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At least 55 records · Page 3
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Scavenger receptor MARCO contributes to macrophage phagocytosis and clearance of tumor cells

Highlights: • The MARCO receptor on macrophages was positively correlated with the ability to phagocyte tumor cells. • Macrophage MARCO affects cytoskeleton rearrangement and pseudopodia formation through the SYK-PI3K-Rac1 pathway. • Integrin is the ligand of MARCO on the tumor cells. Macrophage receptor with collagenous structure (MARCO) is a member of the class A scavenger receptor family which is expressed on the cell surface of macrophages. It is well known that MARCO avidly binds to unopsonized pathogens, leading to its ingestion by macrophages. However, the role of MARCO in the recognition and phagocytosis of tumor cells by macrophages remains poorly understood. In this study, we used lentiviral technology to knockdown and overexpress MARCO and investigated the ability of macrophages to phagocytose tumor cells. Our results showed that MARCO expression was correlated with the ability of macrophages to carry on phagocytosis. MARCO knockdown led to significant decreases in the number of engulfment pseudopodia of macrophages and inhibition of the phagocytosis of tumor cells. On the other hand, MARCO overexpression elevated activity of SYK, PI3K and Rac1 in macrophages, which led to changes in macrophage morphology and enhanced phagocytosis by promoting formation of stress fibers and pseudopodia. By Co-IP analysis we showed that MARCO directly binds to the β5 integrin of SL4 tumor cells. In summary, these results demonstrated the important role for MARCO in demonstrated tumor cells uptake and clearance by macrophages.

60 APPLIED LIFE SCIENCES↗
NASA NTRS2000

Centrifugation of Cultured Osteoblasts And Macrophages as a Model To Study How Gravity Regulates The Function of Skeletal Cells

Mechanical loading helps define the architecture of weight-bearing bone via the tightly regulated process of skeletal turnover. Turnover occurs by the concerted activity of osteoblasts, responsible for bone formation. and osteoclasts, responsible for bone resorption. Osteoclasts are specialized megakaryon macrophages, which differentiate from monocytes in response to resorption stimuli, such as reduced weight-bearing. Habitation in space dramatically alters musculoskeletal loading, which modulates both cell function and bone structure. Our long-term objective is to define the molecular and cellular mechanisms that mediate skeletal adaptations to altered gravity environments. Our experimental approach is to apply hypergravity loads by centrifugation to rodents and cultured cells. As a first step, we examined the influence of centrifugation on the structure of cancellous bone in rats to test the ability of hypergravity to change skeletal architecture. Since cancellous bone undergoes rapid turnover we expected the most dramatic structural changes to occur in the shape of trabeculae of weight-bearing, cancellous bone. To define the cellular responses to hypergravity loads, we exposed cultured osteoblasts and macrophages to centrifugation. The intraosseous and intramedullary pressures within long bones in vivo reportedly range from 12-40 mm Hg, which would correspond to 18-59 gravity (g) in our cultures. We assumed that hydrostatic pressure from the medium above the cell layer is at least one major component of the mechanical load generated by centrifuging cultured cells. and therefore we exposed the cells to 10-50g. In osteoblasts, we examined the structure of their actin and microtubule networks, production of prostaglandin E2 (PGE2), and cell survival. Analysis of the shape of the cytoskeletal networks provides evidence for the ability of centrifugation to affect cell structure, while the production of PGE2 serves as a convenient marker for mechanical stimulation. We examined cell survival, reasoning that osteoblasts might mold skeletal structure in a hypergravity environment in part by regulating apoptosis and thus the duration of osteoblast productivity. Finally, we tested the influence of centrifugation on microbial activation of a macrophage cell line (RAW264.7). In response to the appropriate hormonal stimulation, this cell line is reportedly capable of undergoing differentiation to express osteoclast markers. In addition, a component of the cell wall of gram-negative bacteria, lipopolysaccaride (LPS), stimulates the formation of osteoclasts in vivo. Thus we tested the influence on centrifugation on RAW264.7 cells stimulated with LPS to provide an index of the function of osteoclast precursors.

Globus, Ruth K.↗
DOE OSTI2021

Antibody:CD47 ratio regulates macrophage phagocytosis through competitive receptor phosphorylation

Cancer immunotherapies often modulate macrophage effector function by introducing either targeting antibodies that activate Fcγ receptors (FcγRs) or blocking antibodies that disrupt inhibitory SIRPα-CD47 engagement. However, how these competing signals are integrated is poorly understood, raising questions about how to effectively titrate immune responses. Here, we find that macrophage phagocytic decisions are regulated by the ratio of activating ligand to inhibitory ligand over a broad range of absolute molecular densities. Using both endogenous and chimeric receptors, we show that activating:inhibitory ligand ratios of at least 10:1 are required to promote phagocytosis of model antibody-opsonized CD47-inhibited targets and that lowering that ratio reduces FcγR phosphorylation because of inhibitory phosphatases recruited to CD47-bound SIRPα. We demonstrate that ratiometric signaling is critical for phagocytosis of tumor cells and can be modified by blocking SIRPα, indicating that balancing targeting and blocking antibodies may be important for controlling macrophage phagocytosis in cancer immunotherapy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Molecular Imaging of Matrix Metalloproteinase-2 in Atherosclerosis Using a Smart Multifunctional PET/MRI Nanoparticle

Matrix metalloproteinases from macrophages are important intraplaque components that play pivotal roles in plaque progression and regression. This study sought to develop a novel multifunctional positron emission tomography (PET) and magnetic resonance imaging (MRI) contrast agents based on MMP-2 cleavable nanoparticles to noninvasive assessment of MMP-2 activity in mouse carotid atherosclerotic plaques. Macrophage-rich vascular lesions were induced by carotid ligation plus high-fat diet and streptozotocin-induced diabetes in CL57/BL6 mice. To render iron oxide nanoparticles (IONP) specific for the extracellular MMP-2, the magnetic nanoparticle base material has been derivatized with 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) for the nuclear tracer 64 Cu labeling and the MMP-2-cleavable peptide modified with polyethylene glycol 2000, yielding a multi-modality reporter ( 64 Cu-NOTA-IONP@MMP2c-PEG2K, MMP2cNPs) for PET/MR imaging. Small animal PET imaging and biodistribution data revealed that MMP2cNPs exhibited remarkable plaque uptake (3.06 ± 0.87% ID/g and 1.83 ± 0.28% ID/g at 4 and 12 h, respectively). And MMP2cNPs were rapidly cleared from the contralateral normal carotid artery, resulting in excellent plaque-to-normal carotid artery contrasts. Furthermore, in vivo MRI showed a preferential accumulation of MMP2cNPs in atherosclerotic lesions compared with the non-cleavable reference compound, MMP2ncNPs. In addition, histological analyses revealed iron accumulations in the carotid atherosclerotic plaque, in colocalization with MMP-2 expression and macrophages. Using a combination of innovative imaging modalities, in this study, we demonstrate the feasibility of applying the novel smart MMP2cNPs as a PET/MR hybrid imaging contrast agent for detection of MMP-2 in atherosclerotic plaque in vivo.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1995

Staphylococcal enterotoxins bind H-2Db molecules on macrophages

We screened a panel of monoclonal antibodies against selected macrophage cell surface molecules for their ability to inhibit enterotoxin binding to major histocompatibility complex class II-negative C2D (H-2b) macrophages. Two monoclonal antibodies, HB36 and TIB126, that are specific for the alpha 2 domain of major histocompatibility complex class I, blocked staphylococcal enterotoxins A and B (SEA and SEB, respectively) binding to C2D macrophages in a specific and concentration-dependent manner. Inhibitory activities were haplotype-specific in that SEA and SEB binding to H-2k or H-2d macrophages was not inhibited by either monoclonal antibody. HB36, but not TIB126, inhibited enterotoxin-induced secretion of cytokines by H-2b macrophages. Lastly, passive protection of D-galactosamine-sensitized C2D mice by injection with HB36 antibody prevented SEB-induced death. Therefore, SEA and SEB binding to the alpha 2 domain of the H-2Db molecule induces biological activity and has physiological consequences.

Non-NASA Center↗
NASA NTRS1996

Characterization of the early pulmonary inflammatory response associated with PTFE fume exposure

Heating of polytetrafluoroethylene (PTFE) has been described to release fumes containing ultrafine particles (approximately 18 nm diam). These fumes can be highly toxic in the respiratory tract inducing extensive pulmonary edema with hemorrhagic inflammation. Fischer-344 rats were exposed to PTFE fumes generated by temperatures ranging from 450 to 460 degrees C for 15 min at an exposure concentration of 5 x 10(5) particles/cm3, equivalent to approximately 50 micrograms/m3. Responses were examined 4 hr post-treatment when these rats demonstrated 60-85% neutrophils (PMNs) in their lung lavage. Increases in abundance for messages encoding the antioxidants manganese superoxide dismutase and metallothionein (MT) increased 15- and 40-fold, respectively. For messages encoding the pro- and anti-inflammatory cytokines: inducible nitric oxide synthase, interleukin 1 alpha, 1 beta, and 6 (IL-1 alpha, IL-1 beta, and IL-6), macrophage inflammatory protein-2, and tumor necrosis factor-alpha (TNF alpha) increases of 5-, 5-, 10-, 40-, 40-, and 15-fold were present. Vascular endothelial growth factor, which may play a role in the integrity of the endothelial barrier, was decreased to 20% of controls. In situ sections were hybridized with 33P cRNA probes encoding IL-6, MT, surfactant protein C, and TNF alpha. Increased mRNA abundance for MT and IL-6 was expressed around all airways and interstitial regions with MT and IL-6 demonstrating similar spatial distribution. Large numbers of activated PMNs expressed IL-6, MT, and TNF alpha. Additionally, pulmonary macrophages and epithelial cells were actively involved. These observations support the notion that PTFE fumes containing ultrafine particles initiate a severe inflammatory response at low inhaled particle mass concentrations, which is suggestive of an oxidative injury. Furthermore, PMNs may actively regulate the inflammatory process through cytokine and antioxidant expression.

Non-NASA Center↗
DOE OSTI2021

HIF1α promotes tumor chemoresistance via recruiting GDF15-producing TAMs in colorectal cancer

Chemoresistance is a tremendous challenge to efficacy of systemic chemotherapy which is the preferred treatment for the advanced CRC patients. More tumor-associated macrophages (TAMs) are recruited into the CRC tumor under chemotherapy, which are highly implicated in the chemoresistance development, but the underlying molecular mechanism is unclear. Here, we present that activated HIF1α signaling in CRC cells under chemotherapy drives the expression of HMGB1to promotes macrophage infiltration and in turn chemoresistance development. Chemotherapeutic treatment with 5-FU leads to increased recruitment of macrophages into tumors, which display tumor-protective alternative activation. Mechanistically, tumor HIF1α signaling activated by chemo-induced ROS drives the transcription of HMGB1 to promote more macrophage infiltration into CRC tumor. Furthermore, high levels of GDF15 produced by TAMs impair the chemosensitity of tumor cells via enhancing fatty acids β-oxidation. Together, our current study reveals a new insight into the cross-talking between tumor cells and immune cells, and provides novel drug targets for clinic treatments for CRC.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Comprehensive characterization of hepatocyte-derived extracellular vesicles identifies direct miRNA-based regulation of hepatic stellate cells and DAMP-based hepatic macrophage IL-1ß and IL-17 upregulation in alcoholic hepatitis mice.

Extracellular vesicles (EVs) have been growingly recognized as biomarkers and mediators of alcoholic liver disease (ALD) in human and mice. Here we characterized hepatocyte-derived EVs (HC-EVs) and their cargo for their biological functions in a novel murine model that closely resembles liver pathology observed in patients with alcoholic hepatitis (AH), the most severe spectrum of ALD. The number of circulating EVs and HC-EVs were significantly increased by 10-fold in AH mice compared to control mice. The miRNA(miR)-seq analysis detected 20 up-regulated and 4 down-regulated miRNAs (P < 0.001-0.05) in AH-HC-EVs. Treatment of murine primary hepatic stellate cells (HSCs) with AH-HC-EVs induced ?-SMA (P < 0.05) and Col1a1 (P < 0.001). Smad7, Nr1d2, and Rora genes which regulate HSC activation were predicted targets of 17 miRs up-regulated in AH-HC-EVs. Among them were miR-27a and miR-181 which upon transfection in HSCs, repressed Nr1d2, the HSC quiescence marker. AH-HC-EVs were also enriched in organelle proteins and mitochondrial DNA (10-fold, P < 0.05) and upregulated IL-1??and IL-17 production by hepatic macrophages (HMs) from AH mice in a TLR9-dependent manner. These results demonstrate HC-EV release is intensified in AH and suggest that AH-HC-EVs orchestrate liver fibrogenesis by directly targeting HSC quiescence transcripts via a unique set of miRNAs and by amplifying HSC activation via DAMP-based induction of profibrogenic IL-1? and IL-17 by HMs.

AH, hepatocyte-derived EVs, miRNAs, DAMPs, HSC act↗
DOE OSTI2021

Engineered extracellular vesicle mimetics from macrophage promotes hair growth in mice and promotes human hair follicle growth

Recent studies clearly show that cell-derived extracellular vesicles (EVs, including exosomes) can promote hair growth. However, large-scale production of EVs remains a big hurdle. Recently, extracellular vesicle mimetics (EMs) engineered by extrusion through various membranes are emerging as a complementary approach for large-scale production. In this study, to investigate their ability to induce hair growth, we generated macrophage-engineered EMs (MAC-EMs) that activated the human dermal papilla (DP) cells in vitro. MAC-EMs intradermally injected into the skin of C57BL/6 mice were retained for up to 72 h. Microscopy imaging revealed that MAC-EMs were predominately internalized into hair follicles. The MAC-EMs treatment induced hair regrowth in mice and hair shaft elongation in a human hair follicle, suggesting the potential of MAC-EMs as an alternative to EVs to overcome clinical limitation.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Macrophage-derived MLKL in alcohol-associated liver disease: Regulation of phagocytosis

Mixed lineage kinase domain-like pseudokinase (MLKL), a key terminal effector of necroptosis, also plays a role in intracellular vesicle trafficking that is critical for regulating liver inflammation and injury in alcohol-associated liver disease (ALD). Although receptor interacting protein kinase 3 (Rip3) -/- mice are completely protected from ethanol-induced liver injury, Mlkl -/- mice are only partially protected. Therefore, we hypothesized that cell-specific functions of MLKL may contribute to ethanol-induced injury. Bone marrow transplants between Mlkl -/- mice and littermates were conducted to distinguish the role of myeloid versus nonmyeloid Mlkl in the Gao-binge model of ALD. Ethanol-induced hepatic injury, steatosis, and inflammation were exacerbated in Mlkl -/- →wild-type (WT) mice, whereas Mlkl deficiency in nonmyeloid cells (WT→Mlkl -/- ) had no effect on Gao-binge ethanol-induced injury. Importantly, Mlkl deficiency in myeloid cells exacerbated ethanol-mediated bacterial burden and accumulation of immune cells in livers. Mechanistically, challenging macrophages with lipopolysaccharide (LPS) induced signal transducer and activator of transcription 1–mediated expression and phosphorylation of MLKL, as well as translocation and oligomerization of MLKL to intracellular compartments, including phagosomes and lysosomes but not plasma membrane. Importantly, pharmacological or genetic inhibition of MLKL suppressed the phagocytic capability of primary mouse Kupffer cells (KCs) at baseline and in response to LPS with/without ethanol as well as peripheral monocytes isolated from both healthy controls and patients with alcohol-associated hepatitis. Further, in vivo studies revealed that KCs of Mlkl -/- mice phagocytosed fewer bioparticles than KCs of WT mice. Together, these data indicate that myeloid MLKL restricts ethanol-induced liver inflammation and injury by regulating hepatic immune cell homeostasis and macrophage phagocytosis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Iron Heterogeneity in Early Active Multiple Sclerosis Lesions

Objective: Multiple sclerosis (MS) is a heterogeneous inflammatory demyelinating disease. Iron distribution is altered in MS patients' brains, suggesting iron liberation within active lesions amplifies demyelination and neurodegeneration. Whether the amount and distribution of iron are similar or different among different MS immunopatterns is currently unknown. Methods: We used synchrotron X-ray fluorescence imaging, histology, and immunohistochemistry to compare the iron quantity and distribution between immunopattern II and III early active MS lesions. We analyzed archival autopsy and biopsy tissue from 21 MS patients. Results: Immunopattern II early active lesions contain 64% more iron (95% confidence interval [CI] = 17-127, p = 0.004) than immunopattern III lesions, and 30% more iron than the surrounding periplaque white matter (95% CI = 3–64%, p = 0.03). Iron in immunopattern III lesions is 28% lower than in the periplaque white matter (95% CI = -40 to -14%, p < 0.001). When normalizing the iron content of early active lesions to that of surrounding periplaque white matter, the ratio is significantly higher in immunopattern II (p < 0.001). Microfocused X-ray fluorescence imaging shows that iron in immunopattern II lesions localizes to macrophages, whereas macrophages in immunopattern III lesions contain little iron. Interpretation: Iron distribution and content are heterogeneous in early active MS lesions. Iron accumulates in macrophages in immunopattern II, but not immunopattern III lesions. This heterogeneity in the two most common MS immunopatterns may be explained by different macrophage polarization, origin, or different demyelination mechanisms, and paves the way for developing new or using existing iron-sensitive magnetic resonance imaging techniques to differentiate among immunopatterns in the general nonbiopsied MS patient population.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Leisingera sp. ANG59 & Pseudoalteromonas sp. JC28 Genomes Overview

The Hawaiian bobtail squid, Euprymna scolopes, has a symbiotic bacterial consortium in the accessory nidamental gland (ANG), a female reproductive organ that protects eggs against fouling microorganisms. To test the antibacterial activity of ANG community members, 19 bacterial isolates were screened for their ability to inhibit Gram-negative and Gram-positive bacteria, of which two strains were inhibitory. These two antibacterial isolates, Leisingera sp. ANG59 and Pseudoalteromonas sp. JC28, were subjected to further genomic characterization. Genomic analysis of Leisingera sp. ANG59 revealed a biosynthetic gene cluster encoding the antimicrobial compound indigoidine. The genome of Pseudoalteromonas sp. JC28 had a 14-gene cluster with >95% amino acid identity to a known bromoalterochromide (BAC) cluster. Chemical analysis confirmed production of known BACs, BAC-A/A′ (compounds 1a/1b), as well as two new derivatives, BAC-D/D′ (compounds 2a/2b). Extensive nuclear magnetic resonance (NMR) analyses allowed complete structural elucidation of compounds 2a/2b, and the absolute stereochemistry was unambiguously determined using an optimized Marfey’s method. The BACs were then investigated for in vitro antibacterial, antifungal, and nitric oxide (NO) inhibitory activity. Compounds 1a/1b were active against the marine bacteria Bacillus algicola and Vibrio fischeri, while compounds 2a/2b were active only against B. algicola. Compounds 1a/1b inhibited NO production via lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage cells and also inhibited the pathogenic fungus Fusarium keratoplasticum, which, coupled with their antibacterial activity, suggests that these polyketide-nonribosomal peptides may be used for squid egg defense against potential pathogens and/or fouling microorganisms. These results indicate that BACs may provide Pseudoalteromonas sp. JC28 an ecological niche, facilitating competition against nonsymbiotic microorganisms in the host’s environment.​

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

microRNAs (miR 9, 124, 155 and 224) transdifferentiate mouse macrophages to neurons

Highlights: • miR 9, miR 124 and miR155 could reprogramme macrophages to mature neurons. • miR-induced neuronal reprogramming involves stem cell-like intermediate phenotype. • miR 9, miR 124 and miR155 could also reprogramme CD133+ stem cells to mature neurons. Development is an irreversible process of differentiating the undifferentiated cells to functional cells. Brain development involves generation of cells with varied phenotype and functions, which is limited during adulthood, stress, damage/degeneration. Cellular reprogramming makes differentiation reversible process with reprogramming somatic/stem cells to alternative fate with/without stem cells. Exogenously expressed transcription factors or small molecule inhibitors have driven reprogramming of stem/somatic cells to neurons providing alternative approach for pre-clinical/clinical testing and therapeutics. Here in, we report a novel approach of microRNA (miR)- induced trans-differentiation of macrophages (CD11b high) to induced neuronal cells (iNCs) (neuronal markershigh- Nestin, Nurr1, Map2, NSE, Tubb3 and Mash1) without exogenous use of transcription factors. miR 9, 124, 155 and 224 successfully transdifferentiated macrophages to neurons with transient stem cell-like phenotype. We report trans differentiation efficacy 18% and 21% with miR 124 and miR 155. in silico(String 10.0, miR gator, mESAdb, TargetScan 7.0) and experimental analysis indicate that the reprogramming involves alteration of pluripotencygenes like Oct4, Sox2, Klf4, Nanog and pluripotency miR, miR 302. iNCs also shifted to G0 phase indicating manipulation of cell cycle by these miRs. Further, CD133+ intermediate cells obtained during current protocol could be differentiated to iNCs using miRs. The syanpsin{sup +} neurons were functionally active and displayed intracellular Ca{sup +2} evoke on activation. miRs could also transdifferentiate bone marrow-derived macrophages and peripheral blood mononuclear cells to neuronal cells. The current protocol could be employed for direct in vivo reprogramming of macrophages to neurons without teratoma formation for transplantation and clinical studies.

60 APPLIED LIFE SCIENCES↗
NASA NTRS2007

Toxicity of Lunar and Martian Dust Simulants to Alveolar Macrophages Isolated from Human Volunteers

NASA is planning to build a habitat on the Moon and use the Moon as a stepping stone to Mars. JSC-1, an Arizona volcanic ash that has mineral properties similar to lunar soil, is used to produce lunar environments for instrument and equipment testing. NASA is concerned about potential health risks to workers exposed to these fine dusts in test facilities. The potential toxicity of JSC-1 and a Martian soil simulant (JSC-Mars-1, a Hawaiian volcanic ash) was evaluated using human alveolar macrophages (HAM) isolated from volunteers; titanium dioxide and quartz were used as reference dusts. This investigation is a prerequisite to studies of actual lunar dust. HAM were treated in vitro with these test dusts for 24 h; assays of cell viability and apoptosis showed that JSC-1 and TiO2 were comparable, and more toxic than saline control, but less toxic than quartz. HAM treated with JSC-1 or JSC-Mars 1 showed a dose-dependent increase in cytotoxicity. To elucidate the mechanism by which these dusts induce apoptosis, we investigated the involvement of the scavenger receptor (SR). Pretreatment of cells with polyinosinic acid, an SR blocker, significantly inhibited both apoptosis and necrosis. These results suggest HAM cytotoxicity may be initiated by interaction of the dust particles with SR. Besides being cytotoxic, silica is known to induce shifting of HAM phenotypes to an immune active status. The immunomodulatory effect of the simulants was investigated. Treatment of HAM with either simulant caused preferential damage to the suppressor macrophage subpopulation, leading to a net increase in the ratio of activator (RFD1+) to suppressor (RFD1+7+) macrophages, a result similar to treatment with silica. It is recommended that appropriate precautions be used to minimize exposure to these fine dusts in large-scale engineering applications.

Latch, Judith N.↗
NASA NTRS1992

Effects of dimethyl prostaglandin A1 on herpes simplex virus and human immunodeficiency virus replication

We have investigated the direct effect of dimethyl prostaglandin A1 (dmPGA1) on the replication of herpes simplex virus (HSV) and human immunodeficiency virus type 1 (HIV-1). dmPGA1 significantly inhibited viral replication in both HSV and HIV infection systems at concentrations of dmPGA1 that did not adversely alter cellular DNA synthesis. The 50% inhibitory concentration (ID50) for several HSV type 1 (HSV-1) strains ranged from 3.8 to 5.6 micrograms/ml for Vero cells and from 4.6 to 7.3 micrograms/ml for human foreskin fibroblasts. The ID50s for two HSV-2 strains varied from 3.8 to 4.5 micrograms/ml for Vero cells; the ID50 was 5.7 micrograms/ml for human foreskin fibroblasts. We found that closely related prostaglandins did not have the same effect on the replication of HSV; dmPGE2 and dmPGA2 caused up to a 60% increase in HSV replication compared with that in untreated virus-infected cells. HIV-1 replication in acutely infected T cells (VB line) and chronically infected macrophages was assessed by quantitative decreases in p24 concentration. The effective ID50s were 2.5 micrograms/ml for VB cells acutely infected with HIV-1 and 5.2 micrograms/m for chronically infected macrophages. dmPGA1 has an unusual broad-spectrum antiviral activity against both HSV and HIV-1 in vitro and offers a new class of potential therapeutic agents for in vivo use.

Non-NASA Center↗
NASA NTRS1994

Differential RNA regulation by staphylococcal enterotoxins A and B in murine macrophages

Staphylococcal enterotoxin A (SEA) is significantly better than enterotoxin B (SEB) in activating tumor necrosis factor (TNF) secretion by B6MP102 cells. Both toxins bound to B6MP102 cells; however, SEB competed less effectively with SEA than SEA competed with SEB. This suggested that receptors unique to SEA were present on B6MP102 cells. Signal transduction occurred in response to both toxins. Within 30 s after addition, SEA and SEB significantly increased the F-actin concentration in B6MP102 cells. However, only SEA induced increased TNF mRNA levels. B6MP102 cells incubated with interferon-gamma and SEB secreted TNF. However, enhanced mRNA expression was delayed and the concentration of TNF secreted was less than that of B6MP102 cells stimulated with SEA. Although these data suggest that receptors unique to SEA are present on B6MP102 cells, they also indicate that staphylococcal enterotoxins differentially regulate TNF at the RNA level, perhaps because of differences in binding to the plasma membrane.

NASA Discipline Cell Biology↗
DOE OSTI2021

Effects of varying gelatin coating concentrations on RANKL induced osteoclastogenesis

Here, we assessed the effects of varying concentrations of gelatin coating on Receptor Activator of Nuclear Factor κ-B Ligand (RANKL)-induced RAW264.7 murine macrophage differentiation into osteoclast (OC) via osteoclastogenesis. The microstructures of coating surfaces with different concentrations of gelatin were examined by scanning electron microscopy and atomic force microscopy. Increased gelatin coating concentrations led to decreased gel rigidity but increased surface adhesion force attenuated OC differentiation and the decreased actin ring formation in RANKL-induced osteoclastogenesis. The decreased actin ring formation is associated with decreased lysosomal-associated membrane protein 1 (LAMP1) activity and bone resorption in the differentiated OCs with different gelatin coating concentrations as compared to the cells differentiated without gelatin coatings. In addition, increasing concentrations of gelatin coating attenuated the medium TGF-β1 protein levels and the expression levels of TGF-β and type-I (R1) and type-II (R2) TGF-β receptors in OCs, suggesting the gelatin-induced suppression of TGF-β signaling for the regulation of RNAKL-induced OC differentiation. Taken together, these findings showed that changes in gelatin coating concentrations, which were associated with altered gel thickness and substrate rigidity, might attenuate TGF-β signaling events to modulate OC differentiation and concomitant actin ring formation and bone matrix resorption in RANKL-induced osteoclastogenesis.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

Early Unloading After ACL Rupture and Prior to Surgical Restabilization in Mice Slows Post-Traumatic Osteoarthritis Progression

Purpose: People who sustain joint injuries such as anterior cruciate ligament (ACL) rupture often go on to develop post-traumatic osteoarthritis (PTOA). ACL injuries are often treated with ACL reconstruction, but there is typically a gap of several weeks between injury and surgery. However, it is unclear how loading or unloading of the injured joint during the early postinjury period affects the progression of PTOA. The goal of this study was to determine how unloading between noninvasive ACL injury and surgical restabilization of the injured joint affects PTOA progression in mice. Findings: Mice were subjected to noninvasive ACL injury or no injury followed by 1 week of hindlimb unloading (HLU) or normal cage activity. After 1 week of HLU or cage activity, mice underwent restabilization surgery or no surgery. ACL injury resulted in considerable epiphyseal trabecular bone loss regardless of HLU or cage activity. HLU groups exhibited significantly reduced chondrophyte/osteophyte formation, OA scoring, and synovitis at day 42. Single-cell RNA sequencing revealed that 1 week of HLU resulted in more neutrophils and less monocytes-macrophages in the injured joint. Conclusions: This study establishes that 1 week of HLU after ACL injury effectively slowed PTOA progression, suggesting that the early inflammatory response and joint instability play a key role in PTOA initiation and progression, and neutrophils and monocytes-macrophages play roles in the modulation. However, subsequent joint restabilization surgery caused greater inflammatory protease activity in the joint and exacerbated the loss of epiphyseal trabecular bone but did not significantly diminish OA score or synovitis.

ACL injury↗