aim of this study was to evaluate [68Ga]Ga-FAPI-46 PET/CT staging in this setting. Methods: Twenty-seven patients with locally recurrent or locally advanced pancreatic adenocarcinoma (LRPAC n = 15, LAPAC n = 12) in stable disease or partial remission after chemotherapy underwent FAPI PET/CT and received consolidation CRT in stage M0 with follow-up FAPI PET/CT every three months until systemic progression. Quantitative PET parameters SUVmax, SUVmean, FAPI-derived tumor volume and total lesion FAPI-uptake were measured in baseline and follow-up PET/CT scans. Contrast-enhanced CT (ceCT) and PET/CT data were evaluated blinded and staged according to TNM classification. Results: FAPI PET/CT modified staging compared to ceCT alone in 23 of 27 patients in baseline, resulting in major treatment alterations in 52% of all patients (30%: target volume adjustment due to N downstaging, 15%: switch to palliative systemic chemotherapy only due to diffuse metastases, 7%: abortion of radiotherapy due to other reasons). Regarding follow-up scans, major treatment alterations after performing FAPI PET/CT were noted in eleven of 24 follow-up scans (46%) with switch to systemic chemotherapy or best supportive care due to M upstaging and ablative radiotherapy of distant lymph node and oligometastasis. Unexpectedly, in more than 90 % of the follow-up scans, radiotherapy did not induce local fibrosis related FAPI uptake. During the first follow-up, all quantitative PET metrics decreased, and irradiated lesions showed significantly lower FAPI uptake in locally controlled disease (SUVmax p = 0.047, SUVmean p = 0.0092) compared to local failure. Conclusion: Compared to ceCT, FAPI PET/CT led to major therapeutic alterations in patients with LRPAC and LAPAC prior to and after radiotherapy, which might help identify patients benefiting from adjustments in every treatment stage. FAPI PET/CT should be considered a useful diagnostic tool in LRPAC or LAPAC before and after CRT.

Cytosolic protein translation regulates cell asymmetry and function in early TCR activation of human CD8+ T lymphocytes.

Introduction: CD8+ cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide-MHC-I complex by the T-cell receptor (TCR) and co-stimulation. This cognate interaction promotes the organisation of intimate cell-cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell-cell contact, promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux, providing the necessary energy through oxidative phosphorylation, and its own protein translation on 70S ribosomes. However, the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Methods: Here, we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton, mitochondria, and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 70S by puromycin. Results: We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Discussion: Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs.

Expression and function of the major histocompatibility complex (MHC) class I chain-related A (MICA)*010 in NK cell killing activity.

The major histocompatibility complex (MHC) class I chain-related A (MICA) plays an important role in stress cell recognition. High polymorphisms of MICA are relevant to NKG2D binding capacity, responses of NK cells and tumor progression. In this study, MICA genotyping of 97 cholangiocarcinoma patients was performed using PCR-SSP. MICA*010 was positively associated with a corrected p-value of < 0.001 (RR=2.16 (95 % CI, 1.48-3.14)). MICA*010 was previously reported as a non-expressed allele. Thus, the expression of MICA*010 on the cell surface was studied on both MICA*010 transfected cells (HEK 293 T and L929 cells) and stimulated primary monocytes obtained from homozygous MICA*010 individuals using different clones of antibodies (1H10, 1D10, 1C3.1, 1C3.2, 6D4 and 3H5) for detection. Surprisingly, the expression of MICA*010 could be observed on both transfected cells and stimulated monocytes and effectively bound to the NKG2D-Fc fusion protein. The functional study of various MICA alleles revealed the high relative killing activity of NK cells induced by the MICA*010 transfected C1R cells, not following the previously reported rule of the M129V substitution. The structural analysis highlighted the amino acid at position 36 as another important amino acid relevant to preserving the structural integrity of the MICA protein and NKG2D binding. Our data propose a new aspect of functional MICA contributing motifs and that MICA*010 has a potential effect on NK cell functions and might be applicable to other fields of immune responses.

The History of Macrophage Activation Syndrome in Autoimmune Diseases.

In 1979, it became recognized in the literature that what we call hemophagocytic lymphohistiocytosis (HLH) was a nonmalignant disease of histiocytes. Subsequently a familial form and a secondary form of HLH were differentiated. When HLH is secondary to an autoimmune disease, rheumatologists refer to this entity as macrophage activation syndrome (MAS) to differentiate it from HLH itself. Although the first cases of MAS likely appeared in the literature in the 1970s, it was not until 1985 that the term activated macrophages was used to describe patients with systemic juvenile idiopathic arthritis (sJIA) complicated by MAS and the term macrophage activation syndrome first appeared in the title of a paper in 1993.MAS is one of the many types of secondary HLH and should not be confused with primary HLH. Experience has taught that MAS secondary to different autoimmune diseases is not equal. In the 30 years since initial description in patients with sJIA, the clinical spectrum, diseases associated with MAS, therapy, and understanding the pathogenesis have all made significant gains. The diagnostic/classification criteria for MAS secondary to sJIA, SLE, RA, and KD differ based on the different laboratory abnormalities associated with each (Ahn et al., J Rheumatol 44:996-1003, 2017; Han et al., Ann Rheum Dis 75:e44, 2016; Ravelli et al., Ann Rheum Dis 75:481-489, 2016; Borgia et al., Arthritis Rheumatol 70:616-624, 2018). These examples include the thrombocytosis associated with sJIA, a chronic generalized activation of the immune system, leading to elevations of fibrinogen and sIL-2R, low platelet count associated with SLE, and more acute inflammation associated with KD. Therefore, individual diagnostic criteria are required, and they all differ from the diagnostic criteria for HLH, which are based on a previously non-activated immune system (Ahn et al., J Rheumatol 44:996-1003, 2017; Han et al., Ann Rheum Dis 75:e44, 2016; Ravelli et al., Ann Rheum Dis 75:481-489, 2016; Borgia et al., Arthritis Rheumatol 70:616-624, 2018; Henter et al., Pediatr Blood Cancer 48:124-131, 2007). This helps to explain why the HLH diagnostic criteria do not perform well in MAS.The initial treatment remains high-dose steroids and IVIG followed by the use of a calcineurin inhibitor for resistant cases. IVIG can be used if there is a concern about malignancy to wait for appropriate investigations or with steroids. Interluekin-1 inhibition is now the next therapy if there is a failure to respond to steroids and calcineurin inhibitors. Advances in understanding the mechanisms leading to MAS, which has been greatly aided by the use of mouse models of MAS and advances in genome sequencing, offer a bright future for more specific therapies. More recent therapies are directed to specific cytokines involved in the pathogenesis of MAS and can lead to decreases in the morbidity and mortality associated with MAS. These include therapies directed to inhibiting the JAK/STAT pathway and/or specific cytokines, interleukin-18 and gamma interferon, which are currently being studied in MAS. These more specific therapies may obviate the need for nonspecific immunosuppressive therapies including high-dose prolonged steroids, calcineurin inhibitors, and etoposide.

Genetics of Macrophage Activation Syndrome in Systemic Juvenile Idiopathic Arthritis.

Macrophage activation syndrome (MAS) is a life-threatening episode of hyperinflammation driven by excessive activation and expansion of T cells (mainly CD8) and hemophagocytic macrophages producing proinflammatory cytokines. MAS has been reported in association with almost every rheumatic disease, but it is by far most common in systemic juvenile idiopathic arthritis (SJIA). Clinically, MAS is similar to familial or primary hemophagocytic lymphohistiocytosis (pHLH), a group of rare autosomal recessive disorders linked to various genetic defects all affecting the perforin-mediated cytolytic pathway employed by NK cells and cytotoxic CD8 T lymphocytes. Decreased cytolytic activity in pHLH patients leads to prolonged survival of target cells associated with increased production of proinflammatory cytokines that overstimulate macrophages. The resulting cytokine storm is believed to be responsible for the frequently fatal multiorgan system failure seen in MAS. Whole exome sequencing as well as targeted sequencing of pHLH-associated genes in patients with SJIA-associated MAS demonstrated increased "burden" of rare protein-altering variants affecting the cytolytic pathway compared to healthy controls, suggesting that as in pHLH, genetic variability in the cytolytic pathway contributes to MAS predisposition. Functional studies of some of the novel variants have shown that even in a heterozygous state, their presence partially reduces cytolytic activity that may lead to increased cytokine production.

Kawasaki Disease-Associated Cytokine Storm Syndrome.

Kawasaki disease (KD) is a hyperinflammatory syndrome manifesting as an acute systemic vasculitis characterized by fever, nonsuppurative conjunctival injection, rash, oral mucositis, extremity changes, and cervical lymphadenopathy. KD predominantly affects young children and shares clinical features and immunobiology with other hyperinflammation syndromes including systemic juvenile idiopathic arthritis (sJIA) and multisystem inflammatory syndrome in children (MIS-C). Cytokine storm syndrome (CSS) is an acute complication in ~2% of KD patients; however, the incidence is likely underestimated as many clinical and laboratory features of both diseases overlap. CSS should be entertained when a child with KD is unresponsive to IVIG therapy with recalcitrant fever. Early recognition and prompt institution of immunomodulatory treatment can substantially reduce the mortality and morbidity of CSS in KD. Given the known pathogenetic role of IL-1ß in both syndromes, the early use of IL-1 blockers in refractory KD with CSS deserves consideration.

Murine Models of Secondary Cytokine Storm Syndromes.

Hemophagocytic lymphohistiocytosis (HLH) comprises a broad spectrum of life-threatening cytokine storm syndromes, classified into primary (genetic) or secondary (acquired) HLH. The latter occurs in a variety of medical conditions, including infections, malignancies, autoimmune and autoinflammatory diseases, acquired immunodeficiency, and metabolic disorders. Despite recent advances in the field, the pathogenesis of secondary HLH remains incompletely understood. Considering the heterogeneity of triggering factors and underlying diseases in secondary HLH, a large diversity of animal models has been developed to explore pivotal disease mechanisms. To date, over 20 animal models have been described that each recapitulates certain aspects of secondary HLH. This review provides a comprehensive overview of the existing models, highlighting relevant findings, discussing the involvement of different cell types and cytokines in disease development and progression, and considering points of interest toward future therapeutic strategies.

Cytokine Storm and Sepsis-Induced Multiple Organ Dysfunction Syndrome.

There is extensive overlap of clinical features among familial or primary HLH (pHLH), reactive or secondary hemophagocytic lymphohistiocytosis (sHLH) [including macrophage activation syndrome (MAS) related to rheumatic diseases], and hyperferritinemic sepsis-induced multiple organ dysfunction syndrome (MODS); however, the distinctive pathobiology that causes hyperinflammatory process in each condition requires careful considerations for therapeutic decision-making. pHLH is defined by five or more of eight HLH-2004 criteria [1], where genetic impairment of natural killer (NK) cells or CD8+ cytolytic T cells results in interferon gamma (IFN-γ)-induced hyperinflammation regardless of triggering factors. Cytolytic treatments (e.g., etoposide) or anti-IFN-γ monoclonal antibody (emapalumab) has been effectively used to bridge the affected patients to hematopoietic stem cell transplant. Secondary forms of HLH also have normal NK cell number with decreased cytolytic function of varying degrees depending on the underlying and triggering factors. Although etoposide was uniformly used in sHLH/MAS in the past, the treatment strategy in different types of sHLH/MAS is increasingly streamlined to reflect the triggering/predisposing conditions, severity/progression, and comorbidities. Accordingly, in hyperferritinemic sepsis, the combination of hepatobiliary dysfunction (HBD) and disseminated intravascular coagulation (DIC) reflects reticuloendothelial system dysfunction and defines sepsis-associated MAS. It is demonstrated that as the innate immune response to infectious organism prolongs, it results in reduction in T cells and NK cells with subsequent lymphopenia even though normal cytolytic activity continues (Figs. 30.1, 30.2, 30.3, and 30.4). These changes allow free hemoglobin and pathogens to stimulate inflammasome activation in the absence of interferon-γ (IFN-γ) production that often responds to source control, intravenous immunoglobulin (IVIg), plasma exchange, and interleukin 1 receptor antagonist (IL-1Ra), similar to non-EBV, infection-induced HLH.

Etoposide Therapy of Cytokine Storm Syndromes.

Etoposide has revolutionized the treatment of primary as well as secondary hemophagocytic lymphohistiocytosis (HLH), and it is, together with corticosteroids, the most widely used therapy for HLH. In the early 1980s, long-term survival in primary HLH was <5% but with the etoposide-/dexamethasone-based protocols HLH-94 and HLH-2004, in combination with stem cell transplantation, 5-year survival increased dramatically to around 60% in primary HLH, and based on analyses from the HLH-2004 study, there is likely room for further improvement. Biologically, etoposide administration results in potent selective deletion of activated T cells as well as efficient suppression of inflammatory cytokine production. Moreover, etoposide has also been reported to promote programmed cell death (apoptosis) rather than proinflammatory lytic cell death (pyroptosis), conceivably ameliorating subsequent systemic inflammation, i.e., a treatment very suitable for cytokine storm syndromes (CSS). The combination of etoposide and corticosteroids may also be beneficial in cases of severe or refractory secondary HLH (sHLH) with imminent organ failure, such as infection-associated HLH caused by Epstein-Barr virus (EBV) or malignancy-triggered HLH. In CSS associated with rheumatic diseases (macrophage activation syndrome, MAS or MAS-HLH), etoposide is currently used as second- or third-line therapy. Recent studies suggest that etoposide perhaps should be part of an aggressive therapeutic intervention for patients with severe refractory or relapsing MAS, in particular if there is CNS involvement. Importantly, awareness of sHLH must be further increased since treatment of sHLH is often delayed, thereby missing the window of opportunity for a timely, effective, and potentially life-saving HLH-directed treatment.

Anti-Interferon-γ Therapy for Cytokine Storm Syndromes.

A vast body of evidence provides support to a central role of exaggerated production of interferon-γ (IFN-γ) in causing hypercytokinemia and signs and symptoms of hemophagocytic lymphohistiocytosis (HLH). In this chapter, we will describe briefly the roles of IFN-γ in innate and adaptive immunity and in host defense, summarize results from animal models of primary HLH and secondary HLH with particular emphasis on targeted therapeutic approaches, review data on biomarkers associated with activation of the IFN-γ pathway, and discuss initial efficacy and safety results of IFN-γ neutralization in humans.

IL-1 Family Blockade in Cytokine Storm Syndromes.

Interleukin-1 is a prototypic proinflammatory cytokine that is elevated in cytokine storm syndromes (CSSs), such as secondary hemophagocytic lymphohistiocytosis (sHLH) and macrophage activation syndrome (MAS). IL-1 has many pleotropic and redundant roles in both innate and adaptive immune responses. Blockade of IL-1 with recombinant human interleukin-1 receptor antagonist has shown efficacy in treating CSS. Recently, an IL-1 family member, IL-18, has been demonstrated to be contributory to CSS in autoinflammatory conditions, such as in inflammasomopathies (e.g., NLRC4 mutations). Anecdotally, recombinant IL-18 binding protein can be of benefit in treating IL-18-driven CSS. Lastly, another IL-1 family member, IL-33, has been postulated to contribute to CSS in an animal model of disease. Targeting of IL-1 and related cytokines holds promise in treating a variety of CSS.

Salvage Therapy and Allogeneic Hematopoietic Cell Transplantation for the Severe Cytokine Storm Syndrome of Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) can be considered as a severe cytokine storm syndrome disorder. HLH typically manifests as a life-threatening inflammatory syndrome characterized by fevers, cytopenias, hepatosplenomegaly, and various other accompanying manifestations such as coagulopathy, hepatitis or liver failure, seizures or altered mental status, and even multi-organ failure. Standard up-front treatments do not always bring HLH into remission or maintain adequate response, and salvage or alternative therapies are often needed. For patients with genetic diseases that cause HLH, curative allogeneic hematopoietic cell transplantation is usually offered to prevent future episodes of life-threatening HLH. Here, we will discuss the options and approaches for salvage therapy and hematopoietic cell transplantation for patients with HLH.

Hypoxia Reversion and STING Pathway Activation through Large Mesoporous Nanozyme for Near-Infrared-II Light Amplified Tumor Polymetallic-Immunotherapy.

cGAS/STING pathway, which is highly related to tumor hypoxia, is considered as a potential target for remodeling the immunosuppressive microenvironment of solid tumors. Metal ions, such as Mn2+, activate the cGAS/STING pathway, but their efficacy in cancer therapy is limited by insufficient effect on immunogenic tumor cell death of a single ion. Here, we evaluate the association between tumor hypoxia and cGAS/STING inhibition and report a polymetallic-immunotherapy strategy based on large mesoporous trimetal-based nanozyme (AuPdRh) coordinated with Mn2+ (Mn2+@AuPdRh) to activate cGAS/STING signaling for robust adaptive antitumor immunity. Specifically, the inherent CAT-like activity of this polymetallic Mn2+@AuPdRh nanozyme decomposes the endogenous H2O2 into O2 to relieve tumor hypoxia induced suppression of cGAS/STING signaling. Moreover, the Mn2+@AuPdRh nanozyme displays a potent near-infrared-II photothermal effect and strong POD-mimic activity; and the generated hyperthermia and •OH radicals synergistically trigger immunogenic cell death in tumors, releasing abundant dsDNA, while the delivered Mn2+ augments the sensitivity of cGAS to dsDNA and activates the cGAS-STING pathway, thereby triggering downstream immunostimulatory signals to kill primary and distant metastatic tumors. Our study demonstrates the potential of metal-based nanozyme for STING-mediated tumor polymetallic-immunotherapy and may inspire the development of more effective strategies for cancer immunotherapy.

Activated dormant stem cells recover spermatogenesis in chemoradiotherapy-induced infertility.

Male infertility is a recognized side effect of chemoradiotherapy. Extant spermatogonial stem cells (SSCs) may act as originators for any subsequent recovery. However, which type of SSCs, the mechanism by which they survive and resist toxicity, and how they act to restart spermatogenesis remain largely unknown. Here, we identify a small population of Set domain-containing protein 4 (Setd4)-expressing SSCs that occur in a relatively dormant state in the mouse seminiferous tubule. Extant beyond high-dose chemoradiotherapy, these cells then activate to recover spermatogenesis. Recovery fails when Setd4+ SSCs are deleted. Confirmed to be of fetal origin, these Setd4+ SSCs are shown to facilitate early testicular development and also contribute to steady-state spermatogenesis in adulthood. Upon activation, chromatin remodeling increases their genome-wide accessibility, enabling Notch1 and Aurora activation with corresponding silencing of p21 and p53. Here, Setd4+ SSCs are presented as the originators of both testicular development and spermatogenesis recovery in chemoradiotherapy-induced infertility.

Targeting undruggable phosphatase overcomes trastuzumab resistance by inhibiting multi-oncogenic kinases.

AIMS: Resistance to targeted therapy is one of the critical obstacles in cancer management. Resistance to trastuzumab frequently develops in the treatment for HER2+ cancers. The role of protein tyrosine phosphatases (PTPs) in trastuzumab resistance is not well understood. In this study, we aim to identify pivotal PTPs affecting trastuzumab resistance and devise a novel counteracting strategy. METHODS: Four public datasets were used to screen PTP candidates in relation to trastuzumab responsiveness in HER2+ breast cancer. Tyrosine kinase (TK) arrays were used to identify kinases that linked to protein tyrosine phosphate receptor type O (PTPRO)-enhanced trastuzumab sensitivity. The efficacy of small activating RNA (saRNA) in trastuzumab-conjugated silica nanoparticles was tested for PTPRO upregulation and resistance mitigation in cell models, a transgenic mouse model, and human cancer cell line-derived xenograft models. RESULTS: PTPRO was identified as the key PTP which influences trastuzumab responsiveness and patient survival. PTPRO de-phosphorated several TKs, including the previously overlooked substrate ERBB3, thereby inhibiting multiple oncogenic pathways associated with drug resistance. Notably, PTPRO, previously deemed "undruggable," was effectively upregulated by saRNA-loaded nanoparticles. The upregulated PTPRO simultaneously inhibited ERBB3, ERBB2, and downstream SRC signaling pathways, thereby counteracting trastuzumab resistance. CONCLUSIONS: Antibody-conjugated saRNA represents an innovative approach for targeting "undruggable" PTPs.

Deep learning ensemble approach with explainable AI for lung and colon cancer classification using advanced hyperparameter tuning.

Lung and colon cancers are leading contributors to cancer-related fatalities globally, distinguished by unique histopathological traits discernible through medical imaging. Effective classification of these cancers is critical for accurate diagnosis and treatment. This study addresses critical challenges in the diagnostic imaging of lung and colon cancers, which are among the leading causes of cancer-related deaths worldwide. Recognizing the limitations of existing diagnostic methods, which often suffer from overfitting and poor generalizability, our research introduces a novel deep learning framework that synergistically combines the Xception and MobileNet architectures. This innovative ensemble model aims to enhance feature extraction, improve model robustness, and reduce overfitting.Our methodology involves training the hybrid model on a comprehensive dataset of histopathological images, followed by validation against a balanced test set. The results demonstrate an impressive classification accuracy of 99.44%, with perfect precision and recall in identifying certain cancerous and non-cancerous tissues, marking a significant improvement over traditional approach.The practical implications of these findings are profound. By integrating Gradient-weighted Class Activation Mapping (Grad-CAM), the model offers enhanced interpretability, allowing clinicians to visualize the diagnostic reasoning process. This transparency is vital for clinical acceptance and enables more personalized, accurate treatment planning. Our study not only pushes the boundaries of medical imaging technology but also sets the stage for future research aimed at expanding these techniques to other types of cancer diagnostics.

Quantum chemical calculations of nitrosamine activation and deactivation pathways for carcinogenicity risk assessment.

N-nitrosamines and nitrosamine drug substance related impurities (NDSRIs) became a critical topic for the development and safety of small molecule medicines following the withdrawal of various pharmaceutical products from the market. To assess the mutagenic and carcinogenic potential of different N-nitrosamines lacking robust carcinogenicity data, several approaches are in use including the published carcinogenic potency categorization approach (CPCA), the Enhanced Ames Test (EAT), in vivo mutagenicity studies as well as read-across to analogue molecules with robust carcinogenicity data. We employ quantum chemical calculations as a pivotal tool providing insights into the likelihood of reactive ion formation and subsequent DNA alkylation for a selection of molecules including e.g., carcinogenic N-nitrosopiperazine (NPZ), N-nitrosopiperidine (NPIP), together with N-nitrosodimethylamine (NDMA) as well as non-carcinogenic N-nitrosomethyl-tert-butylamine (NTBA) and bis (butan-2-yl) (nitros)amine (BBNA). In addition, a series of nitroso-methylaminopyridines is compared side-by-side. We draw comparisons between calculated reaction profiles for structures representing motifs common to NDSRIs and those of confirmed carcinogenic and non-carcinogenic molecules with in vivo data from cancer bioassays. Furthermore, our approach enables insights into reactivity and relative stability of intermediate species that can be formed upon activation of several nitrosamines. Most notably, we reveal consistent differences between the free energy profiles of carcinogenic and non-carcinogenic molecules. For the former, the intermediate diazonium ions mostly react, kinetically controlled, to the more stable DNA adducts and less to the water adducts via transition-states of similar heights. Non-carcinogenic molecules yield stable carbocations as intermediates that, thermodynamically controlled, more likely form the statistically preferred water adducts. In conclusion, our data confirm that quantum chemical calculations can contribute to a weight of evidence approach for the risk assessment of nitrosamines.

Efficacy and safety evaluation of cross-reactive Fibroblast activation protein scFv-based CAR-T cells.

Introduction: Fibroblast activation protein (FAP) overexpression on cancer-associated fibroblasts (CAFs) is associated with poor prognosis and worse clinical outcomes. Selective ablation of pro-tumorgenic FAP+ stromal cells with CAR-T cells may be a new therapeutic strategy. However, the clinical use of FAP-CAR T cells is suggested to proceed with caution for occasional poor efficacy and induction of on-target off-tumor toxicity (OTOT), including lethal osteotoxicity and cachexia. Hence, more investigations and preclinical trials are required to optimize the FAP-CAR T cells and to approve their safety and efficacy. Methods: In this study, we designed second-generation CAR T cells targeting FAP with 4-1BB as a co-stimulatory molecule, and tested their cytotoxicity against FAP-positive cells (hFAP-HT1080 cells and a variety of primary CAFs) in vitro and in Cell line-derived xenograft (CDX) and a patient-derived xenograft (PDX) model. Results: Results showed that our FAP-CAR T cells were powerfully potent in killing human and murine FAP-positive tumor cells and CAFs in multiple types of tumors in BALB/c and C57BL/6 mice and in patient-derived xenografts (PDX) model. And they were proved to be biologically safe and exhibit low-level OTOT. Discussion: Taken together, the human/murine cross-reactive FAP-CAR T cells were powerfully potent in killing human and murine FAP positive tumor cells and CAFs. They were biologically safe and exhibit low-level OTOT, warranting further clinical investigation into our FAP-CAR T cells.