Cells, Volume 12, Issue 16 (August-2 2023) – 103 articles

Infectious diseases, particularly Tuberculosis (TB) caused by Mycobacterium tuberculosis, pose a significant global health challenge, with 1.6 million reported deaths in 2021, making it the most fatal disease caused by a single infectious agent. The rise of drug-resistant infectious diseases adds to the urgency of finding effective and safe intervention therapies. Antisense therapy uses antisense oligonucleotides (ASOs) that are short, chemically modified, single-stranded deoxyribonucleotide molecules complementary to their mRNA target. Due to their designed target specificity and inhibition of a disease-causing gene at the mRNA level, antisense therapy has gained interest as a potential therapeutic approach. This type of therapy is currently utilized in numerous diseases, such as cancer and genetic disorders. Currently, there are limited but steadily increasing studies available that report on the use of ASOs as treatment for infectious diseases. This review explores the sustainability of FDA-approved and preclinically tested ASOs as a treatment for infectious diseases and the adaptability of ASOs for chemical modifications resulting in reduced side effects with improved drug delivery; thus, highlighting the potential therapeutic uses of ASOs for treating infectious diseases. Full article

Studies about radiation-induced human cataractogenesis are generally limited by (1) the poor number of epithelial lens cell lines available (likely because of the difficulties of cell sampling and amplification) and (2) the lack of reliable biomarkers of the radiation-induced aging process. We have developed a mechanistic model of the individual response to radiation based on the nucleoshuttling of the ATM protein (RIANS). Recently, in the frame of the RIANS model, we have shown that, to respond to permanent endo- and exogenous stress, the ATM protein progressively agglutinates around the nucleus attracted by overexpressed perinuclear ATM-substrate protein. As a result, perinuclear ATM crowns appear to be an interesting biomarker of aging. The radiobiological characterization of the two human epithelial lens cell lines available and the four porcine epithelial lens cell lines that we have established showed delayed RIANS. The BFSP2 protein, found specifically overexpressed around the lens cell nucleus and interacting with ATM, may be a specific ATM-substrate protein facilitating the formation of perinuclear ATM crowns in lens cells. The perinuclear ATM crowns were observed inasmuch as the number of culture passages is high. Interestingly, 2 Gy X-rays lead to the transient disappearance of the perinuclear ATM crowns. Altogether, our findings suggest a strong influence of the ATM protein in radiation-induced cataractogenesis. Full article

Protein turnover, a highly regulated process governed by the ubiquitin–proteasome system (UPS), is essential for maintaining cellular homeostasis. Dysregulation of the UPS has been implicated in various diseases, including viral infections and cancer, making the proteins in the UPS attractive targets for therapeutic intervention. However, the functional and structural redundancies of UPS enzymes present challenges in identifying precise drug targets and achieving target selectivity. Consequently, only 26S proteasome inhibitors have successfully advanced to clinical use thus far. To overcome these obstacles, engineered peptides and proteins, particularly engineered ubiquitin, have emerged as promising alternatives. In this review, we examine the impact of engineered ubiquitin on UPS and non-UPS proteins, as well as on viral enzymes. Furthermore, we explore their potential to guide the development of small molecules targeting novel surfaces, thereby expanding the range of druggable targets. Full article

The factors influencing mother-to-child cell trafficking and persistence over children’s lives have yet to be established. The quantification of maternal microchimerism was previously reported through HLA-based approaches, which introduced bias regarding the tolerogenic environment. We aimed to identify cells of maternal origin irrespective of the HLA repertoire and to ascertain the determinants of microchimeric cells. This case–control study enrolled 40 male infants attending pediatric surgery from January 2022 to October 2022. Female cells were quantified in infants’ tonsil tissue by using cytogenetic fluorescent in situ hybridization (FISH) coupled with optimized automated microscopy. Out of the 40 infants, half (47.4%) had been breastfed for more than one month, a quarter for less a month, and 10 children (26.3%) were never breastfed. XX cells were observed in male tonsils in two-thirds of participants at a median density of 5 cells per 100,000 cells. In univariate analyses, child age was negatively associated with a high female cell density. In exploratory multivariate analyses, previous breastfeeding is a likely determinant of the persistence of these cells in the host, as well as the rank among siblings. Part of the benefit of breastmilk for child health may therefore be driven by breastfeeding-related microchimerism. Full article

Background: Alzheimer’s disease (AD) is a neurodegenerative disease that remains uncured. Its pathogenesis is characterized by the formation of β-amyloid (Aβ) plaques. The use of antigen-specific regulatory T cells (Tregs) through adoptive transfer has shown promise for the treatment of many inflammatory diseases, although the effectiveness of polyspecific Tregs is limited. Obtaining a sufficient number of antigen-specific Tregs from patients remains challenging. Aims and Methods: To address this problem, we used an antibody-like single-chain variable fragment from a phage library and subsequently generated a chimeric antigen receptor (CAR) targeting β-amyloid. Results: The β-amyloid-specific CARs obtained were stimulated by both recombinant and membrane-bound Aβ isolated from the murine brain. The generated CAR-Tregs showed a normal Treg phenotype, were antigen-specific activatable, and had suppressive capacity. Conclusion: This study highlights the potential of CAR technology to generate antigen-specific Tregs and presents novel approaches for developing functional CARs. Full article

The liver is a vital organ responsible for metabolic and digestive functions, protein synthesis, detoxification, and numerous other necessary functions. Various acute, chronic, and neoplastic disorders affect the liver and hamper its biological functions. Most of the untreated liver diseases lead to inflammation and fibrosis which develop into cirrhosis. The human amniotic membrane (hAM), the innermost layer of the fetal placenta, is composed of multiple layers that include growth-factor rich basement membrane, epithelial and mesenchymal stromal cell layers. hAM possesses distinct beneficial anti-fibrotic, anti-inflammatory and pro-regenerative properties via the secretion of multiple potent trophic factors and/or direct differentiation into hepatic cells which place hAM-based therapies as potential therapeutic strategies for the treatment of chronic liver diseases. Decellularized hAM is also an ideal scaffold for liver tissue engineering as this biocompatible niche provides an excellent milieu for cell proliferation and hepatocytic differentiation. Therefore, the current review discusses the therapeutic potential of hAM and its derivatives in providing therapeutic solutions for liver pathologies including acute liver failure, metabolic disorders, liver fibrosis as well as its application in liver tissue engineering. Full article

Although melanoma accounts for only 5.3% of skin cancer, it results in >75% of skin-cancer-related deaths. To avoid disfiguring surgeries on the head and neck associated with surgical excision, there is a clear unmet need for other strategies to selectively remove cutaneous melanoma lesions. Mohs surgery is the current treatment for cutaneous melanoma lesions and squamous and basal cell carcinoma. While Mohs surgery is an effective way to remove melanomas in situ, normal tissue is also excised to achieve histologically negative margins. This paper describes a novel combination therapy of nonthermal plasma (NTP) which emits a multitude of reactive oxygen species (ROS) and the injection of a pharmaceutical agent. We have shown that the effects of NTP are augmented by the DNA-damaging prodrug, tirapazamine (TPZ), which becomes a free radical only in conditions of hypoxemia, which is often enhanced in the tumor microenvironment. In this study, we demonstrate the efficacy of the combination therapy through experiments with B16-F10 and 1205 Lu metastatic melanoma cells both in vitro and in vivo. We also show the safety parameters of the therapy with no significant effects of the therapy when applied to porcine skin. We show the need for the intratumor delivery of TPZ in combination with the surface treatment of NTP and present a model of a medical device to deliver this combination therapy. The importance of functional gap junctions is indicated as a mechanism to promote the therapeutic effect. Collectively, the data support a novel therapeutic combination to treat melanoma and the development of a medical device to deliver the treatment in situ. Full article

The anti-inflammatory effects of depolymerizing microtubule-targeting agents on leukocytes are known for a long time, but the potential involvement of the vascular endothelium and the underlying mechanistic basis is still largely unclear. Using the recently synthesized depolymerizing microtubule-targeting agent pretubulysin, we investigated the anti-inflammatory potential of pretubulysin and other microtubule-targeting agents with respect to the TNF-induced leukocyte adhesion cascade in endothelial cells, to improve our understanding of the underlying biomolecular background. We found that treatment with pretubulysin reduces inflammation in vivo and in vitro via inhibition of the TNF-induced adhesion of leukocytes to the vascular endothelium by down-regulation of the pro-inflammatory cell adhesion molecules ICAM-1 and VCAM-1 in a JNK-dependent manner. The underlying mechanism includes JNK-induced deregulation and degradation of the histone acetyltransferase Bromodomain-containing protein 4. This study shows that depolymerizing microtubule-targeting agents, in addition to their established effects on leukocytes, also significantly decrease the inflammatory activation of vascular endothelial cells. These effects are not based on altered pro-inflammatory signaling cascades, but require deregulation of the capability of cells to enter constructive transcription for some genes, setting a baseline for further research on the prominent anti-inflammatory effects of depolymerizing microtubule-targeting agents. Full article

MicroRNA (miRNA) 107 expression is downregulated but Wnt3a protein and β-catenin are upregulated in degenerated intervertebral disc (IVD). We investigated mir-107/Wnt3a-β-catenin signaling in vitro and in vivo following hyperbaric oxygen (HBO) intervention. Our results showed 96 miRNAs were upregulated and 66 downregulated in degenerated nucleus pulposus cells (NPCs) following HBO treatment. The 3′ untranslated region (UTR) of the Wnt3a mRNA contained the “seed-matched-sequence” for miR-107. MiR-107 was upregulated and a marked suppression of Wnt3a was observed simultaneously in degenerated NPCs following HBO intervention. Knockdown of miR-107 upregulated Wnt3a expression in hyperoxic cells. HBO downregulated the protein expression of Wnt3a, phosphorylated LRP6, and cyclin D1. There was decreased TOP flash activity following HBO intervention, whereas the FOP flash activity was not affected. HBO decreased the nuclear translocation of β-catenin and decreased the secretion of MMP-3 and -9 in degenerated NPCs. Moreover, rabbit serum KS levels and the stained area for Wnt3a and β-catenin in repaired cartilage tended to be lower in the HBO group. We observed that HBO inhibits Wnt3a/β-catenin signaling-related pathways by upregulating miR-107 expression in degenerated NPCs. HBO may play a protective role against IVD degeneration and could be used as a future therapeutic treatment. Full article

Phytoplasmas are intracellular plant pathogens that heavily rely on host cell nutrients for survival and propagation due to their limited ability to synthesize essential substrates. The endoplasmic reticulum (ER), which plays a vital role in various cellular processes, including lipid and protein biosynthesis, is an attractive target for numerous intracellular pathogens to exploit. This study investigated the impact of potato purple top (PPT) phytoplasma infection on the ER in tomato plants. Abnormal accumulation of ER-resident proteins, disrupted ER network structures, and formation of protein aggregates in the phloem were observed using confocal microscopy and transmission electron microscopy, indicating a phytoplasma-infection-induced disturbance in ER homeostasis. The colocalization of phytoplasmas with the accumulated ER-resident proteins suggests an association between ER stress, unfolded protein response (UPR) induction, and phytoplasma infection and colonization, with the ER stress response likely contributing to the host plant’s defense mechanisms. Quantitative real-time PCR revealed a negative correlation between ER stress/UPR activation and PPT phytoplasma titer, implying the involvement of UPR in curbing phytoplasma proliferation. Inducing ER stress and activating the UPR pathway effectively decreased phytoplasma titer, while suppressing the ER-resident protein, binding immunoglobulin protein (BiP) increased phytoplasma titer. These results highlight the ER as an intracellular battleground where phytoplasmas exploit host components for survival and multiplication, while host plants deploy defense mechanisms to counteract the invasion. Understanding the intricate interactions between phytoplasmas and plant hosts at the subcellular level, particularly within the ER, provides valuable insights for developing new strategies to control phytoplasma diseases. Full article

Ethanol induces neuroinflammation, which is believed to contribute to the pathogenesis of alcohol use disorder (AUD). Toll-like receptors (TLRs) are a group of pattern recognition receptors (PRRs) expressed on both immune cells, including microglia and astrocytes, and non-immune cells in the central nervous system (CNS). Studies have shown that alcohol activates TLR4 signaling, resulting in the induction of pro-inflammatory cytokines and chemokines in the CNS. However, the effect of alcohol on signaling pathways downstream of TLR4, such as MyD88 and TRIF (TICAM) signaling, has not been evaluated extensively. In the current study, we treated male wild-type, TLR4-, MyD88-, and TRIF-deficient mice using a chronic plus binge mouse model of AUD. Evaluation of mRNA expression by qRT-PCR revealed that ethanol increased IL-1β, TNF-α, CCL2, COX2, FosB, and JunB in the cerebellum in wild-type and TRIF-deficient mice, while ethanol generally did not increase the expression of these molecules in TLR4- and MyD88-deficient mice. Furthermore, IRF3, IRF7, and IFN-β1, which are associated with the TRIF-dependent signaling cascade, were largely unaffected by alcohol. Collectively, these results suggest that the TLR4 and downstream MyD88-dependent signaling pathways are essential in ethanol-induced neuroinflammation in this mouse model of AUD. Full article

Organoids are three-dimensional constructs generated by placing cells in scaffolds to facilitate the growth of cultures with cell–cell and cell–matrix interactions close to the in vivo situation. Organoids may contain different types of cells, including cancer cells, progenitor cells, or differentiated cells. As distinct culture conditions have significant effects on cell metabolism, we explored the expansion of cells and expression of marker genes in bladder cancer cells expanded in two different common scaffolds. The cells were seeded in basement membrane extract (BME; s.c., Matrigel^®) or in a cellulose-derived hydrogel (GrowDex^®, GD) and cultured. The size of organoids and expression of marker genes were studied. We discovered that BME facilitated the growth of significantly larger organoids of cancer cell line RT112 (p < 0.05), cells from a solid tumor (p < 0.001), and a voiding urine sample (p < 0.001). Expression of proliferation marker Ki76, transcription factor TP63, cytokeratin CK20, and cell surface marker CD24 clearly differed in these different tumor cells upon expansion in BME when compared to cells in GD. We conclude that the choice of scaffold utilized for the generation of organoids has an impact not only on cell growth and organoid size but also on protein expression. The disadvantages of batch-to-batch-variations of BME must be balanced with the phenotypic bias observed with GD scaffolds when standardizing organoid cultures for clinical diagnoses. Full article

Evidence suggests that patients with long COVID can experience neuropsychiatric, neurologic, and cognitive symptoms. However, these clinical data are mostly associational studies complicated by confounding variables, thus the mechanisms responsible for persistent symptoms are unknown. Here we establish an animal model of long-lasting effects on the brain by eliciting mild disease in K18-hACE2 mice. Male and female K18-hACE2 mice were infected with 4 × 10³ TCID50 of SARS-CoV-2 and, following recovery from acute infection, were tested in the open field, zero maze, and Y maze, starting 30 days post infection. Following recovery from SARS-CoV-2 infection, K18-hACE2 mice showed the characteristic lung fibrosis associated with SARS-CoV-2 infection, which correlates with increased expression of the pro-inflammatory kinin B1 receptor (B1R). These mice also had elevated expression of B1R and inflammatory markers in the brain and exhibited behavioral alterations such as elevated anxiety and attenuated exploratory behavior. Our data demonstrate that K18-hACE2 mice exhibit persistent effects of SARS-CoV-2 infection on brain tissue, revealing the potential for using this model of high sensitivity to SARS-CoV-2 to investigate mechanisms contributing to long COVID symptoms in at-risk populations. These results further suggest that elevated B1R expression may drive the long-lasting inflammatory response associated with SARS-CoV-2 infection. Full article

One of the hallmarks of microgravity-induced effects in several cellular models is represented by the alteration of oxidative balance with the consequent accumulation of reactive oxygen species (ROS). It is well known that male germ cells are sensitive to oxidative stress and to changes in gravitational force, even though published data on germ cell models are scarce. We previously studied the effects of simulated microgravity (s-microgravity) on a 2D cultured TCam-2 seminoma-derived cell line, considered the only human cell line available to study in vitro mitotically active human male germ cells. In this study, we used a corresponding TCam-2 3D cell culture model that mimics cell–cell contacts in organ tissue to test the possible effects induced by s-microgravity exposure. TCam-2 cell spheroids were cultured for 24 h under unitary gravity (Ctr) or s-microgravity conditions, the latter obtained using a random positioning machine (RPM). A significant increase in intracellular ROS and mitochondria superoxide anion levels was observed after RPM exposure. In line with these results, a trend of protein and lipid oxidation increase and increased pCAMKII expression levels were observed after RPM exposure. The ultrastructural analysis via transmission electron microscopy revealed that RPM-exposed mitochondria appeared enlarged and, even if seldom, disrupted. Notably, even the expression of the main enzymes involved in the redox homeostasis appears modulated by RPM exposure in a compensatory way, with GPX1, NCF1, and CYBB being downregulated, whereas NOX4 and HMOX1 are upregulated. Interestingly, HMOX1 is involved in the heme catabolism of mitochondria cytochromes, and therefore the positive modulation of this marker can be associated with the observed mitochondria alteration. Altogether, these data demonstrate TCam-2 spheroid sensitivity to acute s-microgravity exposure and indicate the capability of these cells to trigger compensatory mechanisms that allow them to overcome the exposure to altered gravitational force. Full article

The therapeutic efficacy of mesenchymal stromal cells (MSCs) has been shown to rely on their immunomodulatory and regenerative properties. In order to obtain sufficient numbers of cells for clinical applications, MSCs have to be expanded ex vivo. Expansion media with xenogeneic-free (XF) growth-promoting supplements like human platelet lysate (PL) or serum- and xenogeneic-free (SF/XF) formulations have been established as safe and efficient, and both groups provide different beneficial qualities. In this study, MSCs were expanded in XF or SF/XF media as well as in mixtures thereof. MSCs cultured in these media were analyzed for phenotypic and functional properties. MSC expansion was optimal with SF/XF conditions when PL was present. Metabolic patterns, consumption of growth factors, and secretome of MSCs differed depending on the type and concentration of supplement. The lactate per glucose yield increased along with a higher proportion of PL. Many factors in the supernatant of cultured MSCs showed distinct patterns depending on the supplement (e.g., FGF-2, TGFβ, and insulin only in PL-expanded MSC, and leptin, sCD40L PDGF-AA only in SF/XF-expanded MSC). This also resulted in changes in cell characteristics like migratory potential. These findings support current approaches where growth media may be utilized for priming MSCs for specific therapeutic applications. Full article

Tripartite motif-containing protein 32 (TRIM32) is a member of the tripartite motif family and is highly conserved from flies to humans. Via its E3 ubiquitin ligase activity, TRIM32 mediates and regulates many physiological and pathophysiological processes, such as growth, differentiation, muscle regeneration, immunity, and carcinogenesis. TRIM32 plays multifunctional roles in the maintenance of skeletal muscle. Genetic variations in the TRIM32 gene are associated with skeletal muscular dystrophies in humans, including limb–girdle muscular dystrophy type 2H (LGMD2H). LGMD2H-causing genetic variations of TRIM32 occur most frequently in the C-terminal NHL (ncl-1, HT2A, and lin-41) repeats of TRIM32. LGMD2H is characterized by skeletal muscle dystrophy, myopathy, and atrophy. Surprisingly, most patients with LGMD2H show minimal or no dysfunction in other tissues or organs, despite the broad expression of TRIM32 in various tissues. This suggests more prominent roles for TRIM32 in skeletal muscle than in other tissues or organs. This review is focused on understanding the physiological roles of TRIM32 in skeletal muscle, the pathophysiological mechanisms mediated by TRIM32 genetic variants in LGMD2H patients, and the correlations between TRIM32 and Duchenne muscular dystrophy (DMD). Full article

Besides its importance as a livestock species, pig is increasingly being used as an animal model for biomedical research. Macrophages play critical roles in immunity to pathogens, tissue development, homeostasis and tissue repair. These cells are also primary targets for replication of viruses such as African swine fever virus, classical swine fever virus, and porcine respiratory and reproductive syndrome virus, which can cause huge economic losses to the pig industry. In this article, we review the current status of knowledge on porcine macrophages, starting by reviewing the markers available for their phenotypical characterization and following with the characteristics of the main macrophage populations described in different organs, as well as the effect of polarization conditions on their phenotype and function. We will also review available cell lines suitable for studies on the biology of porcine macrophages and their interaction with pathogens. Full article

Fabry disease (FD) is a lysosomal disorder caused by α-galactosidase A deficiency, resulting in the accumulation of globotriaosylceramide (Gb-3) and its metabolite globotriaosylsphingosine (Lyso-Gb-3). Cardiovascular complications and hypertrophic cardiomyopathy (HCM) are the most frequent manifestations of FD. While an echocardiogram and cardiac MRI are clinical tools to assess cardiac involvement, hypertrophic pattern variations and fibrosis make it crucial to identify biomarkers to predict early cardiac outcomes. This study aims to investigate potential biomarkers associated with HCM in FD: transforming growth factor-β1 (TGF-β1), TGF-β active form (a-TGF-β), vascular endothelial growth factor (VEGF-A), and fibroblast growth factor (FGF2) in 45 patients with FD, categorized into cohorts based on the HCM severity. TGF-β1, a-TGF-β, FGF2, and VEGF-A were elevated in FD. While the association of TGF-β1 with HCM was not gender-related, VEGF was elevated in males with FD and HCM. Female patients with abnormal electrocardiograms but without overt HCM also have elevated TGF-β1. Lyso-Gb3 is correlated with TGF-β1, VEGF-A, and a-TGF-β1. Elevation of TGF-β1 provides evidence of the chronic inflammatory state as a cause of myocardial fibrosis in FD patients; thus, it is a potential marker of early cardiac fibrosis detected even prior to hypertrophy. TGF-β1 and VEGF biomarkers may be prognostic indicators of adverse cardiovascular events in FD. Full article

Duchenne muscular dystrophy (DMD) is one of the most devastating myopathies, where severe inflammation exacerbates disease progression. Previously, we demonstrated that adiponectin (ApN), a hormone with powerful pleiotropic effects, can efficiently improve the dystrophic phenotype. However, its practical therapeutic application is limited. In this study, we investigated ALY688, a small peptide ApN receptor agonist, as a potential novel treatment for DMD. Four-week-old mdx mice were subcutaneously treated for two months with ALY688 and then compared to untreated mdx and wild-type mice. In vivo and ex vivo tests were performed to assess muscle function and pathophysiology. Additionally, in vitro tests were conducted on human DMD myotubes. Our results showed that ALY688 significantly improved the physical performance of mice and exerted potent anti-inflammatory, anti-oxidative and anti-fibrotic actions on the dystrophic muscle. Additionally, ALY688 hampered myonecrosis, partly mediated by necroptosis, and enhanced the myogenic program. Some of these effects were also recapitulated in human DMD myotubes. ALY688’s protective and beneficial properties were mainly mediated by the AMPK-PGC-1α axis, which led to suppression of NF-κβ and TGF-β. Our results demonstrate that an ApN mimic may be a promising and effective therapeutic prospect for a better management of DMD. Full article

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the SPTBN2 gene encoding the cytoskeletal protein β-III-spectrin. Previously, we demonstrated that a L253P missense mutation, localizing to the β-III-spectrin actin-binding domain (ABD), causes increased actin-binding affinity. Here we investigate the molecular consequences of nine additional ABD-localized, SCA5 missense mutations: V58M, K61E, T62I, K65E, F160C, D255G, T271I, Y272H, and H278R. We show that all of the mutations, similar to L253P, are positioned at or near the interface of the two calponin homology subdomains (CH1 and CH2) comprising the ABD. Using biochemical and biophysical approaches, we demonstrate that the mutant ABD proteins can attain a well-folded state. However, thermal denaturation studies show that all nine mutations are destabilizing, suggesting a structural disruption at the CH1-CH2 interface. Importantly, all nine mutations cause increased actin binding. The mutant actin-binding affinities vary greatly, and none of the nine mutations increase actin-binding affinity as much as L253P. ABD mutations causing high-affinity actin binding, with the notable exception of L253P, appear to be associated with an early age of symptom onset. Altogether, the data indicate that increased actin-binding affinity is a shared molecular consequence of numerous SCA5 mutations, which has important therapeutic implications. Full article

Schizophrenia is a complex psychiatric condition that may involve immune system dysregulation. Since most putative disease mechanisms in schizophrenia have been derived from genetic association studies and fluid-based molecular analyses, this review aims to summarize the emerging evidence on clinical correlates to immune system dysfunction in this psychiatric disorder. We conclude this review by attempting to develop a unifying hypothesis regarding the relative contributions of microglia and various immune cell populations to the development of schizophrenia. This may provide important translational insights that can become useful for addressing the multifaceted clinical presentation of schizophrenia. Full article

Background: Immunotherapy has recently been incorporated into the spectrum of biliary tract cancer (BTC) treatment. The identification of predictive response biomarkers is essential in order to identify those patients who may benefit most from this novel treatment option. Here, we propose a systematic literature review and a meta-analysis of PD-1, PD-L1, and other immune-related biomarker expression levels in patients with BTC. Methods: Prisma guidelines were followed for this systematic review and meta-analysis. Eligible studies were searched on PubMed. Studies published between 2017 and 2022, reporting data on PD-1/PD-L1 expression and other immune-related biomarkers in patients with BTC, were considered eligible. Results: A total of 61 eligible studies were identified. Despite the great heterogeneity between 39 studies reporting data on PD-L1 expression, we found a mean PD-L1 expression percentage (by choosing the lowest cut-off per study) of 25.6% (95% CI 21.0 to 30.3) in BTCs. The mean expression percentages of PD-L1 were 27.3%, 21.3%, and 27.4% in intrahepatic cholangiocarcinomas (iCCAs—15 studies), perihilar–distal CCAs (p/dCCAs—7 studies), and gallbladder cancer (GBC—5 studies), respectively. Furthermore, 4.6% (95% CI 2.38 to 6.97) and 2.5% (95% CI 1.75 to 3.34) of BTCs could be classified as TMB-H and MSI/MMRd tumors, respectively. Conclusion: From our analysis, PD-L1 expression was found to occur approximately in 26% of BTC patients, with minimal differences based on anatomical location. TMB-H and MSI molecular phenotypes occurred less frequently. We still lack a reliable biomarker, especially in patients with mismatch-proficient tumors, and we must need to make an effort to conceive new prospective biomarker discovery studies. Full article

Serum amyloid A (SAA) proteins belong to a family of acute-phase reactants, playing an integral role in defending the organism from pathological damage. Despite a wealth of data on the regulation of SAA transcripts in teleosts, there is only limited information on these proteins’ abundance in fish. The aim of this study is to characterise SAA protein levels in salmonids using a newly developed antibody specific to salmonid SAA. The salmonid SAA antibody detected SAA and accurately discriminated between stimulated and control specimens from rainbow trout macrophage cell line (RTS-11) in vitro, as well as rainbow trout challenged with Aeromonas salmonicida- or flagellin-stimulated Atlantic salmon in vivo. The presence of SAA protein was analysed in RTS-11 cell line supernatants, liver, and spleen samples using ELISA, immunoblotting, and immunohistochemistry. This study is the first to characterise SAA protein levels in salmonids in vivo and in vitro. The newly developed salmonid SAA antibody was able to discriminate between stimulated and unstimulated specimens, showing that it can be used to study the acute-phase response in salmonids with the potential to be further developed into assays to monitor and evaluate health in wild and farmed fish. Full article

The aromatase-Cre recombinase (Cyp19-Cre) transgenic mouse model has been extensively used for placenta-specific gene inactivation. In a pilot study, we observed unexpected phenotypes using this mouse strain, which prompted an extensive characterization of Cyp19-Cre placental phenotypes using ROSA^mT/mG transgenic reporter mice. The two strains were mated to generate bi-transgenic Cyp19-Cre;ROSA^mT/mG mice following a standard transgenic breeding scheme, and placental and fetal tissues were analyzed on embryonic day 17.5. Both maternal and paternal Cre inheritance were analyzed by mating the respective Cyp19-Cre and ROSA^mT/mG males and females. The genotype results showed the expected percentage of Cyp19-Cre;ROSA^mT/mG fetuses (73%) and Cre mRNA was expressed in all of the Cyp19-Cre placentas. However, surprisingly, only about 50% of the Cyp19-Cre;ROSA^mT/mG placentas showed Cre-mediated recombinase activity as demonstrated by placental enhanced green fluorescent protein (EGFP) expression. Further genetic excision analysis of the placentas revealed consistent results showing the absence of excision of the tdTomato in all of the Cyp19-Cre;ROSA^mT/mG placentas lacking EGFP expression. Moreover, among the EGFP-expressing placentas, there was wide variability in recombination efficiency, even in placentas from the same litter, leading to a mosaic pattern of EGFP expression in different zones and cell types of the placentas. In addition, we observed a significantly higher percentage of Cre recombination activity in placentas with maternal Cre inheritance. Our results show frequent mosaicism, inconsistent recombination activity, and parent-of-origin effects in placentas from Cyp19-Cre;ROSA^mT/mG mice, suggesting that tail-biopsy genotype results may not necessarily indicate the excision of floxed genes in Cyp19-Cre positive placentas. Thus, placenta-specific mutagenesis studies using the Cyp19-Cre model require extensive characterization and careful interpretation of the placental phenotypes for each floxed allele. Full article

Chronic wounds affect more than 2% of the population worldwide, with a significant burden on affected individuals, healthcare systems, and societies. A key regulator of the entire wound healing cascade is transforming growth factor beta (TGF-β), which regulates not only inflammation and extracellular matrix formation but also revascularization. This present work aimed at characterizing wound tissues obtained from acute and chronic wounds regarding angiogenesis, inflammation, as well as ECM formation and degradation, to identify common disturbances in the healing process. Serum and wound tissues from 38 patients (N = 20 acute and N = 18 chronic wounds) were analyzed. The patients’ sera suggested a shift from VEGF/VEGFR to ANGPT/TIE2 signaling in the chronic wounds. However, this shift was not confirmed in the wound tissues. Instead, the chronic wound tissues showed increased levels of MMP9, a known activator of TGF-β. However, regulation of TGF-β target genes, such as CTGF, COL1A1, or IL-6, was absent in the chronic wounds. In wound tissues, all three TGF-β isoforms were expressed with increased levels of TGF-β1 and TGF-β3 and a reporter assay confirmed that the expressed TGF-β was activated. However, Western blots and immunostaining showed decreased canonical TGF-β signaling in the respective chronic wound tissues, suggesting the presence of a TGF-β inhibitor. As a potential regulatory mechanism, the TGF-β proteome profiler array suggested elevated levels of the TGF-β pseudo-receptor BAMBI. Also, tissue expression of BAMBI was significantly increased not only in chronic wounds (10.6-fold) but also in acute wounds that had become chronic (9.5-fold). In summary, our data indicate a possible regulatory role of BAMBI in the development of chronic wounds. The available few in vivo studies support our findings by postulating a therapeutic potential of BAMBI for controlling scar formation. Full article

Vaccines have been hailed as one of the most remarkable medical advancements in human history, and their potential for treating cancer by generating or expanding anti-tumor T cells has garnered significant interest in recent years. However, the limited efficacy of therapeutic cancer vaccines in clinical trials can be partially attributed to the inadequacy of current preclinical mouse models in recapitulating the complexities of the human immune system. In this study, we developed two innovative humanized mouse models to assess the immunogenicity and therapeutic effectiveness of vaccines targeting human papillomavirus (HPV16) antigens and delivering tumor antigens to human CD141⁺ dendritic cells (DCs). Both models were based on the transference of human peripheral blood mononuclear cells (PBMCs) into immunocompromised HLA-A*02-NSG mice (NSG-A2), where the use of fresh PBMCs boosted the engraftment of human cells up to 80%. The dynamics of immune cells in the PBMC-hu-NSG-A2 mice demonstrated that T cells constituted the vast majority of engrafted cells, which progressively expanded over time and retained their responsiveness to ex vivo stimulation. Using the PBMC-hu-NSG-A2 system, we generated a hyperplastic skin graft model expressing the HPV16-E7 oncogene. Remarkably, human cells populated the skin grafts, and upon vaccination with a DNA vaccine encoding an HPV16-E6/E7 protein, rapid rejection targeted to the E7-expressing skin was detected, underscoring the capacity of the model to mount a vaccine-specific response. To overcome the decline in DC numbers observed over time in PBMC-hu-NSG-A2 animals, we augmented the abundance of CD141⁺ DCs, the specific targets of our tailored nanoemulsions (TNEs), by transferring additional autologous PBMCs pre-treated in vitro with the growth factor Flt3-L. The Flt3-L treatment bolstered CD141⁺ DC numbers, leading to potent antigen-specific CD4⁺ and CD8⁺ T cell responses in vivo, which caused the regression of pre-established triple-negative breast cancer and melanoma tumors following CD141⁺ DC-targeting TNE vaccination. Notably, using HLA-A*02-matching PBMCs for humanizing NSG-A2 mice resulted in a delayed onset of graft-versus-host disease and enhanced the efficacy of the TNE vaccination compared with the parental NSG strain. In conclusion, we successfully established two humanized mouse models that exhibited strong antigen-specific responses and demonstrated tumor regression following vaccination. These models serve as valuable platforms for assessing the efficacy of therapeutic cancer vaccines targeting HPV16-dysplastic skin and diverse tumor antigens specifically delivered to CD141⁺ DCs. Full article

Activated lymphocyte-derived DNA (ALD-DNA) has been reported to drive the polarization of macrophages toward M2b, producing inflammatory cytokines and inducing inflammation, correspondingly playing an essential role in the development of systemic lupus erythematosus (SLE). Recently, accumulating evidence has pinpointed metabolic adaptation as the crucial cell-intrinsic determinant for inflammatory response, in which glucose metabolism is the key event. However, whether and how glucose metabolism was involved in ALD-DNA-induced macrophage inflammatory response and SLE development remains unclear. Herein, we performed glucose metabolomic analyses of ALD-DNA-stimulated macrophages and uncovered increased glycolysis and diminished pentose phosphate pathway (PPP), as well as enhanced glycogenesis. In ALD-DNA-stimulated macrophages, increased glycolysis resulted in higher lactate production, whereas diminished PPP efficiently led to lower levels of nicotinamide adenine dinucleotide phosphate (NADPH) with higher levels of reactive oxygen species (ROS). While blockade of lactate generation exerted no significant effect on macrophage inflammation in response to ALD-DNA, scavenging ROS fundamentally inhibited the inflammatory response of ALD-DNA-stimulated macrophages. Further, cyclic adenosine monophosphate (cAMP), a master for regulating glycogen metabolism, was downregulated by ALD-DNA in macrophages, which subsequently imbalanced glycogen metabolism toward glycogenesis but not glycogenolysis. Administration of cAMP effectively restored glycogenolysis and enhanced PPP, which correspondingly reduced ROS levels and inhibited the inflammatory response of ALD-DNA-stimulated macrophages. Finally, blocking glucose metabolism using 2-deoxy-D-glucose (2-DG) efficiently restricted macrophage inflammatory response and alleviated ALD-DNA-induced lupus disease. Together, our findings demonstrate that ALD-DNA drives the adaptation of glucose metabolism for inducing macrophage inflammatory response in SLE, which might further our understanding of disease pathogenesis and provide clues for interventive explorations. Full article

Age-related macular degeneration (AMD) is the leading cause of vision loss and visual impairment in people over 50 years of age. In the current therapeutic landscape, intravitreal anti-vascular endothelial growth factor (anti-VEGF) therapies have been central to the management of neovascular AMD (also known as wet AMD), whereas treatments for geographic atrophy have lagged behind. Several therapeutic approaches are being developed for geographic atrophy with the goal of either slowing down disease progression or reversing sight loss. Such strategies target the inflammatory pathways, complement cascade, visual cycle or neuroprotective mechanisms to slow down the degeneration. In addition, retinal implants have been tried for vision restoration and stem cell therapies for potentially a dual purpose of slowing down the degeneration and restoring visual function. In particular, therapies focusing on the complement pathway have shown promising results with the FDA approved pegcetacoplan, a complement C3 inhibitor, and avacincaptad pegol, a complement C5 inhibitor. In this review, we discuss the mechanisms of inflammation in AMD and outline the therapeutic landscapes of atrophy AMD. Improved understanding of the various pathway components and their interplay in this complex neuroinflammatory degeneration will guide the development of current and future therapeutic options, such as optogenetic therapy. Full article

Sperm motility and structural integrity are essential for successful fertilization in vivo, and any hindrance of the correct assembly of the axoneme and peri-axonemal structures in the sperm flagellum can lead to fertility problems. While there has been considerable advancement in studying diseases related to the flagellum, the underlying mechanisms that control sperm movement are not yet fully understood. In this study, we reveal that the tetratricopeptide repeat protein 6 (Ttc6) gene, expressed mainly in the testes, plays a crucial role in maintaining male fertility in mice. We further demonstrate that the knockout of Ttc6 in mice results in decreased sperm motility and induces an abnormal circular swimming pattern, consequently leading to male subfertility. Morphological analysis showed an atypical hairpin-like appearance of the spermatozoa, and ultrastructural studies showed unsheathed flagella at the juncture between the midpiece and principal piece. Collectively, these findings suggest that TTC6 plays an essential role in maintaining the stability of the annulus region of the sperm flagellum, thus ensuring the swift and directed motion of sperm. Full article

The transcription factor HOXA5, from the HOX gene family, has long been studied due to its critical role in physiological activities in normal cells, such as organ development and body patterning, and pathological activities in cancer cells. Nonetheless, recent evidence supports the hypothesis of a role for HOXA5 in metabolic diseases, particularly in obesity and type 2 diabetes (T2D). In line with the current opinion that adipocyte and adipose tissue (AT) dysfunction belong to the group of primary defects in obesity, linking this condition to an increased risk of insulin resistance (IR) and T2D, the HOXA5 gene has been shown to regulate adipocyte function and AT remodeling both in humans and mice. Epigenetics adds complexity to HOXA5 gene regulation in metabolic diseases. Indeed, epigenetic mechanisms, specifically DNA methylation, influence the dynamic HOXA5 expression profile. In human AT, the DNA methylation profile at the HOXA5 gene is associated with hypertrophic obesity and an increased risk of developing T2D. Thus, an inappropriate HOXA5 gene expression may be a mechanism causing or maintaining an impaired AT function in obesity and potentially linking obesity to its associated disorders. In this review, we integrate the current evidence about the involvement of HOXA5 in regulating AT function, as well as its association with the pathogenesis of obesity and T2D. We also summarize the current knowledge on the role of DNA methylation in controlling HOXA5 expression. Moreover, considering the susceptibility of epigenetic changes to reversal through targeted interventions, we discuss the potential therapeutic value of targeting HOXA5 DNA methylation changes in the treatment of metabolic diseases. Full article