Research
Potentiation of fentanyl-induced respiratory depression by alcohol is not fully reversed by naloxone
Abstract
The high frequency of opioid overdose deaths often involves co-use of alcohol, which is reported in approximately 30% of fentanyl fatalities. Both substances depress respiratory function, and their combined effects can be lethal. The present study investigated physiological parameters of respiratory-depressant effects of fentanyl when co-administered with alcohol and its sensitivity to naloxone reversal using whole-body plethysmography in male and female Long-Evans rats. Administration of a high, sedative-like dose of alcohol alone or fentanyl alone resulted in no mortality, but fentanyl+alcohol led to mortality rates of 42% and 33% in females and males, respectively. The fentanyl+alcohol combination reduced minute ventilation and increased apneic pauses compared with either drug alone. Lower, binge-like alcohol doses, when combined with fentanyl, also amplified respiratory depression. Pretreatment with naloxone did not fully restore normal respiration. Naloxone administered after fentanyl+alcohol transiently reversed the decrease in minute ventilation but did not reverse apneic pauses. Fentanyl-dependent rats were partially tolerant to fentanyl- and fentanyl+alcohol-induced respiratory depression, but alcohol-dependent rats exhibited sensitization to alcohol- and fentanyl+alcohol-induced apnea. These findings highlight physiological parameters of severe respiratory risks with fentanyl+alcohol co-use, which are inadequately reversed by naloxone, underscoring the need for targeted strategies to manage opioid+alcohol overdoses.
Authors
Emma V. Frye, Lyndsay E. Hastings, Aniah N. Matthews, Adriana Gregory-Flores, Janaina C.M. Vendruscolo, Lindsay A. Kryszak, Shelley N Jackson, Aidan J. Hampson, Nora D. Volkow, Leandro F. Vendruscolo, Renata C.N. Marchette, George F. Koob
Abstract
We hypothesized that performing bone marrow transplant (BMT) using marrow extracted from the vertebral bodies (VB) of an unrelated deceased lung transplant (LTX) donor would be able to establish persistent hematopoiesis, generate immunity, and tolerance. A teenager with severe combined immunodeficiency with lung failure due to recurrent pneumonias underwent LTX in 2016 from a 1/8 HLA allele-matched unrelated donor, followed by BMT 4 months later using T-cell/B-cell-depleted, cryopreserved VB marrow. Rapid engraftment was followed by accelerating immune competence at 6 months, with independence from immunosuppression by 16 months. Donor T-cell (>95%) and myeloid chimerism (7-10%) have persisted for over nine years. At two years post-BMT, circulating T cells were hyporesponsive to host dendritic cells in vitro. T-cell receptor clonotyping revealed the disappearance of host-reactive clones, and T-cell RNA-sequencing exhibited downmodulated signaling pathways for cytotoxicity/rejection, paired with upregulated immunomodulatory pathways, suggesting active suppression. In parallel, host monocytes upregulated certain signaling pathways, indicating active interactions between post-thymic donor T cells and host monocytes. In summary, durable hematopoietic engraftment, immunity, and tolerance were demonstrable for the first time in a recipient of BMT obtained from VB graft.
Authors
Paul Szabolcs, Xiaohua Chen, Marian G. Michaels, Memphis Hill, Evelyn Garchar, Zarreen Amin, Heather M. Stanczak, Shawna McIntyre, Aleksandra Petrovic, Dhivyaa Rajasundaram, Ansuman Chattopadhyay, Jonathan E. Spahr, Peter D. Wearden, Geoffrey Kurland
Abstract
Mutations in LMNA, encoding nuclear lamina protein Lamin A/C, cause premature aging disorders, most notably Hutchinson-Gilford Progeria Syndrome. Despite obvious skull abnormalities in progeroid patients, the disease-causing mechanism remains elusive. The L648R single amino acid substitution blocks prelamin A maturation in mice, modeling a unique human patient. Here, we describe skull deformities in premature aging caused by aberrant suture fusion resembling those of patients with craniosynostosis. Further examinations identify prelamin A accumulation causatively linked to multiple suture synostoses in low bone density. This etiology is distinct from conventional suture fusion mediated by excessive ossification. In addition, the mutation disrupts skeletal stem cell stemness and subsequent stem cell-mediated proliferation and differentiation in osteogenesis. Intrasutural bones present in progeroid patients are highly reminiscent of synostosis caused by stem cell exhaustion. Comparative gene expression profiling further reveals cytoskeletal dynamics associated with skeletogenic cell aging and suture patency in mice and humans. Functional studies demonstrate that abnormal structures of progeric nuclei caused by prelamin A accumulation affect cytoskeleton organization and nucleoskeleton assembly essential for craniofacial skeletogenesis. Pharmacogenetic analyses indicate alleviation of osteogenic defects via actin polymerization. Our findings provide compelling evidence for nuclear and cytoskeletal defects, mediating stem cell-associated osteogenic deformities in progeroid disorders.
Authors
Kai Li, Trunee Hsu, Hitoshi Uchida, Tingxi Wu, Susan Michaelis, Howard J. Worman, Wei Hsu
Abstract
Lung development relies on diverse cell intrinsic and extrinsic mechanisms to ensure proper cellular differentiation and compartmentalization. In addition, it requires precise integration of multiple signaling pathways to temporally regulate morphogenesis and appropriate cell specification. To accomplish this, organogenesis relies on epigenetic and transcriptional regulators to promote cell fate and inhibit alternative cell fates. Using genetic mouse and human embryonic stem cell (hESC) differentiation models, tissue explants, and single-cell transcriptomic analysis, we demonstrated that Bromodomain Containing Protein 4 (BRD4) is required for mammalian lung morphogenesis and cell fate. Endodermal deletion of BRD4 impaired epithelial-mesenchymal crosstalk, leading to disrupted proximal-distal patterning and branching morphogenesis. Moreover, temporal deletion of BRD4 revealed developmental stage-specific defects in airway and alveolar epithelial cell specification with a predominant role in proximal airway cell fate. Similarly, BRD4 promoted lung endodermal cell differentiation into airway lineages in a hESC-derived lung organoid model. Together, these data demonstrated that BRD4 orchestrates early lung morphogenesis and separately regulates cell specification, indicating a multifunctional and evolutionarily conserved role for BRD4 in mammalian lung development.
Authors
Hongbo Wen, Derek C. Liberti, Prashant Chandrasekaran, Shahana Parveen, Kwaku K. Quansah, Mijeong Kim, Ana N. Lange, Abigail T. Marquis, Sylvia N. Michki, Annabelle Jin, MinQi Lu, Ayomikun A. Fasan, Sriyaa Suresh, Shawyon P. Shirazi, Lisa R. Young, Jennifer M.S. Sucre, Maria C. Basil, Rajan Jain, David B. Frank
Abstract
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by immune dysregulation and widespread inflammation. Natural killer (NK) cells display marked functional impairment in SLE, including defective cytotoxicity and cytokine production, but the underlying mechanisms remain poorly defined. Here, we show that mitochondrial dysfunction and impaired mitophagy are key contributors to NK cell abnormalities in SLE. Using complementary structural, metabolic, and proteomic analyses, we found that SLE NK cells accumulate enlarged and dysfunctional mitochondria, exhibit impaired lysosomal acidification, and release mitochondrial DNA into the cytosol—features consistent with defective mitochondrial quality control. Transcriptional and proteomic profiling revealed downregulation of key mitophagy-related genes and pathways. These abnormalities correlated with reduced NK cell degranulation and cytokine production. We then tested whether enhancing mitochondrial quality control could restore NK cell function. The mitophagy activator Urolithin A improved mitochondrial and lysosomal parameters and rescued NK cell effector responses in vitro. Hydroxychloroquine partially restored mitochondrial recycling and reduced cytosolic mtDNA. These findings suggest that defective mitophagy and mitochondrial dysfunction are major contributors to NK cell impairment in SLE and that targeting mitochondrial quality control may represent a promising strategy for restoring immune balance in this disease.
Authors
Natalia W. Fluder, Morgane Humbel, Emeline Recazens, Alexis A. Jourdain, Camillo Ribi, George C. Tsokos, Denis Comte
Abstract
Chronic hyperglycemia changes the expression of various transcription factors and mRNA transcripts, which impair the cellular functionality and delayed wound healing. ZEB2 (zinc finger E-box binding homeobox 2), a key transcription factor maintains the tissue specific macrophage identities, however, its role in regulating macrophage polarization during wound healing under hyperglycemic conditions remains unclear. Here, we have found that persistent hyperglycemia increases ZEB2 expression in wound macrophages via histone acetylation, contributing to chronic inflammation, and delayed wound healing. Exposure to high glucose levels activates P300/CBP, a transcriptional coactivator involved in histone acetylation, enhances ZEB2 expression in wound macrophages. The forced expression of ZEB2 shifts macrophage polarity toward a pro-inflammatory state by upregulating myeloid lineage directed transcription factors (MLDTFs). Conversely, silencing Zeb2 at the wound site reduced hyperglycemia induced macrophage inflammation. Topical application of C646, an inhibitor of P300, at the wound edges of streptozotocin induced high-fat diet fed diabetic mice significantly decreased ZEB2 expression, reduced inflammation and accelerated wound healing. Therefore, targeted inhibition of P300 represents a promising therapeutic strategy for improving diabetic wound healing by modulating ZEB2 driven inflammation in wound macrophages.
Authors
Soumyajit Roy, Debarun Patra, Palla Ramprasad, Shivam Sharma, Parul Katiyar, Ashvind Bawa, Kanhaiya Singh, Kulbhushan Tikoo, Suman Dasgupta, Chandan K. Sen, Durba Pal
Abstract
The survival of patients with acute myelogenous leukemia (AML) carrying mutations in TP53 is dismal. We report the results of a detailed characterization of responses to treatment ex vivo with the MDM2 inhibitor MI219, a p53 protein stabilizer, in AML blasts from 165 patients focusing analyses on TP53 wildtype (WT) patients. In total 33% of AML were absolute resistant to MDM2 inhibitor induced apoptosis, of which 45% carried TP53 mutation and 55% were TP53 WT. We conducted array-based expression profiling of ten resistant and ten sensitive AML cases with WT TP53 status, respectively, at baseline and after 2h and 6h of MDM2 inhibitor treatment. While sensitive cases showed the induction of classical TP53 response genes, this was absent or attenuated in resistant cases. In addition, the sensitive and resistant AML samples at baseline profoundly differed in the expression of inflammation-related and mitochondrial genes. No TP53 mutated AML patient survived. The 4-year survival of AML with defective MDM2 inhibitor induced TP53-mediated apoptosis despite WT TP53 was dismal at 19% when NPM1 was co-mutated and 6% when NPM1 was WT. In summary, we identified prevalent multi-causal defects in TP53-mediated apoptosis in AML resulting in extremely poor patient survival.
Authors
Josephine Dubois, Anthony Palmer, Darren King, Mohamed Rizk, Karan Bedi, Kerby A. Shedden, Sami N. Malek
Abstract
Psoriasis is a chronic inflammatory dermatosis characterized by pathological keratinocyte hyperproliferation and dysregulated immune activation. While ubiquitin-specific peptidase 16 (USP16) has been implicated in modulating multiple cellular signaling pathways, its functional role in psoriatic pathogenesis remains poorly understood. Our investigation revealed pronounced upregulation of USP16 expression in psoriatic epidermis compared to normal controls. Keratinocyte-specific USP16 knockdown demonstrated remarkable therapeutic efficacy, significantly ameliorating characteristic psoriatic phenotypes including epidermal hyperplasia and inflammatory infiltration. RNA sequencing analysis showed that USP16 has substantial effects on cell cycle transition and keratinocytes proliferation. Through KEGG analysis, it was found that USP16 primarily regulates the NLRP3 signaling pathway, leading to enhanced cell proliferation and inflammation. Mechanically, USP16 directly binds to the NLRP3 protein to eliminate K48 ubiquitination modification, enhancing the stability of the NLRP3 protein, activating inflammasome activity. Further studies showed that the therapeutic effect of reducing USP16 on psoriasis progression were counteracted by an NLRP3 activator and keratinocyte-specific NLRP3 overexpression adenovirus. Collectively, these results shed light on how USP16 promotes NLRP3 signaling in keratinocytes, exacerbating psoriasis development. This positive regulation highlights the potential of USP16 as a therapeutic target for psoriasis.
Authors
Nan Wang, Fangqian Guan, Yifan Lin, Bohao Sun, Jindan Dai, Xiejun Xu, Weibo Tang, Yanhua Ren, Xuliang Huang, Wenjie Gao, Xixi Chen, Litai Jin, Weitao Cong, Zhongxin Zhu
Abstract
We provide evidence that human and murine SLFN5 proteins are modulators of Type I IFN responses and the immune response in pancreatic cancer. Blocking expression of Slfn5 in PDAC enhances IFN-responses, suppresses tumor growth, and prolongs survival in immunocompetent mice. Notably, immunophenotypic analysis reveals a reduction in tumor-associated macrophages (TAMs) alongside an increase in tumor infiltrating effector cells in tumors over time. These findings implicate SLFN5 acts as an intracellular immune checkpoint and identify it as a unique therapeutic target for the development of therapies for PDAC and possibly other malignancies.
Authors
Mariafausta Fischietti, Markella Zannikou, Elspeth M. Beauchamp, Diana Saleiro, Aneta H. Baran, Briana N. Hryhorysak, Jamie N. Guillen Magaña, Emely Lopez Fajardo, Gavin T. Blyth, Brandyn A. Castro, Jason M. Miska, Catalina Lee-Chang, Priyam Patel, Elizabeth T. Bartom, Masha Kocherginsky, Frank Eckerdt, Leonidas C. Platanias
Abstract
Alveolar hemorrhage (AH) is a life-threatening condition with high mortality, yet the immunologic mechanisms governing disease severity remain poorly defined. Here, we demonstrate a protective role for T cell–intrinsic β-catenin stabilization in AH using a transgenic mouse model (CAT-Tg) in which β-catenin is stabilized under the Lck promoter. β-Catenin stabilization induced a distinct T cell phenotype marked by expansion of central effector memory cells (CD44+CD122+Eomes+T-bet+) and suppression of proinflammatory signaling, including reduced phosphorylation of STAT1, STAT3, and JAK1. Pristane-induced AH was attenuated in CAT-Tg mice, which exhibited reduced lung injury, decreased proteinuria, and diminished pulmonary proinflammatory cytokine production compared with wild-type controls. Protection was associated with a marked expansion of FOXP3+ regulatory T cells (Tregs). Mechanistically, β-catenin stabilization enhanced lung expression of Amphiregulin and BATF, mediators of Treg stability and tissue repair. Adoptive transfer of CAT-Tg–derived Tregs into wild-type mice conferred superior protection against AH, reducing lung inflammation and proteinuria. Transcriptomic analyses revealed enrichment of tissue repair and immune homeostasis pathways, including PI3K–Akt, angiogenesis, and STAT5 signaling. Collectively, these findings identify β-catenin as a regulator of a protective Amphiregulin–BATF–Treg axis, highlighting a immunomodulatory pathway with therapeutic potential for AH and inflammatory lung disease.
Authors
Fiona Mason, Hui Xiong, Ali Mobeen, Md Saddam Hossain, Sara Mahmudlu, Rosanne Trevail, Mikyal Mobeen, Li Chen, Sunny Lee, Tuncay Delibasi, Jyoti Misra Sen, Mobin Karimi
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