Engineering a soluble extracellular erythropoietin receptor (EPObp) in Pichia pastoris to eliminate microheterogeneity, and its complex with erythropoietin

The extracellular ligand-binding domain (EPObp) of the humanEPO receptor (EPOR) was expressed both in CHO (Chinese HamsterOvary) cells and in Pichia pastoris. The CHO and yeast expressedreceptors showed identical affinity for EPO binding. Expressionlevels in P.pastoris were significantly higher, favoring itsuse as an expression and scale-up production system. Incubationof EPO with a fourfold molar excess of receptor at high proteinconcentrations yielded stable EPO–EPObp complexes. Quantificationof EPO and EPObp in the complex yielded a molar ratio of oneEPO molecule to two receptor molecules. Residues that are responsiblefor EPOR glycosylation and isomerization in Pichia were identifiedand eliminated by site-specific mutagenesis. A thiol modificationwas identified and a method was developed to remove the modifiedspecies from EPObp. EPObp was complexed with erythropoietin(EPO) and purified. The complex crystallized in two crystalforms that diffracted to 2.8 and 1.9 Å respectively. (Form1 and form 2 crystals were independently obtained at AxyS Pharmaceuticals,Inc. and Amgen, Inc. respectively.) Both contained one complexper asymmetric unit with a stoichiometry of two EPObps to oneEPO.     

Myricetin Prevents Alveolar Bone Loss in an Experimental Ovariectomized Mouse Model of Periodontitis

Periodontitis is a common chronic inflammatory disease, which leads to alveolar bone resorption. Healthy and functional alveolar bone, which can support the teeth and enable their movement, is very important for orthodontic treatment. Myricetin inhibited osteoclastogenesis by suppressing the expression of some genes, signaling pathways, and cytokines. This study aimed to investigate the effects of myricetin on alveolar bone loss in an ovariectomized (OVX) mouse model of periodontitis as well as in vitro osteoclast formation and bone resorption. Twenty-four healthy eight-week-old C57BL/J6 female mice were assigned randomly to four groups: phosphate-buffered saline (PBS) control (sham) OVX + ligature + PBS (vehicle), and OVX + ligature + low or high (2 or 5 mg∙kg⁻¹∙day⁻¹, respectively) doses of myricetin. Myricetin or PBS was injected intraperitoneally (i.p.) every other day for 30 days. The maxillae were collected and subjected to further examination, including micro-computed tomography (micro-CT), hematoxylin and eosin (H&E) staining, and tartrate-resistant acid phosphatase (TRAP) staining; a resorption pit assay was also performed in vitro to evaluate the effects of myricetin on receptor activator of nuclear factor κ-B ligand (RANKL)-induced osteoclastogenesis. Myricetin, at both high and low doses, prevented alveolar bone resorption and increased alveolar crest height in the mouse model and inhibited osteoclast formation and bone resorption in vitro. However, myricetin was more effective at high dose than at low dose. Our study demonstrated that myricetin had a positive effect on alveolar bone resorption in an OVX mouse model of periodontitis and, therefore, may be a potential agent for the treatment of periodontitis and osteoporosis.     

Potential Effect of CD271 on Human Mesenchymal Stromal Cell Proliferation and Differentiation

The Low-Affinity Nerve Growth Factor Receptor (LNGFR), also known as CD271, is a member of the tumor necrosis factor receptor superfamily. The CD271 cell surface marker defines a subset of multipotential mesenchymal stromal cells and may be used to isolate and enrich cells derived from bone marrow aspirate. In this study, we compare the proliferative and differentiation potentials of CD271⁺ and CD271⁻ mesenchymal stromal cells. Mesenchymal stromal cells were isolated from bone marrow aspirate and adipose tissue by plastic adherence and positive selection. The proliferation and differentiation potentials of CD271⁺ and CD271⁻ mesenchymal stromal cells were assessed by inducing osteogenic, adipogenic and chondrogenic in vitro differentiation. Compared to CD271⁺, CD271⁻ mesenchymal stromal cells showed a lower proliferation rate and a decreased ability to give rise to osteocytes, adipocytes and chondrocytes. Furthermore, we observed that CD271⁺ mesenchymal stromal cells isolated from adipose tissue displayed a higher efficiency of proliferation and trilineage differentiation compared to CD271⁺ mesenchymal stromal cells isolated from bone marrow samples, although the CD271 expression levels were comparable. In conclusion, these data show that both the presence of CD271 antigen and the source of mesenchymal stromal cells represent important factors in determining the ability of the cells to proliferate and differentiate.     

Myeloid Lineage Ablation of Phlpp1 Regulates M-CSF Signaling and Tempers Bone Resorption in Female Mice

Prior work demonstrated that Phlpp1 deficiency alters trabecular bone mass and enhances M-CSF responsiveness, but the cell types and requirement of Phlpp1 for this effect were unclear. To understand the function of Phlpp1 within myeloid lineage cells, we crossed Phlpp1 floxed mice with mice harboring LysM-Cre. Micro-computed tomography of the distal femur of 12-week-old mice revealed a 30% increase in bone volume per total volume of Phlpp1 female conditional knockouts, but we did not observe significant changes within male Phlpp1 cKO_LysM mice. Bone histomorphmetry of the proximal tibia further revealed that Phlpp1 cKO_LysM females exhibited elevated osteoclast numbers, but conversely had reduced levels of serum markers of bone resorption as compared to littermate controls. Osteoblast number and serum markers of bone formation were unchanged. In vitro assays confirmed that Phlpp1 ablation enhanced osteoclast number and area, but limited bone resorption. Additionally, reconstitution with exogenous Phlpp1 suppressed osteoclast numbers. Dose response assays demonstrated that Phlpp1^−/− cells are more responsive to M-CSF, but reconstitution with Phlpp1 abrogated this effect. Furthermore, small molecule-mediated Phlpp inhibition enhanced osteoclast numbers and size. Enhanced phosphorylation of Phlpp substrates—including Akt, ERK1/2, and PKCζ—accompanied these observations. In contrast, actin cytoskeleton disruption occurred within Phlpp inhibitor treated osteoclasts. Moreover, Phlpp inhibition reduced resorption of cells cultured on bovine bone slices in vitro. Our results demonstrate that Phlpp1 deficiency within myeloid lineage cells enhances bone mass by limiting bone resorption while leaving osteoclast numbers intact; moreover, we show that Phlpp1 represses osteoclastogenesis and controls responses to M-CSF.     

Distinct Immune Signatures Indicative of Treatment Response and Immune-Related Adverse Events in Melanoma Patients under Immune Checkpoint Inhibitor Therapy

To identify potential early biomarkers of treatment response and immune-related adverse events (irAE), a pilot immune monitoring study was performed in stage IV melanoma patients by flow cytometric analysis of peripheral blood mononuclear cells (PBMC). Overall, 17 patients were treated with either nivolumab or pembrolizumab alone, or with a combination of nivolumab and ipilimumab every three weeks. Of 15 patients for which complete response assessment was available, treatment responders (n = 10) as compared to non-responders (n = 5) were characterized by enhanced PD-1 expression on CD8⁺ T cells immediately before treatment (median ± median absolute deviation/MAD 26.7 ± 10.4% vs. 17.2 ± 5.3%). Responders showed a higher T cell responsiveness after T cell receptor ex vivo stimulation as determined by measurement of programmed cell death 1 (PD-1) expression on CD3⁺ T cells before the second cycle of treatment. The percentage of CD8⁺ effector memory (CD8⁺CD45RA⁻CD45RO⁺CCR7⁻) T cells was higher in responders compared to non-responders before and immediately after the first cycle of treatment (median ± MAD 39.2 ± 7.3% vs. 30.5 ± 4.1% and 37.7 ± 4.6 vs. 24.0 ± 6.4). Immune-related adverse events (irAE) were accompanied by a higher percentage of activated CD4⁺ (CD4⁺CD38⁺HLADR⁺) T cells before the second treatment cycle (median ± MAD 14.9 ± 3.9% vs. 5.3 ± 0.4%). In summary, PBMC immune monitoring of immune-checkpoint inhibition (ICI) treatment in melanoma appears to be a promising approach to identify early markers of treatment response and irAEs.     

Protocadherin-7 Regulates Osteoclast Differentiation through Intracellular SET-Binding Domain-Mediated RhoA and Rac1 Activation

Protocadherin-7 (Pcdh7) is a member of the non-clustered protocadherin δ1 subgroup of the cadherin superfamily. Although the cell-intrinsic role of Pcdh7 in osteoclast differentiation has been demonstrated, the molecular mechanisms of Pcdh7 regulating osteoclast differentiation remain to be determined. Here, we demonstrate that Pcdh7 contributes to osteoclast differentiation by regulating small GTPases, RhoA and Rac1, through its SET oncoprotein binding domain. Pcdh7 is associated with SET along with RhoA and Rac1 during osteoclast differentiation. Pcdh7-deficient (Pcdh7^−/−) cells showed abolished RANKL-induced RhoA and Rac1 activation, and impaired osteoclast differentiation. Impaired osteoclast differentiation in Pcdh7^−/− cells was restored by retroviral transduction of full-length Pcdh7 but not by a Pcdh7 mutant that lacks SET binding domain. The direct crosslink of the Pcdh7 intracellular region induced the activation of RhoA and Rac1, which was not observed when Pcdh7 lacks the SET binding domain. Additionally, retroviral transduction of the constitutively active form of RhoA and Rac1 completely restored the impaired osteoclast differentiation in Pcdh7^−/− cells. Collectively, these results demonstrate that Pcdh7 controls osteoclast differentiation by regulating RhoA and Rac1 activation through the SET binding domain.     

Combined Blockade of TIGIT and CD39 or A2AR Enhances NK-92 Cell-Mediated Cytotoxicity in AML

This study aimed to characterize different natural killer (NK) cell phenotypes on bone marrow and peripheral blood cells from acute myeloid leukemia (AML) patients and healthy donors (HDs). Our data show that CD56^dimCD16⁻ and CD56^brightCD16⁻ NK cells represent the predominant NK cell subpopulations in AML, while the CD56^dimCD16⁺ NK cells are significantly reduced compared to HDs. Moreover, TIGIT⁺ and PVRIG⁺ cells cluster on the CD56^dimCD16⁺ subset whereas CD39⁺ and CD38⁺ cells do so on CD56^brightCD16⁻ NK cells in AML. Furthermore, functional effects of (co-)blockade of TIGIT and CD39 or A2AR on NK cell functionality were analyzed. These experiments revealed that the single blockade of the TIGIT receptor results in an increased NK-92 cell-mediated killing of AML cells in vitro. Combined targeting of CD39 or A2AR significantly augments the anti-TIGIT-mediated lysis of AML cells. Our data indicate that distinct NK cell subsets in AML exhibit different immunosuppressive patterns (via the TIGIT/PVRIG receptors and the purinergic pathway). In summary, we conclude that TIGIT, CD39, and A2AR constitute relevant inhibitory checkpoints of NK cells in AML patients. A combinatorial blockade synergistically strengthens NK-92 cell-mediated cytotoxicity. As inhibitors of TIGIT, CD39, and A2AR are clinically available, studies on their combined use could be conducted in the near future.     

The CD200 Regulates Inflammation in Mice Independently of TNF-α Production

Inflammatory bowel disease is characterized by the infiltration of immune cells and chronic inflammation. The immune inhibitory receptor, CD200R, is involved in the downregulation of the activation of immune cells to prevent excessive inflammation. We aimed to define the role of CD200R ligand-CD200 in the experimental model of intestinal inflammation in conventionally-reared mice. Mice were given a dextran sodium sulfate solution in drinking water. Bodyweight loss was monitored daily and the disease activity index was calculated, and a histological evaluation of the colon was performed. TNF-α production was measured in the culture of small fragments of the distal colon or bone marrow-derived macrophages (BMDMs) cocultured with CD200⁺ cells. We found that Cd200^−/− mice displayed diminished severity of colitis when compared to WT mice. Inflammation significantly diminished CD200 expression in WT mice, particularly on vascular endothelial cells and immune cells. The co-culture of BMDMs with CD200⁺ cells inhibited TNF-α secretion. In vivo, acute colitis induced by DSS significantly increased TNF-α secretion in colon tissue in comparison to untreated controls. However, Cd200^−/− mice secreted a similar level of TNF-α to WT mice in vivo. CD200 regulates the severity of DSS-induced colitis in conventionally-reared mice. The presence of CD200⁺ cells decreases TNF-α production by macrophages in vitro. However, during DDS-induced intestinal inflammation secretion of TNF-α is independent of CD200 expression.     

Erythropoietin in Optic Neuropathies: Current Future Strategies for Optic Nerve Protection and Repair

Erythropoietin (EPO) is known as a hormone for erythropoiesis in response to anemia and hypoxia. However, the effect of EPO is not only limited to hematopoietic tissue. Several studies have highlighted the neuroprotective function of EPO in extra-hematopoietic tissues, especially the retina. EPO could interact with its heterodimer receptor (EPOR/βcR) to exert its anti-apoptosis, anti-inflammation and anti-oxidation effects in preventing retinal ganglion cells death through different intracellular signaling pathways. In this review, we summarized the available pre-clinical studies of EPO in treating glaucomatous optic neuropathy, optic neuritis, non-arteritic anterior ischemic optic neuropathy and traumatic optic neuropathy. In addition, we explore the future strategies of EPO for optic nerve protection and repair, including advances in EPO derivates, and EPO deliveries. These strategies will lead to a new chapter in the treatment of optic neuropathy.     

Lipocalin 2 Influences Bone and Muscle Phenotype in the MDX Mouse Model of Duchenne Muscular Dystrophy

Lipocalin 2 (Lcn2) is an adipokine involved in bone and energy metabolism. Its serum levels correlate with bone mechanical unloading and inflammation, two conditions representing hallmarks of Duchenne Muscular Dystrophy (DMD). Therefore, we investigated the role of Lcn2 in bone loss induced by muscle failure in the MDX mouse model of DMD. We found increased Lcn2 serum levels in MDX mice at 1, 3, 6, and 12 months of age. Consistently, Lcn2 mRNA was higher in MDX versus WT muscles. Immunohistochemistry showed Lcn2 expression in mononuclear cells between muscle fibres and in muscle fibres, thus confirming the gene expression results. We then ablated Lcn2 in MDX mice, breeding them with Lcn2^−/− mice (MDXxLcn2^−/−), resulting in a higher percentage of trabecular volume/total tissue volume compared to MDX mice, likely due to reduced bone resorption. Moreover, MDXxLcn2^−/− mice presented with higher grip strength, increased intact muscle fibres, and reduced serum creatine kinase levels compared to MDX. Consistently, blocking Lcn2 by treating 2-month-old MDX mice with an anti-Lcn2 monoclonal antibody (Lcn2Ab) increased trabecular volume, while reducing osteoclast surface/bone surface compared to MDX mice treated with irrelevant IgG. Grip force was also increased, and diaphragm fibrosis was reduced by the Lcn2Ab. These results suggest that Lcn2 could be a possible therapeutic target to treat DMD-induced bone loss.     

CD147 Is Essential for the Development of Psoriasis via the Induction of Th17 Cell Differentiation

Th17 cells play an important role in psoriasis. The differentiation of naïve CD4⁺ T cells into Th17 cells depends on glycolysis as the energy source. CD147/basigin, an integral transmembrane protein belonging to the immunoglobulin superfamily, regulates glycolysis in association with monocarboxylate transporters (MCTs)-1 and -4 in cancer cells and T cells. We examined whether CD147/basigin is involved in the pathogenesis of psoriasis in humans and psoriasis-model mice. The serum level of CD147 was increased in patients with psoriasis, and the expression of CD147 and MCT-1 was elevated in their dermal CD4⁺ RORγt⁺ T cells. In vitro, the potential of naïve CD4⁺ T cells to differentiate into Th17 cells was abrogated in CD147^−/− T cells. Imiquimod (IMQ)-induced psoriatic dermatitis was significantly milder in CD147^−/− mice and bone marrow chimeric mice lacking CD147 in the hematopoietic cells of myeloid lineage. These findings demonstrate that CD147 is essential for the development of psoriasis via the induction of Th17 cell differentiation.     

Repercussions of Bisphenol A on the Physiology of Human Osteoblasts

(1) Background: Bisphenol A (BPA) is an endocrine disruptor that is widely present in the environment and exerts adverse effects on various body tissues. The objective of this study was to determine its repercussions on bone tissue by examining its impact on selected functional parameters of human osteoblasts. (2) Methods: Three human osteoblast lines were treated with BPA at doses of 10⁻⁵, 10⁻⁶, or 10⁻⁷ M. At 24 h post-treatment, a dose-dependent inhibition of cell growth, alkaline phosphatase activity, and mineralization was observed. (4) Results: The expression of CD54 and CD80 antigens was increased at doses of 10⁻⁵ and 10⁻⁶ M, while the phagocytic capacity and the expression of osteogenic genes (ALP, COL-1, OSC, RUNX2, OSX, BMP-2, and BMP-7) were significantly and dose-dependently reduced in the presence of BPA. (5) Conclusions: According to these findings, BPA exerts adverse effects on osteoblasts by altering their differentiation/maturation and their proliferative and functional capacity, potentially affecting bone health. Given the widespread exposure to this contaminant, further human studies are warranted to determine the long-term risk to bone health posed by BPA.     

BMP3 Affects Cortical and Trabecular Long Bone Development in Mice

Bone morphogenetic proteins (BMPs) have a major role in tissue development. BMP3 is synthesized in osteocytes and mature osteoblasts and has an antagonistic effect on other BMPs in bone tissue. The main aim of this study was to fully characterize cortical bone and trabecular bone of long bones in both male and female Bmp3^−/− mice. To investigate the effect of Bmp3 from birth to maturity, we compared Bmp3^−/− mice with wild-type littermates at the following stages of postnatal development: 1 day (P0), 2 weeks (P14), 8 weeks and 16 weeks of age. Bmp3 deletion was confirmed using X-gal staining in P0 animals. Cartilage and bone tissue were examined in P14 animals using Alcian Blue/Alizarin Red staining. Detailed long bone analysis was performed in 8-week-old and 16-week-old animals using micro-CT. The Bmp3 reporter signal was localized in bone tissue, hair follicles, and lungs. Bone mineralization at 2 weeks of age was increased in long bones of Bmp3^−/− mice. Bmp3 deletion was shown to affect the skeleton until adulthood, where increased cortical and trabecular bone parameters were found in young and adult mice of both sexes, while delayed mineralization of the epiphyseal growth plate was found in adult Bmp3^−/− mice.     

A Combination of Chemotherapy and Oncolytic Virotherapy Sensitizes Colorectal Adenocarcinoma to Immune Checkpoint Inhibitors in a cDC1-Dependent Manner

Immune checkpoint therapy has shown great promise in the treatment of cancers with a high mutational burden, such as mismatch repair-deficient colorectal carcinoma (dMMR CRC). However, many patients fail to respond to immune checkpoint therapy. Using a mouse model of dMMR CRC, we demonstrated that tumors can be further sensitized to immune checkpoint therapy by using a combination of low-dose chemotherapy and oncolytic HSV-1. This combination induced the infiltration of CD8⁺ and CD4⁺ T cells into the tumor and the upregulation of gene signatures associated with the chemoattraction of myeloid cell subsets. When combined with immune checkpoint therapy, the combination promoted the infiltration of activated type 1 conventional dendritic cells (cDC1s) into the tumor. Furthermore, we found this combination strategy to be dependent on cDC1s, and its therapeutic efficacy to be abrogated in cDC1-deficient Batf3^−/− mice. Thus, we demonstrated that the adjuvanticity of dMMR CRCs can be improved by combining low-dose chemotherapy and oncolytic HSV-1 in a cDC1-dependent manner.     

Relaxin and Erythropoietin Significantly Reduce Uterine Tissue Damage during Experimental Ischemia–Reperfusion Injury

Successful uterus transplantation, a potential treatment method for women suffering from absolute uterine infertility, is negatively affected by ischemia–reperfusion injury (IRI). The aim of this study is to investigate the protective effect of relaxin (RLX) or/and erythropoietin (EPO) on experimental uterus IRI. Eighty rats, randomly assigned into eight groups (n = 10/group), were pretreated with either saline, 5 μg/kg human relaxin-2, 4000 IU/kg recombinant human erythropoietin or their combination. Ischemia was achieved by clamping the aorta and ovarian arteries for 60 min, following 120 min of reperfusion and tissue sampling. For sham animals, clamping was omitted during surgery. There were no differences in tissue histological score, malondialdehyde (MDA) and superoxide dismutase (SOD) levels, myeloperoxidase (MPO) and TUNEL-positive cell count between all sham-operated rats. Pretreatment with RLX preserved normal tissue morphology, reduced MDA levels, MPO and TUNEL-positive cell count, preserved SOD activity and upregulated NICD and HES1 gene expression when compared to the control group. Pretreatment with EPO reduced MDA levels. In conclusion, pretreatment with RLX, EPO or a combination of both EPO and RLX significantly alleviates uterine tissue damage caused by IRI.     

CTHRSSVVC Peptide as a Possible Early Molecular Imaging Target for Atherosclerosis

The purpose of our work was to select phages displaying peptides capable of binding to vascular markers present in human atheroma, and validate their capacity to target the vascular markers in vitro and in low-density lipoprotein receptor knockout (LDLr^−/−) mouse model of atherosclerosis. By peptide fingerprinting on human atherosclerotic tissues, we selected and isolated four different peptides sequences, which bind to atherosclerotic lesions and share significant similarity to known human proteins with prominent roles in atherosclerosis. The CTHRSSVVC-phage peptide displayed the strongest reactivity with human carotid atherosclerotic lesions (p < 0.05), when compared to tissues from normal carotid arteries. This peptide sequence shares similarity to a sequence present in the fifth scavenger receptor cysteine-rich (SRCR) domain of CD163, which appeared to bind to CD163, and subsequently, was internalized by macrophages. Moreover, the CTHRSSVVC-phage targets atherosclerotic lesions of a low-density lipoprotein receptor knockout (LDLr^−/−) mouse model of atherosclerosis in vivo to High-Fat diet group versus Control group. Tetraazacyclododecane-1,4,7,10-tetraacetic acid-CTHRSSVVC peptide (DOTA-CTHRSSVVC) was synthesized and labeled with ¹¹¹InCl₃ in >95% yield as determined by high performance liquid chromatography (HPLC), to validate the binding of the peptide in atherosclerotic plaque specimens. The results supported our hypothesis that CTHRSSVVC peptide has a remarkable sequence for the development of theranostics approaches in the treatment of atherosclerosis and other diseases.     

Regulatory Cell Populations in Relapsing-Remitting Multiple Sclerosis (RRMS) Patients: Effect of Disease Activity and Treatment Regimens

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) of autoimmune etiology that results from an imbalance between CNS-specific T effector cells and peripheral suppressive mechanisms mediated by regulatory cells (RC). In this research, we collected blood samples from 83 relapsing remitting MS (RRMS) patients and 45 healthy persons (HC), to assess the sizes of their RC populations, including CD4⁺CD25^highFoxp3⁺ (nTregs), CD3⁺CD4⁺HLA⁻G⁺, CD3⁺CD8⁺CD28⁻, CD3⁺CD56⁺, and CD56^bright cells, and how RC are affected by disease activity (acute phase or remission) and types of treatment (methylprednisolone, interferon, or natalizumab). In addition, we isolated peripheral blood mononuclear cells (PBMC) and cultured them with peptides mapping to myelin antigens, to determine RC responsiveness to autoantigens. The results showed decreased levels of nTregs in patients in the acute phase ± methylprednisolone and in remission + natalizumab, but HC levels in patients in remission or receiving interferon. Patients + interferon had the highest levels of CD3⁺CD4⁺HLA⁻G⁺ and CD3⁺CD8⁺CD28⁻ RC, and patients in the acute phase + methylprednisolone the lowest. Patients in remission had the highest levels of CD3⁺CD56⁺, and patients in remission + natalizumab the highest levels of CD56^bright cells. Only nTregs responded to autoantigens in culture, regardless of disease activity or treatment. The highest suppressive activity was exhibited by nTregs from patients in remission. In conclusion, in RRMS disease activity and type of treatment affect different RC populations. nTregs respond to myelin antigens, indicating that it is possible to restore immunological tolerance through nTreg induction.