Mapping and Characterization of HCMV-Specific Unconventional HLA-E-Restricted CD8 T Cell Populations and Associated NK and T Cell Responses Using HLA/Peptide Tetramers and Spectral Flow Cytometry

HCMV drives complex and multiple cellular immune responses, which causes a persistent immune imprint in hosts. This study aimed to achieve both a quantitative determination of the frequency for various anti-HCMV immune cell subsets, including CD8 T, γδT, NK cells, and a qualitative analysis of their phenotype. To map the various anti-HCMV cellular responses, we used a combination of three HLA_peptide tetramer complexes (HLA-E_VMAPRTLIL, HLA-E_VMAPRSLLL, and HLA-A2_NLVPMVATV) and antibodies for 18 surface markers (CD3, CD4, CD8, CD16, CD19, CD45RA, CD56, CD57, CD158, NKG2A, NKG2C, CCR7, TCRγδ, TCRγδ2, CX3CR1, KLRG1, 2B4, and PD-1) in a 20-color spectral flow cytometry analysis. This immunostaining protocol was applied to PBMCs isolated from HCMV⁻ and HCMV⁺ individuals. Our workflow allows the efficient determination of events featuring HCMV infection such as CD4/CD8 ratio, CD8 inflation and differentiation, HCMV peptide-specific HLA-E_UL40 and HLA-A2_pp65CD8 T cells, and expansion of γδT and NK subsets including δ2⁻γT and memory-like NKG2C⁺CD57⁺ NK cells. Each subset can be further characterized by the expression of 2B4, PD-1, KLRG1, CD45RA, CCR7, CD158, and NKG2A to achieve a fine-tuned mapping of HCMV immune responses. This assay should be useful for the analysis and monitoring of T-and NK cell responses to HCMV infection or vaccines.     

The PI-3-Kinase P110α Catalytic Subunit of T Lymphocytes Modulates Collagen-Induced Arthritis

The phosphatidylinositol 3-kinase (PI3K) family of enzymes plays a determinant role in inflammation and autoimmune responses. However, the implication of the different isoforms of catalytic subunits in these processes is not clear. Rheumatoid arthritis (RA) is a chronic, systemic autoimmune inflammatory disease that entails innate and adaptive immune response elements in which PI3K is a potential hub for immune modulation. In a mouse transgenic model with T-cell-specific deletion of p110α catalytic chain (p110α^−/−ΔT), we show the modulation of collagen-induced arthritis (CIA) by this isoform of PI3K. In established arthritis, p110α^−/−ΔT mice show decreased prevalence of illness than their control siblings, higher IgG1 titers and lower levels of IL-6 in serum, together with decreased ex vivo Collagen II (CII)-induced proliferation, IL-17A secretion and proportion of naive T cells in the lymph nodes. In a pre-arthritis phase, at 13 days post-Ag, T-cell-specific deletion of p110α chain induced an increased, less pathogenic IgG1/IgG2a antibodies ratio; changes in the fraction of naive and effector CD4⁺ subpopulations; and an increased number of CXCR5⁺ T cells in the draining lymph nodes of the p110α^−/−ΔT mice. Strikingly, T-cell blasts in vitro obtained from non-immunized p110α^−/−ΔT mice showed an increased expression of CXCR5, CD44 and ICOS surface markers and defective ICOS-induced signaling towards Akt phosphorylation. These results, plus the accumulation of cells in the lymph nodes in the early phase of the process, could explain the diminished illness incidence and prevalence in the p110α^−/−ΔT mice and suggests a modulation of CIA by the p110α catalytic chain of PI3K, opening new avenues of intervention in T-cell-directed therapies to autoimmune diseases.     

ASC Regulates Subcutaneous Adipose Tissue Lipogenesis and Lipolysis via p53/AMPKα Axis

Obesity has become an extensive threat to human health due to associated chronic inflammation and metabolic diseases. Apoptosis-associated speck-like protein (ASC) is a critical link between inflammasome and apoptosis-inducing proteins. In this study, we aimed to clarify the role of ASC in lipid metabolism. With high-fat diet (HFD) and knockout leptin gene mice (ob/ob), we found that ASC expression in subcutaneous adipose tissue (SAT) correlated with obesity. It could also positively regulate the reprogramming of cellular energy metabolism. Stromal vascular fractions (SVF) cells derived from the SAT of Asc^−/− mice or SVF from wild-type (WT) mice transfected with ASC siRNA were used to further investigate the underlying molecular mechanisms. We found ASC deficiency could lead to lipogenesis and inhibit lipolysis in SAT, aggravating lipid accumulation and impairing metabolic balance. In addition, our results showed that p53 and AMPKα expression were inhibited in SAT when ASC level was low. p53 and AMP-activated protein kinase α (AMPKα) were then assessed to elucidate whether they were downstream of ASC in regulating lipid metabolism. Our results revealed that ASC deficiency could promote lipid accumulation by increasing lipogenesis and decreasing lipolysis through p53/AMPKα axis. Regulation of ASC on lipid metabolism might be a novel therapeutic target for obesity.     

Korean Red Ginseng Improves Astrocytic Mitochondrial Function by Upregulating HO-1-Mediated AMPKα–PGC-1α–ERRα Circuit after Traumatic Brain Injury

Heme oxygenase-1 (HO-1) exerts beneficial effects, including angiogenesis and energy metabolism via the peroxisome proliferator-activating receptor-γ coactivator-1α (PGC-1α)–estrogen-related receptor α (ERRα) pathway in astrocytes. However, the role of Korean red ginseng extract (KRGE) in HO-1-mediated mitochondrial function in traumatic brain injury (TBI) is not well-elucidated. We found that HO-1 was upregulated in astrocytes located in peri-injured brain regions after a TBI, following exposure to KRGE. Experiments with pharmacological inhibitors and target-specific siRNAs revealed that HO-1 levels highly correlated with increased AMP-activated protein kinase α (AMPKα) activation, which led to the PGC-1α-ERRα axis-induced increases in mitochondrial functions (detected based on expression of cytochrome c oxidase subunit 2 (MTCO2) and cytochrome c as well as O₂ consumption and ATP production). Knockdown of ERRα significantly reduced the p-AMPKα/AMPKα ratio and PGC-1α expression, leading to AMPKα–PGC-1α–ERRα circuit formation. Inactivation of HO by injecting the HO inhibitor Sn(IV) protoporphyrin IX dichloride diminished the expression of p-AMPKα, PGC-1α, ERRα, MTCO2, and cytochrome c in the KRGE-administered peri-injured region of a brain subjected to TBI. These data suggest that KRGE enhanced astrocytic mitochondrial function via a HO-1-mediated AMPKα–PGC-1α–ERRα circuit and consequent oxidative phosphorylation, O₂ consumption, and ATP production. This circuit may play an important role in repairing neurovascular function after TBI in the peri-injured region by stimulating astrocytic mitochondrial biogenesis.     

Preparation and Characterization of a Chloroperoxidase-like Catalytic Antibody

The small molecule, meso-tetra(α,α,α,α-o-phenylacetamidophenyl) porphyrin (Mr1147.0) was used as complete antigen to elicit MAb through the immunization and cell fusion techniques. The MAb 1F2 obtained was demonstrated to be very pure by MALDI/TOFMS. The subtype of MAb 1F2 is IgG2a, which has a relative molecular weight of 156,678.8 Da.No significant change in the intensity of absorption peaks in UV and CD spectra was observed over a pH range between 6 and 12. The high stability of the abzyme and the tight binding between Fe porphyrin and antibody were also demonstrated. V_max, K_m, κcat, κcat/K_m for abzyme are 5.18 × 10⁻⁸ Ms⁻¹, 1.50 × 10⁻⁸ M, 0.518 s⁻¹, 3.45 × 10⁷ M⁻¹s⁻¹, respectively. The data obtained indicate that catalytic antibody has high catalytic activity. The chloroperoxidase activity of MAb 1F2-Fe porphyrin complex is stable from 10 °C to 60 °C.     

Alicyclobacillus mali FL18 as a Novel Source of Glycosyl Hydrolases: Characterization of a New Thermophilic β-Xylosidase Tolerant to Monosaccharides

A thermo-acidophilic bacterium, Alicyclobacillus mali FL18, was isolated from a hot spring of Pisciarelli, near Naples, Italy; following genome analysis, a novel putative β-xylosidase, AmβXyl, belonging to the glycosyl hydrolase (GH) family 3 was identified. A synthetic gene was produced, cloned in pET-30a(+), and expressed in Escherichia coli BL21 (DE3) RIL. The purified recombinant protein, which showed a dimeric structure, had optimal catalytic activity at 80 °C and pH 5.6, exhibiting 60% of its activity after 2 h at 50 °C and displaying high stability (more than 80%) at pH 5.0–8.0 after 16 h. AmβXyl is mainly active on both para-nitrophenyl-β-D-xylopyranoside (K_M 0.52 mM, k_cat 1606 s⁻¹, and k_cat/K_M 3088.46 mM⁻¹·s⁻¹) and para-nitrophenyl-α-L-arabinofuranoside (K_M 10.56 mM, k_cat 2395.8 s⁻¹, and k_cat/K_M 226.87 mM⁻¹·s⁻¹). Thin-layer chromatography showed its ability to convert xylooligomers (xylobiose and xylotriose) into xylose, confirming that AmβXyl is a true β-xylosidase. Furthermore, no inhibitory effect on enzymatic activity by metal ions, detergents, or EDTA was observed except for 5 mM Cu²⁺. AmβXyl showed an excellent tolerance to organic solvents; in particular, the enzyme increased its activity at high concentrations (30%) of organic solvents such as ethanol, methanol, and DMSO. Lastly, the enzyme showed not only a good tolerance to inhibition by xylose, arabinose, and glucose, but was activated by 0.75 M xylose and up to 1.5 M by both arabinose and glucose. The high tolerance to organic solvents and monosaccharides together with other characteristics reported above suggests that AmβXyl may have several applications in many industrial fields.     

Deficiency of IL-27 Signaling Exacerbates Experimental Autoimmune Uveitis with Elevated Uveitogenic Th1 and Th17 Responses

Human uveitis is an autoimmune disease of the central nervous system that is characterized by ocular inflammation with the involvement of uveitogenic Th1 and Th17 responses. In experimental autoimmune uveitis (EAU), the animal model for human uveitis, both responses are proven to be critical in disease development. Therefore, targeting both Th1 and Th17 cells has therapeutic implication for disease resolution. IL-27 is a multifunctional cytokine that can either promote or inhibit T cell responses and is implicated in both autoimmune and infectious diseases. The aim of this study is to characterize the role of IL-27/IL-27R signaling in regulating uveitogenic Th1/Th17 responses in EAU. By immunizing IL-27Rα^−/− mice and their wild-type (WT) littermates for EAU, we demonstrated that IL-27 signaling deficiency exacerbated EAU with severe ocular inflammation and impairment of visual function. Furthermore, there was a significant increase in the eye-infiltrating Th1 and Th17 cells in IL-27Rα^−/− EAU mice compared to WT. Their retinal antigen-specific Th1 and Th17 responses were also significantly increased, as represented by the elevation of their signature cytokines, IFN-γ and IL-17A, respectively. We also observed the upregulation of another pathogenic cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), from effector T cells in IL-27Rα^−/− EAU mice. Mechanistic studies confirmed that IL-27 inhibited GM-CSF production from Th17 cells. In addition, the induction of IL-10 producing type 1 regulatory T (Tr1) cells was impaired in IL-27Rα^−/− EAU mice. These results identified that IL-27 signaling plays a suppressive role in EAU by regulating multiple CD4⁺ cell subsets, including the effector Th1 and Th17 cells and the regulatory Tr1 cells. Our findings provide new insights for therapeutic potential in controlling uveitis by enhancing IL-27 signaling.     

Effects of Aflatoxin B1 on T-Cell Subsets and mRNA Expression of Cytokines in the Intestine of Broilers

This study was conducted to investigate the effects of aflatoxin B₁ (AFB₁) on T-cell subsets and mRNA expression of cytokines in the small intestine of broilers. One hundred and fifty-six one-day-old healthy Cobb broilers were randomly divided into control group (0 mg/kg AFB₁) and AFB₁ group (0.6 mg/kg AFB₁) with three replicates per group and 26 birds per replicate for 21 days, respectively. At 7, 14, and 21 days of age, the duodenum, jejunum and ileum were sampled for analyzing T cell subsets (CD3⁺, CD3⁺CD4⁺ and CD3⁺CD8⁺) by flow cytometry as well as IL-2, IL-4, IL-6, IL-10, IL-17, IFN-γ and TNF-α mRNA expression by qRT-PCR. The percentages of T-cells in the intra-epithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) of duodenum, jejunum and ileum in the AFB₁ group showed a decreased tendency in comparison to the control group. The mRNA expression of cytokines in the three intestinal segments in the AFB₁ group presented a general decline compared with the control groups. Our data demonstrated that 0.6 mg/kg AFB₁ in the broilers diet could reduce the percentages of T-cell subsets and the expression level of cytokine mRNA in the small intestine, implying that the immune function of the intestinal mucosa might be affected. The reduction of cytokines mRNA expression may be closely associated with the decreased proportions of T cells subsets induced by AFB₁.     

Partial Endothelial Nitric Oxide Synthase Deficiency Exacerbates Cognitive Deficit and Amyloid Pathology in the APPswe/PS1ΔE9 Mouse Model of Alzheimer’s Disease

Increasing evidence implicates endothelial dysfunction in the pathogenesis of Alzheimer’s disease (AD). Nitric oxide (NO) derived from endothelial NO synthase (eNOS) is essential in maintaining cerebrovascular function and can modulate the production and clearance of amyloid beta (Aβ). APPswe/PSdE1 (APP/PS1) mice display age-related Aβ accumulation and memory deficits. In order to make the model more clinically relevant with an element of endothelial dysfunction, we generated APP/PS1/eNOS^+/− mice by crossing complete eNOS deficient (eNOS^−/−) mice and APP/PS1 mice. APP/PS1/eNOS^+/− mice at 8 months of age displayed a more severe spatial working memory deficit relative to age-matched APP/PS1 mice. Moreover, immunohistochemistry and immunoblotting revealed significantly increased Aβ plaque load in the brains of APP/PS1/eNOS^+/− mice, concomitant with upregulated BACE-1 (hence increased Aβ production), downregulated insulin-degrading enzyme (hence reduced Aβ clearance) and increased immunoreactivity and expression of microglia. The present study, for the first time, demonstrated that partial eNOS deficiency exacerbated behavioral dysfunction, Aβ brain deposition, and microglial pathology in APP/PS1 mice, further implicating endothelial dysfunction in the pathogenesis of AD. The present findings also provide the scientific basis for developing preventive and/or therapeutic strategies by targeting endothelial dysfunction.     

HIF-1α Dependent Upregulation of ZIP8, ZIP14, and TRPA1 Modify Intracellular Zn2+ Accumulation in Inflammatory Synoviocytes

Intracellular free zinc ([Zn²⁺]_i) is mobilized in neuronal and non-neuronal cells under physiological and/or pathophysiological conditions; therefore, [Zn²⁺]_i is a component of cellular signal transduction in biological systems. Although several transporters and ion channels that carry Zn²⁺ have been identified, proteins that are involved in Zn²⁺ supply into cells and their expression are poorly understood, particularly under inflammatory conditions. Here, we show that the expression of Zn²⁺ transporters ZIP8 and ZIP14 is increased via the activation of hypoxia-induced factor 1α (HIF-1α) in inflammation, leading to [Zn²⁺]_i accumulation, which intrinsically activates transient receptor potential ankyrin 1 (TRPA1) channel and elevates basal [Zn²⁺]_i. In human fibroblast-like synoviocytes (FLSs), treatment with inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1α (IL-1α), evoked TRPA1-dependent intrinsic Ca²⁺ oscillations. Assays with fluorescent Zn²⁺ indicators revealed that the basal [Zn²⁺]_i concentration was significantly higher in TRPA1-expressing HEK cells and inflammatory FLSs. Moreover, TRPA1 activation induced an elevation of [Zn²⁺]_i level in the presence of 1 μM Zn²⁺ in inflammatory FLSs. Among the 17 out of 24 known Zn²⁺ transporters, FLSs that were treated with TNF-α and IL-1α exhibited a higher expression of ZIP8 and ZIP14. Their expression levels were augmented by transfection with an active component of nuclear factor-κB P65 and HIF-1α expression vectors, and they could be abolished by pretreatment with the HIF-1α inhibitor echinomycin (Echi). The functional expression of ZIP8 and ZIP14 in HEK cells significantly increased the basal [Zn²⁺]_i level. Taken together, Zn²⁺ carrier proteins, TRPA1, ZIP8, and ZIP14, induced under HIF-1α mediated inflammation can synergistically change [Zn²⁺]_i in inflammatory FLSs.     

The Dual Role of the β2-Adrenoreceptor in the Modulation of IL-17 and IFN-γ Production by T Cells in Multiple Sclerosis

Norepinephrine is a neurotransmitter that also has an immunomodulatory effect and is involved in multiple sclerosis (MS) pathogenesis. This study aimed to clarify the role of the β₂-adrenoreceptor in the norepinephrine-mediated modulation of interleukin-17 (IL-17) and interferon-γ (IFN-γ) production, which play a critical pathogenetic role in MS. CD4⁺ T cells obtained from twenty-five relapsing-remitting MS patients and sixteen healthy subjects were cultured ex vivo with norepinephrine and/or β₂-adrenoreceptor antagonist or agonist, followed by a cytokine production analysis using ELISA. Norepinephrine suppressed IL-17 and IFN-γ production by the anti-CD3/anti-CD28-microbead-stimulated CD4⁺ T cells in both groups. Blockade of the β₂-adrenoreceptor with the specific antagonist ICI 118.551 enhanced norepinephrine-mediated IL-17 suppression but decreased its inhibitory effect on IFN-γ production in MS patients. In contrast, the β₂-adrenoreceptor agonist formoterol did not influence norepinephrine’s inhibitory effect on cytokine production in both groups. The blockade of the β₂-adrenoreceptor, even in the absence of exogenous norepinephrine, suppressed IL-17 production but did not influence IFN-γ production in both groups. Conversely, β₂-adrenoreceptor activation by formoterol decreased IFN-γ production and did not affect IL-17 production in both groups. These data illustrate the inhibitory effect of norepinephrine on IL-17 and IFN-γ production by CD4⁺ T cells in MS. The inhibitory effect of norepinephrine on IFN-γ production by CD4⁺ T cells in MS could be mediated via β₂-adrenoreceptor activation.     

Interferon Beta-1a versus Combined Interferon Beta-1a and Oligodendrocyte-Specific FGFR1 Deletion in Experimental Autoimmune Encephalomyelitis

Recombinant beta interferons-1 (IFNβ-1) are used as first line therapies in patients with relapsing multiple sclerosis (MS), a chronic inflammatory and neurodegenerative disease of the CNS. IFNβ-1a/b has moderate effects on the prevention of relapses and slowing of disease progression. Fibroblast growth factors (FGFs) and FGF receptors (FGFRs) are known to play a key role in the pathology of MS and its model EAE. To investigate the effects of short-term treatment with s.c. IFNβ-1a versus the combined application of s.c. IFNβ-1a and oligodendrocyte-specific deletion of FGFR1 (Fgfr1^ind−/− mice) in MOG_35-55-induced EAE. IFNβ-1a (30 mg/kg) was applied s.c. from days 0–7 p.i. of EAE in controls and Fgfr1^ind−/− mice. FGFR signaling proteins associated with inflammation/degeneration in MS/EAE were analyzed by western blot in the spinal cord. Further, FGFR1 in Oli-neu oligodendrocytes were inhibited by PD166866 and treated with IFNβ-1a (400 ng/mL). Application of IFNβ-1a over 8 days resulted in less symptoms only at the peak of disease (days 9–11) compared to controls. Application of IFNβ-1a in Fgfr1^ind−/− mice resulted in less symptoms primarily in the chronic phase of EAE. Fgfr1^ind−/− mice treated with IFNβ-1a showed increased expression of pERK and BDNF. In Oli-neu oligodendrocytes, treatment with PD166866 and IFNβ-1a also showed an increased expression of pERK and BDNF/TrkB. These data suggest that the beneficial effects in the chronic phase of EAE and on signaling molecules associated with ERK and BDNF expression are caused by the modulation of FGFR1 and not by interferon beta-1a. FGFR may be a potential target for therapy in MS.     

Expression of IL-8, IL-6 and IL-1β in Tears as a Main Characteristic of the Immune Response in Human Microbial Keratitis

Corneal infections are frequent and potentially vision-threatening diseases, and despite the significance of the immunological response in animal models of microbial keratitis (MK), it remains unclear in humans. The aim of this study was to describe the cytokine profile of tears in patients with MK. Characteristics of ocular lesions such as size of the epithelial defect, stromal infiltration, and hypopyon were analyzed. Immunological evaluation included determination of interleukine (IL)-1β, IL-6, IL-8, IL-10, IL-12 and tumor necrosis factor (TNF)-α in tear samples obtained from infected eyes of 28 patients with MK and compared with their contralateral non-infected eyes. Additionally, frequency of CD4⁺, CD8⁺, CD19⁺ and CD3⁻CD56⁺ cells was also determined in peripheral blood mononuclear cells in patients with MK, and compared with 48 healthy controls. Non-significant differences were observed in the size of the epithelial defect, stromal infiltration, and hypopyon. Nevertheless, we found an immunological profile apparently related to MK etiology. IL-8 > IL-6 in patients with bacterial keratitis; IL-8 > IL-6 > IL-1β and increased frequency of circulating CD3⁻CD56⁺ NK cells in patients with gram-negative keratitis; and IL-8 = IL-6 > IL-1β in patients with fungal keratitis. Characterization of tear cytokines from patients with MK could aid our understanding of the immune pathophysiological mechanisms underlying corneal damage in humans.     

Chondrocytes from Osteoarthritis Patients Adopt Distinct Phenotypes in Response to Central TH1/TH2/TH17 Cytokines

Chronic low-grade inflammation plays a central role in the pathogenesis of osteoarthritis (OA), and several pro- and anti-inflammatory cytokines have been implicated to mediate and regulate this process. Out of these cytokines, particularly IFNγ, IL-1β, IL-4 and IL-17 are associated with different phenotypes of T helper (T_H) cells and macrophages, both examples of cells known for great phenotypic and functional heterogeneity. Chondrocytes also display various phenotypic changes during the course of arthritis. We set out to study the hypothesis of whether chondrocytes might adopt polarized phenotypes analogous to T_H cells and macrophages. We studied the effects of IFNγ, IL-1β, IL-4 and IL-17 on gene expression in OA chondrocytes with RNA-Seq. Chondrocytes were harvested from the cartilage of OA patients undergoing knee replacement surgery and then cultured with or without the cytokines for 24 h. Total RNA was isolated and sequenced, and GO (Gene Ontology) functional analysis was performed. We also separately investigated genes linked to OA in recent genome wide expression analysis (GWEA) studies. The expression of more than 2800 genes was significantly altered in chondrocytes treated with IL-1β [in the C(IL-1β) phenotype] with a fold change (FC) > 2.5 in either direction. These included a large number of genes associated with inflammation, cartilage degradation and attenuation of metabolic signaling. The profile of genes differentially affected by IFNγ (the C(IFNγ) phenotype) was relatively distinct from that of the C(IL-1β) phenotype and included several genes associated with antigen processing and presentation. The IL-17-induced C(IL-17) phenotype was characterized by the induction of a more limited set of proinflammatory factors compared to C(IL-1β) cells. The C(IL-4) phenotype induced by IL-4 displayed a differential expression of a rather small set of genes compared with control, primarily those associated with TGFβ signaling and the regulation of inflammation. In conclusion, our results show that OA chondrocytes can adopt diverse phenotypes partly analogously to T_H cells and macrophages. This phenotypic plasticity may play a role in the pathogenesis of arthritis and open new therapeutic avenues for the development of disease-modifying treatments for (osteo)arthritis.     

Sphk1 and Sphk2 Differentially Regulate Erythropoietin Synthesis in Mouse Renal Interstitial Fibroblast-like Cells

Erythropoietin (Epo) is a crucial hormone regulating red blood cell number and consequently the hematocrit. Epo is mainly produced in the kidney by interstitial fibroblast-like cells. Previously, we have shown that in cultures of the immortalized mouse renal fibroblast-like cell line FAIK F3-5, sphingosine 1-phosphate (S1P), by activating S1P₁ and S1P₃ receptors, can stabilize hypoxia-inducible factor (HIF)-2α and upregulate Epo mRNA and protein synthesis. In this study, we have addressed the role of intracellular iS1P derived from sphingosine kinases (Sphk) 1 and 2 on Epo synthesis in F3-5 cells and in mouse primary cultures of renal fibroblasts. We show that stable knockdown of Sphk2 in F3-5 cells increases HIF-2α protein and Epo mRNA and protein levels, while Sphk1 knockdown leads to a reduction of hypoxia-stimulated HIF-2α and Epo protein. A similar effect was obtained using primary cultures of renal fibroblasts isolated from wildtype mice, Sphk1−/−, or Sphk2−/− mice. Furthermore, selective Sphk2 inhibitors mimicked the effect of genetic Sphk2 depletion and also upregulated HIF-2α and Epo protein levels. The combined blockade of Sphk1 and Sphk2, using Sphk2−/− renal fibroblasts treated with the Sphk1 inhibitor PF543, resulted in reduced HIF-2α and Epo compared to the untreated Sphk2−/− cells. Exogenous sphingosine (Sph) enhanced HIF-2α and Epo, and this was abolished by the combined treatment with the selective S1P₁ and S1P₃ antagonists NIBR-0213 and TY52156, suggesting that Sph was taken up by cells and converted to iS1P and exported to then act in an autocrine manner through S1P₁ and S1P₃. The upregulation of HIF-2α and Epo synthesis by Sphk2 knockdown was confirmed in the human hepatoma cell line Hep3B, which is well-established to upregulate Epo production under hypoxia. In summary, these data show that sphingolipids have diverse effects on Epo synthesis. While accumulation of intracellular Sph reduces Epo synthesis, iS1P will be exported to act through S1P₁₊₃ to enhance Epo synthesis. Furthermore, these data suggest that selective inhibition of Sphk2 is an attractive new option to enhance Epo synthesis and thereby to reduce anemia development in chronic kidney disease.     

PLG-007 and Its Active Component Galactomannan-α Competitively Inhibit Enzymes That Hydrolyze Glucose Polymers

PLG-007 is a developmental therapeutic compound that has been clinically shown to reduce the magnitude of postprandial glucose excursions and has the potential to be an adjunct treatment for diabetes and inflammatory-related diseases. The present investigation is aimed at understanding the molecular mechanism of action of PLG-007 and its galactomannan (GM) components GMα and GMβ (in a 1:4 mass ratio, respectively) on enzyme (i.e., α-amylase, maltase, and lactase) hydrolysis of glucose polymers using colorimetric assays and ¹³C HSQC NMR spectroscopy. The starch–iodine colorimetric assay indicated that GMα strongly inhibits α-amylase activity (~16-fold more potent than GMβ) and thus is the primary active component in PLG-007. ¹³C HSQC experiments, used to follow the α-amylase-mediated hydrolysis of starch and amylopectin, further demonstrate the α-amylase inhibitory effect of GMα via α-amylase-mediated hydrolysis of starch and amylopectin. Maltohexaose (MT6) was used to circumvent the relative kinetic complexity of starch/amylopectin degradation in Michaelis–Menten analyses. The V_max, K_M, and K_i parameters were determined using peak volume integrals from ¹³C HSQC NMR spectra. In the presence of PLG-007 with α-amylase and MT6, the increase in K_M from 7.5 ± 0.6 × 10⁻³ M (control) to 21 ± 1.4 × 10⁻³ M, with no significant change in V_max, indicates that PLG-007 is a competitive inhibitor of α-amylase. Using K_M values, K_i was estimated to be 2.1 ± 0.9 × 10⁻⁶ M; however, the microscopic K_i value of GMα is expected to be larger as the binding stoichiometry is likely to be greater than 1:1. Colorimetric assays also demonstrated that GMα is a competitive inhibitor of the enzymes maltase and lactase. Overall, this study provides insight as to how PLG-007 (GMα) is likely to function in vivo.     

Mechanisms of Cell Killing Response from Low Linear Energy Transfer (LET) Radiation Originating from 177Lu Radioimmunotherapy Targeting Disseminated Intraperitoneal Tumor Xenografts

Radiolabeled antibodies (mAbs) provide efficient tools for cancer therapy. The combination of low energy β⁻-emissions (500 keV_max; 130 keV_ave) along with a γ-emission for imaging makes ¹⁷⁷Lu (T_1/2 = 6.7 day) a suitable radionuclide for radioimmunotherapy (RIT) of tumor burdens possibly too large to treat with α-particle radiation. RIT with ¹⁷⁷Lu-trastuzumab has proven to be effective for treatment of disseminated HER2 positive peritoneal disease in a pre-clinical model. To elucidate mechanisms originating from this RIT therapy at the molecular level, tumor bearing mice (LS-174T intraperitoneal xenografts) were treated with ¹⁷⁷Lu-trastuzumab comparatively to animals treated with a non-specific control, ¹⁷⁷Lu-HuIgG, and then to prior published results obtained using ²¹²Pb-trastuzumab, an α-particle RIT agent. ¹⁷⁷Lu-trastuzumab induced cell death via DNA double strand breaks (DSB), caspase-3 apoptosis, and interfered with DNA-PK expression, which is associated with the repair of DNA non-homologous end joining damage. This contrasts to prior results, wherein ²¹²Pb-trastuzumab was found to down-regulate RAD51, which is involved with homologous recombination DNA damage repair. ¹⁷⁷Lu-trastuzumab therapy was associated with significant chromosomal disruption and up-regulation of genes in the apoptotic process. These results suggest an inhibition of the repair mechanism specific to the type of radiation damage being inflicted by either high or low linear energy transfer radiation. Understanding the mechanisms of action of β⁻- and α-particle RIT comparatively through an in vivo tumor environment offers real information suitable to enhance combination therapy regimens involving α- and β⁻-particle RIT for the management of intraperitoneal disease.     

ROS/TNF-α Crosstalk Triggers the Expression of IL-8 and MCP-1 in Human Monocytic THP-1 Cells via the NF-κB and ERK1/2 Mediated Signaling

IL-8/MCP-1 act as neutrophil/monocyte chemoattractants, respectively. Oxidative stress emerges as a key player in the pathophysiology of obesity. However, it remains unclear whether the TNF-α/oxidative stress interplay can trigger IL-8/MCP-1 expression and, if so, by which mechanism(s). IL-8/MCP-1 adipose expression was detected in lean, overweight, and obese individuals, 15 each, using immunohistochemistry. To detect the role of reactive oxygen species (ROS)/TNF-α synergy as a chemokine driver, THP-1 cells were stimulated with TNF-α, with/without H₂O₂ or hypoxia. Target gene expression was measured by qRT-PCR, proteins by flow cytometry/confocal microscopy, ROS by DCFH-DA assay, and signaling pathways by immunoblotting. IL-8/MCP-1 adipose expression was significantly higher in obese/overweight. Furthermore, IL-8/MCP-1 mRNA/protein was amplified in monocytic cells following stimulation with TNF-α in the presence of H₂O₂ or hypoxia (p ˂ 0.0001). Synergistic chemokine upregulation was related to the ROS levels, while pre-treatments with NAC suppressed this chemokine elevation (p ≤ 0.01). The ROS/TNF-α crosstalk involved upregulation of CHOP, ERN1, HIF1A, and NF-κB/ERK-1,2 mediated signaling. In conclusion, IL-8/MCP-1 adipose expression is elevated in obesity. Mechanistically, ROS/TNF-α crosstalk may drive expression of these chemokines in monocytic cells by inducing ER stress, HIF1A stabilization, and signaling via NF-κB/ERK-1,2. NAC had inhibitory effect on oxidative stress-driven IL-8/MCP-1 expression, which may have therapeutic significance regarding meta-inflammation.