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International Journal of Molecular... Jun 2024Water is a major requirement for our bodies, and alkaline water has induced an antioxidant response in a model of natural aging. A series of recent reports have shown...
Water is a major requirement for our bodies, and alkaline water has induced an antioxidant response in a model of natural aging. A series of recent reports have shown that aging is related to reduced water intake. Hydrogen-rich water has been suggested to exert a general antioxidant effect in relation to both improving lifestyle and preventing a series of diseases. Here, we wanted to investigate the effect of the daily intake of hydrogen-rich alkaline water (HAW) in counteracting the redox imbalance induced in a model of HO-treated mice. Mice were treated with HO for two weeks and either left untreated or supplied with HAW. The results show that HAW induced a reduction in the ROS plasmatic levels that was consistent with the increase in the circulating glutathione. At the same time, the reduction in plasmatic 8-hydroxy-2'-deoxyguanosine was associated with reduced DNA damage in the whole body. Further analysis of the spleen and bone marrow cells showed a reduced ROS content consistent with a significantly reduced mitochondrial membrane potential and superoxide accumulation and an increase in spontaneous proliferation. This study provides evidence for a clear preventive and curative effect of HAW in a condition of systemic toxic condition and redox imbalance.
Topics: Animals; Mice; Hydrogen Peroxide; Hydrogen; Oxidation-Reduction; Reactive Oxygen Species; Water; Oxidative Stress; Antioxidants; DNA Damage; Male; Membrane Potential, Mitochondrial; 8-Hydroxy-2'-Deoxyguanosine; Glutathione; Dietary Supplements
PubMed: 38928440
DOI: 10.3390/ijms25126736 -
International Journal of Molecular... Jun 2024Disruption of any stage of iron homeostasis, including uptake, utilization, efflux, and storage, can cause progressive damage to peripheral organs. The health hazards...
Disruption of any stage of iron homeostasis, including uptake, utilization, efflux, and storage, can cause progressive damage to peripheral organs. The health hazards associated with occupational exposure to inhalation anesthetics (IA) in combination with chronic iron overload are not well documented. This study aimed to investigate changes in the concentration of essential metals in the peripheral organs of rats after iron overload in combination with IA. The aim was also to determine how iron overload in combination with IA affects tissue metal homeostasis, hepcidin-ferritin levels, and MMP levels according to physiological, functional, and tissue features. According to the obtained results, iron accumulation was most pronounced in the liver (19×), spleen (6.7×), lungs (3.1×), and kidneys (2.5×) compared to control. Iron accumulation is associated with elevated heavy metal levels and impaired essential metal concentrations due to oxidative stress (OS). Notably, the use of IA increases the iron overload toxicity, especially after Isoflurane exposure. The results show that the regulation of iron homeostasis is based on the interaction of hepcidin, ferritin, and other proteins regulated by inflammation, OS, free iron levels, erythropoiesis, and hypoxia. Long-term exposure to IA and iron leads to the development of numerous adaptation mechanisms in response to toxicity, OS, and inflammation. These adaptive mechanisms of iron regulation lead to the inhibition of MMP activity and reduction of oxidative stress, protecting the organism from possible damage.
Topics: Animals; Rats; Hepcidins; Oxidative Stress; Iron; Male; Anesthetics, Inhalation; Iron-Dextran Complex; Ferritins; Iron Overload; Liver; Lung; Kidney; Spleen; Rats, Wistar; Homeostasis; Isoflurane
PubMed: 38928030
DOI: 10.3390/ijms25126323 -
Genes May 2024With the rapid development of gene therapy technology in recent years, its abuse as a method of sports doping in athletics has become a concern. However, there is still...
With the rapid development of gene therapy technology in recent years, its abuse as a method of sports doping in athletics has become a concern. However, there is still room for improvement in gene-doping testing methods, and a robust animal model needs to be developed. Therefore, the purposes of this study were to establish a model of gene doping using recombinant adeno-associated virus vector-9, including the human erythropoietin gene (rAAV9-h), and to establish a relevant testing method. First, it was attempted to establish the model using rAAV9-h on mice. The results showed a significant increase in erythrocyte volume accompanied by an increase in spleen weight, confirming the validity of the model. Next, we attempted to detect proof of gene doping by targeting DNA and RNA. Direct proof of gene doping was detected using a TaqMan-qPCR assay with certain primers/probes. In addition, some indirect proof was identified in RNAs through the combination of a TB Green qPCR assay with RNA sequencing. Taken together, these results could provide the foundation for an effective test for gene doping in human athletes in the future.
Topics: Erythropoietin; Animals; Mice; Doping in Sports; Dependovirus; Humans; Genetic Vectors; Male; Genetic Therapy; Models, Animal
PubMed: 38927645
DOI: 10.3390/genes15060709 -
Antibiotics (Basel, Switzerland) May 2024Slow-growing nontuberculous mycobacteria (NTMs) are highly prevalent and routinely cause opportunistic intracellular infectious disease in immunocompromised hosts.
OBJECTIVES
Slow-growing nontuberculous mycobacteria (NTMs) are highly prevalent and routinely cause opportunistic intracellular infectious disease in immunocompromised hosts.
METHODS
The activity of the triple combination of antibiotics, clarithromycin (CLR), rifabutin (RFB), and clofazimine (CFZ), was evaluated and compared with the activity of single antibiotics as well as with double combinations in an in vitro biofilm assay and an in vivo murine model of subsp. () lung infection.
RESULTS
Treatment of 1-week-old biofilms with the triple combination exerted the strongest effect of all (0.12 ± 0.5 × 10 CFU/mL) in reducing bacterial growth as compared to the untreated (5.20 ± 0.5 × 10/mL) or any other combination (≥0.75 ± 0.6 × 10/mL) by 7 days. The treatment of mice intranasally infected with with either CLR and CFZ or the triple combination provided the greatest reduction in CLR-sensitive bacterial counts in both the lung and spleen compared to any single antibiotic or remaining double combination by 4 weeks posttreatment. After 4 weeks of treatment with the triple combination, there were no resistant colonies detected in mice infected with a CLR-resistant strain. No clear relationships between treatment and spleen or lung organ weights were apparent after triple combination treatment.
CONCLUSIONS
The biofilm assay data and mouse disease model efficacy results support the further investigation of the triple-antibiotic combination.
PubMed: 38927142
DOI: 10.3390/antibiotics13060475 -
ENeuro Jun 2024Microglia carry out important functions as the resident macrophages of the brain. To study their role in health and disease, the research community needs tools to...
Microglia carry out important functions as the resident macrophages of the brain. To study their role in health and disease, the research community needs tools to genetically modify them with maximum completeness in a manner that distinguishes them from closely related cell-types, such as monocytes. While currently available tamoxifen-inducible CreERT2 lines are able to achieve the differentiation from other cells, the field needs improved and publicly available constitutively active Cre lines, especially ones with favorable efficiency and specificity profiles for studies where high recombination efficiency is imperative and where tamoxifen administration is contraindicated. Here, we leverage the microglia-specific gene to generate mice expressing Cre. Using genomic methods, we show correct positioning of the transgene and intact microglia homeostasis in mice. Crossing mice to four different reporters, we demonstrate highly efficient recombination in microglia across differentially sensitive loxP alleles in different genomic contexts, indicating robust applicability of the line. Further, we show that microglia recombine a loxP reporter during early embryonic development, supporting the use of the line for developmental studies. Finally, using immunofluorescence and flow cytometry, we reveal that most border associated macrophages (BAMs) are also targeted whereas only few liver and spleen macrophages and virtually no white blood cell subsets exhibit Cre activity, distinguishing this line from another publicly available Cre line, (MMRRC). mice are immediately available (JAX Stock #036591) and serve as a valuable addition to the community's microglia toolbox by providing highly efficient constitutive Cre activity with excellent specificity, particularly for studies where tamoxifen administration is undesirable. The microglia toolbox is continuously growing with more transgenic lines and most recently even viral tools becoming available. When selecting a Cre driver line, investigators must weigh relative strengths and weaknesses of available lines and carefully make the best choice for their given application. These tradeoffs include (1) availability and ease of employment, (2) chromosomal positioning of Cre with respect to the floxed allele (should not be on the same chromosome for conditional knockout studies), (3) activity level of a given Cre line and thus completeness of recombination across the microglia population, (4) specificity with respect to acceptable off-target cell types and tissues, (5) temporal aspects including earliest onset of Cre expression or inducibility, (6) robustness in disease contexts, and (7) potential perturbation of microglia homeostasis through Cre itself or disruption of the targeting locus. Considering these tradeoffs, it is evident that there may not be a one-size-fits all solution but an application-based preference for a mouse line in the diverse repertoire of microglia tools. mice are an excellent addition to this toolbox.
PubMed: 38926085
DOI: 10.1523/ENEURO.0549-23.2024 -
Brazilian Journal of Biology = Revista... 2024Hepatozoon spp. are the most common haemoparasites reported from reptiles around the world, however, only six species have been described infecting crocodilians. In...
Hepatozoon spp. are the most common haemoparasites reported from reptiles around the world, however, only six species have been described infecting crocodilians. In Brazil, Hepatozoon caimani Carini, 1909 is currently the only recognized species from the caiman hosts. This study provides new data on the diversity of species of Hepatozoon infecting Caiman crocodilus (Linnaeus) using molecular data and phylogenetic analysis, with additional support of morphological data of developmental stages from host blood and tissue. Forty-four individuals were collected and screened for haemogregarines, and blood and tissue samples were analysed by light microscopy with 31 (70.45%) infected. Hepatozoon spp. blood developmental stages included immature and mature gamonts with or without cytoplasmic vacuoles and free gamonts. Additionally, merogonic developmental stages were found in the liver and spleen of infected hosts. Based on the morphological and molecular data, this study identified two possible different species of Hepatozoon, being one of them the H. caimani with intragenotypic divergence.
Topics: Animals; Phylogeny; Brazil; Alligators and Crocodiles; Eucoccidiida; Coccidiosis; Coccidia
PubMed: 38922198
DOI: 10.1590/1519-6984.282989 -
Marine Drugs May 2024Deep seawater (DS), obtained from a depth over 200 m, has health benefits due to its rich nutrients and minerals, and intake of DS has shown diverse immunomodulatory...
Deep seawater (DS), obtained from a depth over 200 m, has health benefits due to its rich nutrients and minerals, and intake of DS has shown diverse immunomodulatory effects in allergies and cancer. Therefore, the immunostimulatory effects of Korean mineral-rich seawaters were examined in a cyclophosphamide (CPA)-induced immunosuppression model. Three samples of Korean seawater, namely DS from the East Sea off the coasts of Pohang (PDS) and Uljin (UDS), and seawater from the West Sea off the coast of Boryeong (BS), were collected. The seawaters were abundant in several minerals (calcium, iron, zinc, selenium, etc.). Mice were orally administered the seawaters for 42 days, followed by CPA-induced immunosuppression. The CPA induction reduced the weight of the spleen and lymph nodes; however, the administration of seawaters increased the weight of the lymphoid organs, accompanied by stimulation of natural killer cells' activity and NF-kB-mediated cytokine production (IFNγ, TNFα, IL1β, IL6, and IL12). The mouse-derived splenocytes showed lymphoproliferation without cytotoxicity in the seawater groups. Histopathological analysis revealed that the seawaters improved the CPA-induced atrophic changes by promoting lymphoproliferation in the spleen and lymph nodes. These results provide useful information for the use of Korean mineral-rich seawaters, particularly PDS and UDS, as alternative immunostimulants under immunosuppressive conditions.
Topics: Animals; Cyclophosphamide; Mice; Seawater; Minerals; Cytokines; Republic of Korea; Immunosuppression Therapy; Spleen; Killer Cells, Natural; Male; Adjuvants, Immunologic; Lymph Nodes; Immunosuppressive Agents; Mice, Inbred BALB C
PubMed: 38921545
DOI: 10.3390/md22060234 -
Cells Jun 2024Recently, we successfully utilized noninvasive magnetic resonance and bioluminescence imaging to track MIN6 cells subcutaneously transplanted in immunocompromised nude...
Recently, we successfully utilized noninvasive magnetic resonance and bioluminescence imaging to track MIN6 cells subcutaneously transplanted in immunocompromised nude mice for up to 64 days. In this study, we further used bioluminescence imaging to investigate the immune rejection of MIN6 cells in immunocompetent C3H mice. A total of 5 × 10 luciferase-transfected MIN6 cells were implanted into the subcutaneous space of each nude or C3H mouse. After transplantation, hypoglycemia and persistent bioluminescence signals were observed in eight of eight (100%) nude mice and five of nine (56%) C3H mice ( < 0.05). We then presensitized a group of C3H mice with C57BL/6 spleen cells just prior to transplantation (n = 14). Interestingly, none of them had hypoglycemia or persistent bioluminescence signals ( < 0.01 vs. C3H mice without presensitization). Histological examination of the grafts revealed a lack or minimal presence of insulin-positive cells in recipients without hypoglycemia and persistent bioluminescence signals. In contrast, recipients with hypoglycemia and persistent bioluminescence signals showed a significant presence of insulin-positive cells in their grafts. Our results indicate that rejection of MIN6 cells occurred in C3H mice and could be enhanced by presensitization with C57BL/6 spleen cells and that bioluminescence imaging is a useful noninvasive tool for detecting rejection of subcutaneously transplanted MIN6 cells.
Topics: Animals; Mice; Graft Rejection; Mice, Inbred C3H; Luminescent Measurements; Mice, Inbred C57BL; Mice, Nude; Cell Line, Tumor; Spleen
PubMed: 38920672
DOI: 10.3390/cells13121044 -
Cells Jun 2024Excessive levels of glutamate activity could potentially damage and kill neurons. Glutamate excitotoxicity is thought to play a critical role in many CNS and retinal...
Excessive levels of glutamate activity could potentially damage and kill neurons. Glutamate excitotoxicity is thought to play a critical role in many CNS and retinal diseases. Accordingly, glutamate excitotoxicity has been used as a model to study neuronal diseases. Immune proteins, such as major histocompatibility complex (MHC) class I molecules and their receptors, play important roles in many neuronal diseases, while T-cell receptors (TCR) are the primary receptors of MHCI. We previously showed that a critical component of TCR, CD3ζ, is expressed by mouse retinal ganglion cells (RGCs). The mutation of CD3ζ or MHCI molecules compromises the development of RGC structure and function. In this study, we investigated whether CD3ζ-mediated molecular signaling regulates RGC death in glutamate excitotoxicity. We show that mutation of CD3ζ significantly increased RGC survival in NMDA-induced excitotoxicity. In addition, we found that several downstream molecules of TCR, including Src (proto-oncogene tyrosine-protein kinase) family kinases (SFKs) and spleen tyrosine kinase (Syk), are expressed by RGCs. Selective inhibition of an SFK member, Hck, or Syk members, Syk or Zap70, significantly increased RGC survival in NMDA-induced excitotoxicity. These results provide direct evidence to reveal the underlying molecular mechanisms that control RGC death under disease conditions.
Topics: Retinal Ganglion Cells; Animals; Glutamic Acid; Signal Transduction; CD3 Complex; Mice; Mice, Inbred C57BL; N-Methylaspartate; Cell Survival; Retina; src-Family Kinases; Syk Kinase
PubMed: 38920637
DOI: 10.3390/cells13121006 -
Frontiers in Immunology 2024Acid ceramidase (Ac) is a lysosomal enzyme catalyzing the generation of sphingosine from ceramide, and Ac inhibitors are currently being investigated as potential cancer...
Acid ceramidase (Ac) is a lysosomal enzyme catalyzing the generation of sphingosine from ceramide, and Ac inhibitors are currently being investigated as potential cancer therapeutics. Yet, the role of the Ac in immune responses, particularly anti-viral immunity, is not fully understood. To investigate the impact of Ac expression on various leukocyte populations, we generated a tamoxifen-inducible global knockout mouse model for the Ac (iAc-KO). Following tamoxifen administration to healthy mice, we extracted primary and secondary lymphoid organs from iAc-KO and wild-type (wt) littermates and subsequently performed extensive flow cytometric marker analysis. In addition, we isolated CD4 T cells from the spleen and lymph nodes for sphingolipid profiling and restimulated them with Dynabeads™ Mouse T-activator CD3/CD28. Intracellular cytokine expression (FACS staining) was analyzed and secreted cytokines detected in supernatants. To study cell-intrinsic effects, we established an model for iAc-KO in isolated CD4 T and B cells. For CD4 T cells of iAc-KO versus wt mice, we observed reduced Ac activity, an increased ceramide level, and enhanced secretion of IFNγ upon CD3/CD28 costimulation. Moreover, there was a marked reduction in B cell and plasma cell and blast numbers in iAc-KO compared to wt mice. To study cell-intrinsic effects and in line with the 3R principles, we established cell culture systems for iAc-KO in isolated B and CD4 T cells. Our findings pinpoint to a key role of the Ac in mature B and antibody-secreting cells and in IFNγ secretion by CD4 T cells.
Topics: Animals; CD4-Positive T-Lymphocytes; Interferon-gamma; Mice; Mice, Knockout; B-Lymphocytes; Acid Ceramidase; Mice, Inbred C57BL; Lymphocyte Count
PubMed: 38919612
DOI: 10.3389/fimmu.2024.1309846