Augmented Response Research Articles

Effects of early treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) on the bronchoalveolar lavage proteome and oxylipids during bovine respiratory syncytial virus (BRSV) infection.

Non-steroidal anti-inflammatory drugs (NSAID) are not recommended for use against pneumonia in humans, but are commonly utilised against bovine respiratory disease. This study aimed to determine if the use of NSAIDs in the early phase of bovine respiratory syncytial virus (BRSV)-infection limits pulmonary inflammation. Four to nine-week old calves were infected with BRSV by aerosol and were treated with either meloxicam intravenously on day (D)4 (n = 5, MEL), acetylsalicylat-DL-lysin intravenously on D4 and D5 (n = 5, ASA), or were left untreated as controls (n = 5, CTR). Clinical signs were monitored daily until necropsy on D7, BRSV-RNA was detected in nasal swabs and bronchoalveolar lavage (BAL) by RT-qPCR, inflammatory cells and proteins were identified in BAL by cytology and label-free quantitative mass spectrometry-based proteomics, respectively, and oxylipids were quantified in BAL and plasma by liquid chromatography tandem mass spectrometry with triple quadrupole mass detectors. The calves developed mild to moderate signs of respiratory disease and, with the exception of one MEL-treated and one ASA-treated calf, limited lung lesions. None of the treatments had a significant effect on virus replication, clinical signs or lung lesion extent. Relative to controls, both treatments initially induced a downregulation of proteins in BAL. Immunoglobulin (Ig)-related proteins, such as the Ig kappa and lambda locus and the joining chain of IgA and IgM, were downregulated in MEL-treated calves compared to controls. In addition, meloxicam induced an increased neutrophil influx in BAL in response to BRSV, possibly related to a reduction in plasma prostaglandin, and to a downregulation of The Liver X Receptor/ Retinoid X Receptor (LXR/RXR), the Farnesoid X Receptor (FXR)/RXR and the 24-Dehydrocholesterol Reductase (DHC24) signalling pathways in the lung. The risk of NSAIDs to increase neutrophil activity during stimulation with BRSV or other toll-like receptor 4 agonists needs to be investigated further. Since augmented neutrophil responses can be detrimental, the results of the present study do not support the use of NSAIDs to prevent the clinical expression of BRSV-infection.

An optical fiber sensor based on a B10H14 derivatives/PMMA film for measuring low concentration formaldehyde in aqueous solutions.

The concentration of formaldehyde in the environment must be precisely monitored, as it is closely linked to human health. In this paper, a decaboryl derivative formaldehyde fluorescent probe (M1) was synthesized for the first time by introducing a 5-amino-isoquinoline group into a decaborane parent. Using theoretical calculations, 1H-NMR, 11B-NMR, HR-MS, and FT-IR, the molecular structure of the probe was determined and its response mechanism to formaldehyde was examined. The fluorescence response of the probe to formaldehyde was then tested, revealing an augmented response to formaldehyde in a solution of 0-600 μM, with a detection limit of 4.18 × 10-6 M. The results show that the formaldehyde fluorescence probe has the advantages of good linearity, strong anti-interference and high sensitivity. On this basis, a fiber optic formaldehyde fluorescence sensor based on an M1/PMMA thin film was constructed in this paper. This fiber optic fluorescence sensor, with its high selectivity, low detection limit, online and remote monitoring, and other advantages, was successfully applied to the detection of formaldehyde in both food and aqueous solutions, with results that were reliable compared to those of acetone. The detection limit of formaldehyde increased to 6.9 × 10-8 M. The potential for its utilization in the chemical, biological, environmental, and other formaldehyde detection fields is quite promising.

Insulin dysregulated horses metabolic responses to forage pellets

Hyperinsulinemia-associated laminitis (HAL) is the primary concern for insulin dysregulated (ID) equids and their insulin response to the consumption of oral, nonstructural carbohydrates (NSC) has been shown to be a risk predictor for HAL development. This randomized, crossover study's objective was to examine the insulinemic responses to 3 forage pellets (1 g/kg BW) (timothy hay, TH, 9.5 % CP & 10 % NSC DM; alfalfa hay, AH, 16.3 % CP & 9.8 % NSC DM; timothy-alfalfa hay; TAH, 17.2 % CP & 9.8 % NSC DM) along with a positive (dehulled oats; OG, 14.7 % CP & 59.7 % NSC DM) and negative dietary challenge control (low-NSC; LNSC, 12.8 % CP & 5.4 % NSC DM) of ID (n = 8; 16.1 ± 2.2 yr; 565.4 ± 99.1 kgs.) and non-ID (NID; n = 7; 17.0 ± 2.8 yr; 583.6 ± 57.9 kgs.) horses. ID horses had higher positive incremental area under the curve for insulin (IAUCi) (ID: 890 ± 925 µIU/mL*minute vs. NID: 225 ± 228 µIU/mL*minute), peak (ID: 101.5 ± 80.72 µIU/mL vs. NID: 25.7 ± 7.2 µIU/mL), and delta (ID: 45.5 ± 77.1 µIU/mL vs. NID: 4.9 ± 5.3 µIU/mL) insulin for all forage pellets compared to NID (p < 0.01). ID horses IAUCi for the forage pellets was not different compared to the LNSC (218 ± 327 µIU/mL*minute) but was different from OG (10,522 ± 4,565 µIU/mL*minute). ID horses’ lack of an augmented insulinemic response to the low NSC forage pellets (fed in small amounts) indicates that they could be a safe feedstuff for ID animals.

Visceral fat sympathectomy ameliorates systemic and local stress response related to chronic sleep restriction.

Disturbance of sleep homeostasis encompasses health issues, including metabolic disorders like obesity, diabetes, and augmented stress vulnerability. Sleep and stress interact bidirectionally to influence the central nervous system and metabolism. Murine models demonstrate that decreased sleep time is associated with an increased systemic stress response, characterized by endocrinal imbalance, including the elevated activity of hypothalamic-pituitary-adrenal axis, augmented insulin, and reduced adiponectin, affecting peripheral organs physiology, mainly the white adipose tissue (WAT). Within peripheral organs, a local stress response can also be activated by promoting the formation of corticosterone. This local amplifying glucocorticoid signaling is favored through the activation of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). In WAT, 11β-HSD1 activity is upregulated by the sympathetic nervous system, suggesting a link between sleep loss, augmented stress response, and a potential WAT metabolic disturbance. To gain more understanding about this relationship, metabolic and stress responses of WAT-sympathectomized rats were analyzed to identify the contribution of the autonomic nervous system to stress response-related metabolic disorders during chronic sleep restriction. Male Wistar rats under sleep restriction were allowed just 6 h of daily sleep over eight weeks. Results showed that rats under sleep restriction presented higher serum corticosterone, increased adipose tissue 11β-HSD1 activity, weight loss, decreased visceral fat, augmented adiponectin, lower leptin levels, glucose tolerance impairment, and mildly decreased daily body temperature. In contrast, sympathectomized rats under sleep restriction exhibited decreased stress response (lower serum corticosterone and 11β-HSD1 activity). In addition, they maintained weight loss, explained by a reduced visceral fat pad, leptin, and adiponectin, improved glucose management, and persisting decline in body temperature. These results suggest autonomic nervous system is partially responsible for the WAT-exacerbated stress response and its metabolic and physiological disturbances.

Influence of maternal immune activation on autism-like symptoms and coping strategies in male offspring

Maternal immune activation (MIA) caused by exposure to pathogens or inflammation during critical periods of gestation increased susceptibility to neurodevelopmental disorders, including autism, in the offspring. In the present work, we aimed to provide characterization of the long-term consequences on anxiety-like behavior and cardiovascular stress response of MIA in the offspring. This study aimed to evaluate the effect of MIA by lipopolysaccharide (LPS) in adult male offspring. In our study, the animals were subjected to a range of behavioral and physiological tests, including the elevated plus maze, social interaction, cat odor response, open field behavior, contextual fear conditioning, and cardiovascular responses during restraint stress. In the offspring of MIA, our study unveiled distinct anxious behaviors. This was evident by fewer entries into the open arms of the maze, diminished anti-thigmotaxis in the open field, and a decrease in social interaction time. Moreover, these rats showed heightened sensitivity to cat odor, exhibited prolonged freezing during fear conditioning, and presented elevated 22 Hz ultrasonic vocalizations. Notably, during restraint stress, these animals manifested an augmented blood pressure response, and this was associated with an increase in c-fos expression in the locus coeruleus compared to the control group. These findings collectively underline the extensive behavioral and physiological alterations stemming from MIA. This study deepens our understanding of the significance of maternal health in predisposing offspring to neurobehavioral deficits and psychiatric disorders.

Insights into the Unanticipated Chemical Reactivity of Functionalized Nanosheets Derived from TiB2.

Titanium diboride (TiB2) is a member of the AlB2-type layered metal boride family; the materials of this family are receiving renewed research interest owing to their amenability to nanoscaling. Earlier, we showed that TiB2 can be nanoscaled to yield quasi 2D nanostructures following a dissolution-recrystallization approach. This approach yielded nanosheets that were chemically functionalized with oxy-functional groups. Also, these nanosheets could inherently form a gel-like substance. In this work, we show that these functionalized nanosheets can interact with ascorbic acid in a way that first imparts a characteristic orange hue to the original yellowish nanosheet dispersion. Second, this interaction results in the loss of gel-like behavior of the nanosheet dispersion. We utilize several spectroscopic techniques such as UV-visible, FT-IR, NMR, EPR, XPS, and XANES to unravel this unexplored chemical interaction. The findings show that both titania as well as oxy-boron species react with ascorbic acid, leading to a profound modification of the nanosheets. This modification results in an augmented electrochemical response, implying that the modified nanosheets can be used in novel applications. This study is therefore a step toward gaining an even deeper understanding of the chemical opportunities that these nanoscaled borides can provide.

Molecular cloning, tissue expression pattern, responses to different fatty acids and potential functions of lysophosphatidylcholine acyltransferase 1 (LPCAT1) in large yellow croaker (Larimichthys crocea)

In farmed fish, diets rich in palm oil have been observed to promote abnormal lipid build-up in the liver, subsequently leading to physiological harm and disease onset. Emerging research suggests that integrating phospholipids into the feed could serve as a potent countermeasure against hepatic impairments induced by vegetable oil consumption. Phosphatidylcholine is the most abundant type among phospholipids. In the metabolic processes of mammal, lysophosphatidylcholine acyltransferase 1 (LPCAT1), crucial for phosphatidylcholine remodeling, demonstrates a marked affinity towards palmitic acid (PA). Nonetheless, aspects concerning the cloning, tissue-specific distribution, and affinity of the LPCAT1 gene to diverse oil sources have yet to be elucidated in the large yellow croaker (Larimichthys crocea). Within the scope of this study, we successfully isolated and cloned the cDNA of the LPCAT1 gene from the large yellow croaker. Subsequent analysis revealed distinct gene expression patterns of LPCAT1 across ten different tissues of the species. The fully sequenced coding DNA sequence (CDS) of LPCAT1 spans 1503 bp and encodes a sequence of 500 amino acids. Comparative sequence alignment indicates that LPCAT1 shares a 69.75 % amino acid similarity with its counterparts in other species. Although LPCAT1 manifests across various tissues of the large yellow croaker, its predominance is markedly evident in the liver and gills. Furthermore, post exposure of the large yellow croaker's hepatocytes to varied fatty acids, PA has a strong response to LPCAT1. Upon the addition of appropriate lysolecithin to palm oil feed, the mRNA expression of LPCAT1 in the liver cells of the large yellow croaker showed significant variations compared to other subtypes. Concurrently, the mRNA expression of pro-inflammatory genes il-1β, il-6, il-8, tnf-α and ifn-γ in the liver tissue of the large yellow croaker decreased. Interestingly, they exhibit the same trend of change. In conclusion, we have cloned the LPCAT1 gene on fish successfully and find the augmented gene response of LPCAT1 in hepatocytes under PA treatment first. The results of this study suggest that LPCAT1 may be associated with liver inflammation in fish and offer new insights into mitigating liver diseases in fish caused by palm oil feed.

P134: Immediate stress responses to music during psychomotor stimulation in 2 study cases with dementia.

Background:The use of music in older people with advanced dementia is possible because perception, sensitivity, emotion, and memory of music may remain intact after other types of memory disappear. Previous literature is controversial about stress biomarkers response to music introduction in therapy routines for people with severe cognitive impairment and neural-behavioural disorders. Particularly, for these patients, it is possible that they feel lower pleasure levels with music-based therapies.Objective:To characterize the immediate physiological effects of listening to music during psychomotor stimulation in an old participant with combined dementia and depression disorder and in a participant with a dementia diagnosis.Methods:Two study cases with dementia diagnosis participated in this study (P1: 84yrs; male Parkinson; FAB=9; P2: 85 yrs; female; Alzheimer; FAB=11; depression diagnosis) and were submitted to psychomotor stimulation (2 sessions). The first 20 min. of each session was dedicated to psychomotor stimulation without music (A), followed by 20 minutes with music (B). Heart rate was monitored (H10 Polar sensor) in a continuous mode. Cortisol levels were collected at the beginning of the session (T0) and then repeated at periods A and B (μg/dL). The range between minimum and maximum HR values (beats per minute- bpm) and mean values for cortisol levels were considered for the stress response analysis.Results:Salivary cortisol levels were higher at T0 for P1 (0.393 vs 0.203). During period A, the P1 slightly decreased their values (↓0,076) and P2 had no changes. After introducing music, both P1 and P2 increased cortisol levels (↑0,085; 0,162↑). For both P1 and P2, a wide range of HR was detected during period B (P1: 13 vs 23 bpm) vs (P2: 15 vs 41 bpm).Conclusion:Immediate responses to the music inclusion in a psychomotor intervention caused an augmented stress response in elderly participants with dementia, especially in P2. In specific, the depression diagnosis in this participant may be associated with a low capacity to handle emotions during new experiences, causing a higher stress response.

Abstract A025: Combination of anti-CTLA4 and radiation induce synergistic tumor control in radiorecurrent prostate cancer

Abstract Background: Advanced prostate cancer (PCa) is a leading cause in cancer death and can elicit significant morbidity and mortality. A common treatment modality for advanced PCa is radiation therapy (RT). Currently, salvage of local disease recurrence after RT is a major clinical problem. Emerging evidence indicates the importance of the immune system in governing RT response. In support of this, immune checkpoint inhibitors (ICIs), which enhance immune activation, have demonstrated clinical therapeutic promise in combination with RT in certain advanced cancers. Irradiation (IR) combined with ICIs may prime the immune system to recognize and target recurrent cancer. Purpose: Investigate the therapeutic efficacy of ICIs in combination with RT for radiorecurrent PCa in a syngeneic pre-clinical model. Methods: TRAMP-C2 cells were treated with 10 Gy of radiation over 5 fractions (similar to clinical hypofractionated (HF) schedule) to generate TRAMP-C2 HF cells. Immune-competent mice were transplanted subcutaneously with TRAMP-C2 HF cells. Once tumors reached palpable size, mice were administered either ICIs (αPDL1 or αCTLA4) alone or in combination with RT. Tumor volume was monitored to determine the treatment effect. Correlative studies on excised tumors and secondary lymphoid organs included flow cytometry and NanoString gene expression panel to evaluate immunological mechanisms that contribute to anti-tumor effects[SL1]. Results: TRAMP-C2 HF cells were validated for radiation resistance and exhibited a more aggressive phenotype (reduction in senescence, increase in clonogenicity) similar to clinically recurrent PCa. Radiation resistance of TRAMP-C2 HF tumors was validated in vivo. Administration of αPDL1 and αCTLA4 as monotherapy or in combination did not achieve significant tumor growth delay compared to control. IR alone produced an observable tumor growth delay compared to ICIs alone. The combination of αPDL1 and IR did not yield additional growth delay compared to IR alone. Strikingly, significant tumor growth delay was seen with the combination of IR and αCTLA4 compared to IR alone, while also resulting in complete cure in a third of the mice. NanoString analysis of lymph nodes from mice treated with αCTLA4 and IR vs. IR alone demonstrated differential expression of genes regulating CD molecules, interleukins, and T-cell functions. Furthermore, enrichment of CD45 and CD8a RNA following αCTLA4 and IR was observed. Validation by flow cytometry is currently ongoing. Conclusion: We generated the first syngeneic radiorecurrent PCa model and demonstrated that combining αCTLA4 and IR results in synergistic tumor response. Combined therapy resulted in augmented immune response, most notably enhancement of CD8 T cell activity. Significance: These findings contribute to our understanding of immunological events associated with RT and ICIs in the context of radiorecurrent PCa and support new avenues for salvage therapy in clinical trials. Citation Format: Hanzhi Wang, Xiaoyong Huang, Stepahnie D White, Linsey Gong, Tera N Petchiny, Hans Chung, Emmanouil Fokas, Robert S Kerbel, Stanley K Liu. Combination of anti-CTLA4 and radiation induce synergistic tumor control in radiorecurrent prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A025.

Mitochondrial-targeted antioxidant attenuates preeclampsia-like phenotypes induced by syncytiotrophoblast-specific Gαq signaling.

Syncytiotrophoblast stress is theorized to drive development of preeclampsia, but its molecular causes and consequences remain largely undefined. Multiple hormones implicated in preeclampsia signal via the Gαq cascade, leading to the hypothesis that excess Gαq signaling within the syncytiotrophoblast may contribute. First, we present data supporting increased Gαq signaling and antioxidant responses within villous and syncytiotrophoblast samples of human preeclamptic placenta. Second, Gαq was activated in mouse placenta using Cre-lox and DREADD methodologies. Syncytiotrophoblast-restricted Gαq activation caused hypertension, kidney damage, proteinuria, elevated circulating proinflammatory factors, decreased placental vascularization, diminished spiral artery diameter, and augmented responses to mitochondrial-derived superoxide. Administration of the mitochondrial-targeted antioxidant Mitoquinone attenuated maternal proteinuria, lowered circulating inflammatory and anti-angiogenic mediators, and maintained placental vascularization. These data demonstrate a causal relationship between syncytiotrophoblast stress and the development of preeclampsia and identify elevated Gαq signaling and mitochondrial reactive oxygen species as a cause of this stress.

Response to salinity stress in four Olea europaea L. genotypes: A multidisciplinary approach

Despite a drought- and erosion-tolerant root system, olive trees are vulnerable to abiotic stress due to limited genetic variability. Though some olive cultivars are moderately tolerant to salinity stress, soil salinity is increasing in the semi-arid and arid regions where olive cultivation is common, significantly reducing overall production. In response, breeding programs may rely on proper selection markers for abiotic stresses, including salinity, but these are generally lacking for olive. Here, physiological and biochemical parameters were measured in four Olea europaea genotypes (Frantoio, Leccino, Lecciana, and Oliana) subjected to different intensities of salinity stress (0 mM, 100 mM and 200 mM NaCl). At moderate and high salt concentrations, Na+ exclusion, higher photosynthetic productivity and tissue water content in the tolerant cultivar Frantoio were linked with increased production of polyphenols, with more favorable K+/Na+ values (quercetin and rutin), mitigation of oxidative stress (oleuropein) and increased water absorption (luteolin). In Frantoio and Leccino, a significant change of the proteome repertoire occurred, with overrepresentation of components regulating cellular metabolism, ion transport, redox insult and dissipation of excess photochemical energy. Conversely, Lecciana and Oliana showed increased sensitivity to salinity stress in terms of photosynthetic parameters and elevated internal Na+ concentrations, together with the lowest number of differentially represented proteins. These results highlighted olive germplasm strategies to cope with osmotic stress, suggested a physiological and molecular basis for the augmented responsiveness of tolerant cultivars and identified specific biomarkers as useful targets for future breeding programs.

Abstract C080: Ultra-high concentration nitric oxide (UNO) enhances anti-CTLA-4 treatment activity and induces a durable anti-tumor immune response

Abstract Background: CTLA-4 blockade is a highly effective therapeutic modality but only in a subset of cancer patients. We previously demonstrated that intratumoral administration of ultra-high concentration nitric oxide (UNO) augmented response to the immune checkpoint inhibitor, anti-mPD-1. Specifically, more than half of the mice treated with 10-minutes UNO (50,000 ppm) and anti-mPD-1 were primary and secondary tumor-free at Day 100 post-UNO treatment (vs 25% of control mice treated with anti-mPD-1 alone). In addition, the combination of UNO and anti-mCTLA-4 assessed in an immunocompetent breast cancer model (4T1) demonstrated prolonged survival in mice compared to single-agent anti-mCTLA-4. In CT26 and 4T1 mice, the immunodominant AH-1 antigen is associated with anti-tumor immune response. In this study, we assessed the systemic levels of CD8+ T-cells recognizing the AH-1 antigen following UNO treatment and the association with anti-tumor response. Methods: Here we explored the effects of UNO treatment in combination with anti-mCTLA-4 therapy. Colon carcinoma (CT26) tumor-bearing mice were treated with intra-tumoral 100,000 ppm UNO for 5 minutes in combination with 5 mg/kg (IP) anti-mCTLA-4. Effects on tumor growth and anti-tumor immune response were assessed. AH-1 specific CD8+ T cells were quantified by flow cytometry. Results: Combining 100,000 ppm UNO with anti-mCTLA4 in CT26 tumor-bearing mice resulted in considerable growth delay and regression of treated tumors. Furthermore, the combination induced a strong systemic response that led to the rejection of a distant metastasis-like tumor inoculated on the contralateral flank. Specifically, at Day 7 post-treatment, all UNO treated mice exhibited tumor-specific CD8+ T-cells in the blood and to a higher degree than in untreated mice. Moreover, systemic levels of tumor-specific CD8+ T-cells were higher in mice treated with the combination of UNO and anti-mCTLA4 compared to mice treated with monotherapy anti-mCTLA-4, indicating an increase in immune surveillance following UNO treatment. Finally, cured mice treated with combination therapy displayed higher systemic levels of antigen-specific CD8+ T-cells compared to untreated or naïve mice. Conclusion: UNO stimulates a higher tumor-specific T-cell response than anti-mCTLA-4 and synergizes with this drug to generate an even more significant targeted immune response. Citation Format: Yogev Sela, Matan Goldshtein, Jedidiah M. Monson, Hila Confino, Amir Avniel, Mark D. Pegram, Selena Chaisson. Ultra-high concentration nitric oxide (UNO) enhances anti-CTLA-4 treatment activity and induces a durable anti-tumor immune response [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C080.

Phenotype Molding of Monocytes in the Multiple Myeloma Microenvironment

Human monocytes are mononuclear phagocytes that comprise a major population of innate immune cells. Various monocyte-derived cells, including osteoclasts, dendritic cells, and macrophages, have been implicated in immunosuppression within the multiple myeloma (MM) tumor microenvironment (TME). Despite recent developments in understanding the link between monocyte-derived cells and immunosuppression in myeloma, there are still critical knowledge gaps in the association between monocytes themselves and immunosuppression within the context of MM. Specifically, the functional and developmental alterations of monocytes in the MM TME have not been explored based on the single-cell studies. In the present study, we generated a comprehensive single-cell map of peripheral blood (PB) and bone marrow (BM) monocytes from both healthy donors (HDs) and MM patients. Specifically, Heparin-anticoagulated PB samples or BM aspirates were collected from seven newly diagnosed MM (NDMM) patients and healthy donors (HDs). Paired BM aspirates were obtained from 10 MM patients at the time of diagnosis and after induction therapy. Differential expression analysis showed that type I interferon (IFN) response was upregulated in MM monocytes. And BM monocytes derived from MM patients exhibited impaired T cell attraction abilities and demonstrated an elevated capacity for suppressing T cell function. Moreover, immune checkpoint molecules, including CD274 (encoding PD-L1) and PDCD1LG2 (encoding PD-L2), were significantly upregulated in MM monocytes. Additionally, a positive correlation was observed between type I IFN response and T cell suppression capacity of MM monocytes. Trajectory analyses revealed an IFN-focused disruption of developmental pathways of both BM and PB monocytes in MM. Lastly, we included 10 MM patients at the time of diagnosis and after induction therapy to monitor transcriptional changes over time. Our results revealed that anti-tumor therapy successfully mitigated the excessive type-I interferon response of BM monocytes in MM. We document that MM monocytes exhibited an augmented type-I IFN response compared to monocytes from HDs, while their overall heterogeneity remained unchanged. Furthermore, we extended the knowledge on immunosuppression of the MM TME that the excessive type-I IFN response in MM had a significant impact on the differentiation of both BM and PB monocytes, leading to T-cell immunosuppression, specifically in BM monocytes. In the cohort that we longitudinally followed, we observed a decrease in the type-I IFN response across nearly all monocyte subsets in MM BM monocytes after induction therapy, accompanied by a reduction in the expression of the T-cell suppression gene set. In sum, our findings indicates that monocytes in the BM promotes the immunosuppression of the MM TME and represents potential therapeutic targets for modulating the MM TME.

Clonal Hematopoiesis Is Associated with Severe COVID-19 Enhancing Inflammatory Responses in Myeloid Cells

Background Clonal hematopoiesis (CH) is prevalent among aged populations and highlighted because its association with not only hematological malignancies but various benign diseases (e.g. cardiovascular diseases). Of particular interest among these diseases is COVID-19. To date, the association between severe COVID-19 and CH-related gene mutations or mosaic chromosomal alterations (mCAs) was examined using biobank samples taken long before the pandemic and the association has not been tested in a sufficiently large cohort of patients on the basis of CH evaluated at the time of COVID-19 diagnosis. Moreover, no study examined the joint effect of gene mutations and mCAs. The mechanism of the association between severe COVID-19 and CH has not been addressed either. Methods To address these issues we enrolled a total of 4,541 patients with COVID-19, of whom 54% had a severe infection. We performed targeted-capture sequencing of 42 CH-related genes and SNP-array analysis to detect CH-related mutations and mCAs. We also performed RNA-seq (n=987) of whole blood samples and plasma proteomic analysis (n=1,200) using the Olink technology to investigate the effect of biological consequence of CH, which was applied to understand the underlying mechanism of sever COVID-19. Results We identified gene mutations and mCAs in 16.3% and 6.2% of COVID-19 patients, respectively. They coexisted in 2.4% patients. As reported in CH in normal individuals, most frequent alterations included mutations in DNMT3A (7.6%), TET2 (4.1%), ASXL1 (1.4%), PPM1D (1.7%), and mCAs such as 14qUPD, and del(20q). To evaluate the risk of severe COVID-19 conferred by CH, we compared the age-stratified frequencies of CH between severe and non-severe COVID-19 patients (Figure, a). Focusing on CH in ≧10% clonal fractions, CH was significantly enriched in severe COVID-19 in elderly patients, we detected a significant enrichment of cases with both gene mutations and mCAs and isolated gene mutations, but not that of isolated mCAs, in severe cases. Multivariable logistic regression analysis with adjustment for age and sex revealed the significant association of CH defined either CH-mutations or mCA with severe COVID-19 with an odds ratio (OR) of 1.34 [95%CI=1.03-1.84] in patients aged ≥65 years. Particularly, CH defined by combined gene mutations and mCAs conferred a remarkable increase in the risk of severe COVID-19 [OR=12.1, 95%CI=1.61-90.6], suggesting the presence of their synergistic impact on severe COVID-19. To elucidate the underlying mechanism of the observed association between severe COVID-19 and CH, we next evaluated the transcriptomic/proteomic features of severe COVID-19 and CH (Figure, b). In severe COVID-19, multiple inflammatory pathways including IFN-α/γ, TNF-α, and IL-6 responses were consistently up-regulated in both transcriptomic and proteomic analysis. Similar to these features of severe COVID-19, CH was also associated with inflammatory response and IFN-α/γ responses in RNA expression while the impact of CH on protein expression was not prominent. To further clarify the transcriptomic features of severe COVID-19 and CH, we performed deconvolution analysis of gene expression to estimate the fraction of each cell component and cell-type-specific expression profiles. Severe COVID-19 was characterized by elevated fractions of neutrophils, activated dendritic cells (DCs), and decreased fractions of several types of lymphocytes, while only a marginal increase of activated DCs was observed in CH. Cell-type-specific pathway analysis revealed up-regulations of inflammatory responses and IL-6/TNF-a signaling in neutrophils/monocytes were associated with both severe COVID-19 and CH independently, suggesting that CH might be involved in the development of severe COVID-19 through the augmentation of inflammatory responses in these myeloid cells, which are further augmented by increased neutrophils induced by COVID-19 independent of CH. Conclusion CH was significantly associated with an increased risk of severe COVID-19, particularly when the patients had both gene mutations and mCAs, where the augmented inflammatory responses by myeloid cells can be a mechanism of severe COVID-19 risk conferred by CH. Our findings highlight the link between the mechanism of severe COVID-19 and the biological effects of CH.

Sitagliptin Induces Tolerogenic Human Dendritic Cells

Sitagliptin, an anti-diabetic drug, is a dipeptidyl peptidase (DPP)-4/CD26 inhibitor with additional anti-inflammatory and immunomodulatory properties. In this study, we investigated for the first time the effect of sitagliptin on the differentiation and functions of human dendritic cells generated from monocytes (MoDCs) for 4 days using the standard GM-CSF/IL-4 procedure. LPS/IFN-γ treatment for an additional 24 h was used for maturation induction of MoDCs. Sitagliptin was added at the highest non-cytotoxic concentration (500 µg/mL) either at the beginning (sita 0d protocol) or after MoDC differentiation (sita 4d protocol). Sitagliptin impaired differentiation and maturation of MoDCs as judged with the lower expression of CD40, CD83, CD86, NLRP3, and HLA-DR, retention of CD14 expression, and inhibited production of IL-β, IL-12p70, IL-23, and IL-27. In contrast, the expression of CD26, tolerogenic DC markers (ILT4 and IDO1), and production of immunoregulatory cytokines (IL-10 and TGF-β) were increased. Generally, the sita 0d protocol was more efficient. Sitagliptin-treated MoDCs were poorer allostimulators of T-cells in MoDC/T-cell co-culture and inhibited Th1 and Th17 but augmented Th2 and Treg responses. Tolerogenic properties of sitagliptin-treated MoDCs were additionally confirmed by an increased frequency of CD4+CD25+CD127- FoxP3+ Tregs and Tr1 cells (CD4+IL-10+FoxP3-) in MoDC/T-cell co-culture. The differentiation of IL-10+ and TGF-β+ Tregs depended on the sitagliptin protocol used. A Western blot analysis showed that sitagliptin inhibited p65 expression of NF-kB and p38MAPK during the maturation of MoDCs. In conclusion, sitagliptin induces differentiation of tolerogenic DCs, and the effect is important when considering sitagliptin for treating autoimmune diseases and allotransplant rejection.

Heterotypic Stressors Unmask Behavioral Influences of PMAT Deficiency in Mice.

Certain life stressors having enduring physiological and behavioral consequences, in part by eliciting dramatic signaling shifts in monoamine neurotransmitters. High monoamine levels can overwhelm selective transporters like the serotonin transporter. This is when polyspecific transporters like plasma membrane monoamine transporter (PMAT, Slc29a4) are hypothesized to contribute most to monoaminergic signaling regulation. Here, we employed two distinct counterbalanced stressors-fear conditioning and swim stress-in mice to systematically determine how reductions in PMAT function affect heterotypic stressor responsivity. We hypothesized that male heterozygotes would exhibit augmented stressor responses relative to female heterozygotes. Decreased PMAT function enhanced context fear expression, an effect unexpectedly obscured by a sham stress condition. Impaired cued fear extinction retention and enhanced context fear expression in males were conversely unmasked by a sham swim condition. Abrogated corticosterone levels in male heterozygotes that underwent swim stress after context fear conditioning did not map onto any measured behaviors. In sum, male heterozygous mouse fear behaviors proved malleable in response to preceding stressor or sham stress exposure. Combined, these data indicate that reduced male PMAT function elicits a form of stress-responsive plasticity. Future studies should assess how PMAT is differentially affected across sexes and identify downstream consequences of the stress-shifted corticosterone dynamics.

Reinforced antimyeloma therapy via dual-lymphoid activation mediated by a panel of antibodies armed with bridging-BiTE

AbstractImmunotherapy using bispecific antibodies including bispecific T-cell engager (BiTE) has the potential to enhance the efficacy of treatment for relapsed/refractory multiple myeloma. However, myeloma may still recur after treatment because of downregulation of a target antigen and/or myeloma cell heterogeneity. To strengthen immunotherapy for myeloma while overcoming its characteristics, we have newly developed a BiTE-based modality, referred to as bridging-BiTE (B-BiTE). B-BiTE was able to bind to both a human immunoglobulin G–Fc domain and the CD3 molecule. Clinically available monoclonal antibodies (mAbs) were bound with B-BiTE before administration, and the mAb/B-BiTE complex induced antitumor T-cell responses successfully while preserving and supporting natural killer cell reactivity, resulting in enhanced antimyeloma effects via dual-lymphoid activation. In contrast, any unwanted off-target immune-cell reactivity mediated by mAb/B-BiTE complexes or B-BiTE itself appeared not to be observed in vitro and in vivo. Importantly, sequential immunotherapy using 2 different mAb/B-BiTE complexes appeared to circumvent myeloma cell antigen escape, and further augmented immune responses to myeloma relative to those induced by mAb/B-BiTE monotherapy or sequential therapy with 2 mAbs in the absence of B-BiTE. Therefore, this modality facilitates easy and prompt generation of a broad panel of bispecific antibodies that can induce deep and durable antitumor responses in the presence of clinically available mAbs, supporting further advancement of reinforced immunotherapy for multiple myeloma and other refractory hematologic malignancies.

Identifying potential immuno-oncology targets in salivary gland mucoepidermoid carcinoma based on inflammatory status and treatment response.

Mucoepidermoid carcinoma is a rare salivary gland malignant tumour. This study aimed to investigate inflammatory and immune signatures of mucoepidermoid carcinoma by identifying potential proteo-transcriptomic biomarkers towards the development of precision immuno-oncology treatment strategies. A total of 30 biopsies obtained from patients diagnosed with mucoepidermoid carcinoma between 2013 and 2022 were analysed after H&E staining for scoring of histological inflammatory stroma subtypes and inflammatory hotspots with QuPath. Multiplex immunofluorescence staining and NanoString nCounter PanCancer IO 360™ panel were used to assess stroma and tumour inflammation signatures in high grade mucoepidermoid carcinoma cases in the tumour microenvironment via proteomics and transcriptomics, respectively. Inflammatory cells within the histological inflammatory stroma inflammatory (HIS-INF/hot) tumour neighbourhoods were greater compared to the histological inflammatory stroma-immune desert (HIS-ID/cold) (p = 0.001). A similar trend was observed between treatment non-responders and responders in stroma neighbourhoods (p = 0.0625) and in stroma-to-interface inflammatory hotspots (p = 0.0081), indicating an augmented inflammatory response in hot tumours and non-responders. Furthermore, there were striking differences in the expression of pan-immune leukocyte marker CD45 between responders and non responders particularly in the tumour neighbourhoods (p = 0.0341), but such were not robust for PD-1 and macrophage fractions. Additionally, transcriptomic analysis revealed key differences in leukocyte activation profiles between responders and non-responders. This preliminary report unveils the importance of assessing immune leukocyte cellular fractions and pathways for future prognostic biomarker discoveries in mucoepidermoid carcinoma as per the involvement of CD45-driven inflammatory and immune mediators in high grade mucoepidermoid carcinoma in non-responders to treatment. These findings will potentially contribute to the development of novel personalised immunotherapies.

Subgingival titanium wire implantation induces weak inflammatory responses but does not promote substantial T cell activation.

Titanium is a biocompatible material commonly used for dental treatments. However, the detailed mechanism underlying the weak biological activity of titanium has not been elucidated. We investigated both the inflammatory responses and T cell activation induced by solid titanium in the gingiva in mice. Both titanium and nickel wire implantation promoted neutrophil infiltration into the gingiva on day 2. Nickel, but not titanium, wire implantation enhanced proinflammatory cytokine expression and dendritic cell activity in gingival tissue by day 2. Nickel wire implantation enhanced the activity of T cells in draining lymph nodes on day 5. Moreover, T cell and neutrophil infiltration and elevated proinflammatory cytokine expression in the gingival tissue were still observed on day 5. However, no such augmented biological responses were observed after titanium wire implantation. These findings suggest that, unlike nickel, solid titanium does not induce sufficient inflammatory responses leading to T cell activation in gingival tissue.

Brahma-related gene 1 acts as a profibrotic mediator and targeting it by micheliolide ameliorates peritoneal fibrosis

BackgroundProgressive peritoneal fibrosis is a worldwide public health concern impacting patients undergoing peritoneal dialysis (PD), yet there is no effective treatment. Our previous study revealed that a novel compound, micheliolide (MCL) inhibited peritoneal fibrosis in mice. However, its mechanism remains unclear. Brahma-related gene 1 (BRG1) is a key contributor to organ fibrosis, but its potential function in PD-related peritoneal fibrosis and the relationship between MCL and BRG1 remain unknown.MethodsThe effects of MCL on BRG1-induced fibrotic responses and TGF-β1-Smads pathway were examined in a mouse PD model and in vitro peritoneal mesothelial cells. To investigate the targeting mechanism of MCL on BRG1, coimmunoprecipitation, MCL-biotin pulldown, molecular docking and cellular thermal shift assay were performed.ResultsBRG1 was markedly elevated in a mouse PD model and in peritoneal mesothelial cells cultured in TGF-β1 or PD fluid condition. BRG1 overexpression in vitro augmented fibrotic responses and promoted TGF-β1-increased-phosphorylation of Smad2 and Smad3. Meanwhile, knockdown of BRG1 diminished TGF-β1-induced fibrotic responses and blocked TGF-β1-Smad2/3 pathway. MCL ameliorated BRG1 overexpression-induced peritoneal fibrosis and impeded TGF-β1-Smad2/3 signaling pathway both in a mouse PD model and in vitro. Mechanically, MCL impeded BRG1 from recognizing and attaching to histone H3 lysine 14 acetylation by binding to the asparagine (N1540) of BRG1, in thus restraining fibrotic responses and TGF-β1-Smad2/3 signaling pathway. After the mutation of N1540 to alanine (N1540A), MCL was unable to bind to BRG1 and thus, unsuccessful in suppressing BRG1-induced fibrotic responses and TGF-β1-Smad2/3 signaling pathway.ConclusionOur research indicates that BRG1 may be a crucial mediator in peritoneal fibrosis and MCL targeting N1540 residue of BRG1 may be a novel therapeutic strategy to combat PD-related peritoneal fibrosis.