Receptor-expressing Cells Research Articles

Stable and Minimum Size Solubilization of Membrane Proteins with Cocktails of Phospholipid Analogues.

Membrane proteins (MPs) play important roles in various cellular processes and are major targets for drugs. Solubilization of MPs is often needed for structural and biophysical studies. For high-resolution nuclear magnetic resonance measurements, there is a size limit of the sample (<100 kDa), and a high thermal stability at an increased temperature is required. Furthermore, lipid bilayer-like environments are desirable to preserve the native states of MPs. However, existing solubilization techniques do not fulfill these requirements at the same time. In this study, we combined two phospholipid analogues as a solubilizer and stabilizer to isolate MPs. This method maintained bacteriorhodopsin (bR) extracted from purple membranes in its native state for 7 d at 40 °C. The solubility was comparable to that of conventional detergents for MPs, and the thermal stability of the solubilizate was the best among them. The increase in the molecular size caused by the solubilization of bR was only 20 kDa, indicating that 20 phospholipid analogue molecules were sufficient to solubilize one bR molecule. 15N-1H heteronuclear single quantum coherence spectra of solubilized 2H- and 15N-labeled bR gave ∼80% of the expected peaks. In addition, the lysate of human neuropeptide Y2 receptor-expressing mammalian cells exhibited ligand recognition for 7 d at 37 °C, suggesting that this technique can be used for ligand screening. Moreover, the structure of the single membrane-spanning M2 protein of the influenza A virus expressed in Escherichia coli was stably maintained for 7 d at 40 °C. Thus, our method is promising for various MP studies.

A Preliminary Study on the Correlation Between TILs and CTCs and Clinical Value in Lung Cancer Patients Analyzed by Lipid Magnetic Microspheres

The purpose of this study was to analyze the advantages of TCR gene and its relationship with tumor infiltrating lymphocytes (til) and circulating tumor cells (CTCs). The properties of EpCAM and EGFR lipid magnetic microspheres were analyzed. The levels of Programmed cell death 1 ligand 1, B7-H3 and B7-H4 in peripheral blood were measured by quantitative immunofluorescence (QIF). T cell receptor expression profile and clone proliferation were detected. The prepared EpCAM and EGFR lipid magnetic microspheres showed good stability and specificity. The therapeutic method proposed in this study is expected to be applied.

Human decidua basalis mesenchymal stem/stromal cells enhance anticancer properties of human natural killer cells, in vitro

IntroductionMesenchymal stem cells/stromal cells from the Decidua Basalis of the human placenta (DBMSCs) express wide range of effector molecules that modulate the functions of their target cells. These properties make them potential candidate for use in cellular therapy. In this study, we have investigated the consequences of interaction between DBMSCs and natural killer (NK) cells for both cell types.MethodsDBMSCs were cultured with IL-2-activated and resting non-activated NK cells isolated from healthy human peripheral blood and various functional assays were performed including, NK cell proliferation and cytolytic activities. Flow cytometry and microscopic studies were performed to examine the expression of NK cell receptors that mediate these cytolytic activities against DBMSCs. Moreover, the mechanism underlying these effects was also investigated.ResultsOur findings revealed that, co-culture of DBMSCs and NK cells resulted in inhibition of proliferation of resting NK cells, while proliferation of IL-2 activated NK cells was increased. Contrarily, treatment of DBMSC’s with comparatively high numbers of IL-2 activated NK cells, resulted in their lysis, whereas treatment with low numbers resulted in reduction in their proliferation. Cytolytic activity of NK cells against DBMSCs was mediated by several activating NK cell receptors. In spite of the expression of HLA class I molecules by DBMSCs, they were still lysed by NK cells, excluding their involvement in cytolytic activity. In addition, preconditioning NK cells by DBMSCs, enhanced their ability to suppress tumor cell proliferation and in severe cases resulted in their partial lysis. Lysis and decrease of tumor cell proliferation is associated with increased expression of important molecules involved in anticancer activities.DiscussionWe conclude that DBMSCs exhibit dualfunctions on NK cells that enhance their anticancer therapeutic potential.

T cell and soluble co-inhibitory receptor expression in patients with visceral leishmaniasis are markers of treatment response and clinical outcome

Abstract Background Co-inhibitory receptors (immune checkpoints) regulate activated immune cells. Their expression on T cells can limit host defense. We hypothesized that chronic Leishmania donovani infection in patients with visceral leishmaniasis (VL) leads to expression of co-inhibitory receptors that could be markers of treatment response and clinical outcome. Method A prospective cohort of 21 subjects with VL (7 with HIV coinfection) and 10 controls was established to measure T cell expression of co-inhibitory receptors (PD-1, Tim-3, LAG-3, CTLA-4, and TIGIT) by flow cytometry in discarded remnants of diagnostic splenic or bone marrow aspirates and peripheral blood collected before and after treatment. Plasma levels of soluble co-inhibitory proteins (sPD-1, sTim-3, sLAG-3, and sCTLA-4), and selected cytokines were determined by immunoassay. Results Expression of co-inhibitory receptors in peripheral blood T cells generally reflected findings in spleen and bone marrow aspirates. PD-1 and Tim-3 were upregulated in CD4+ T cells in HIV-negative and HIV-positive subjects with VL compared to controls. CD8+ T cells from HIV-negative subjects with VL displayed a similar pattern. Plasma levels of sPD-1 and sTim-3 were also greater in VL patients than controls. CD8+ and CD4+ T cells co-expressing PD-1 and Tim-3 showed considerable decline with treatment. Mortality in HIV-negative VL patients was associated with increased CD8+ T cells co-expressing Tim-3 and PD-1, triple positive CD4+ and CD8+ T cells (PD-1+Tim-3+LAG-3+), and elevated sLAG3. Conclusion Tim-3 and PD-1 expression on CD4+ and CD8+ T cells, and increased plasma sLAG-3, were markers of treatment response and clinical outcome in patients with VL.

Structure–function relationship of dynorphin B variants using naturally occurring amino acid substitutions

Dynorphins (Dyn) represent the subset of endogenous opioid peptides with the highest binding affinity to kappa opioid receptors (KOPrs). Activation of the G-protein-coupled pathway of KOPrs has strong anticonvulsant effects. Dyn also bind to mu (MOPrs) and delta opioid receptors (DOPrs) with lower affinity and can activate the β-arrestin pathway. To fully exploit the therapeutic potential of dynorphins and reduce potential unwanted effects, increased selectivity for KOPrs combined with reduced activation of the mTOR complex would be favorable. Therefore, we investigated a series of dynorphin B (DynB) variants, substituted in one or two positions with naturally occurring amino acids for differential opioid receptor activation, applying competitive radio binding assays, GTPγS assays, PRESTO-Tango, and Western blotting on single-opioid receptor-expressing cells. Seven DynB derivatives displayed at least 10-fold increased selectivity for KOPrs over either MOPrs or DOPrs. The highest selectivity for KOPrs over MOPrs was obtained with DynB_G3M/Q8H, and the highest selectivity for KOPrs over DOPrs was obtained with DynB_L5S. Increased selectivity for KOPr over MOPr and DOPr was based on a loss of affinity or potency at MOPr and DOPr rather than a higher affinity or potency at KOPr. This suggests that the investigated amino acid exchanges in positions 3, 5, and 8 are of higher importance for binding and activation of MOPr or DOPr than of KOPr. In tests for signal transduction using the GTPγS assay, none of the DynB derivatives displayed increased potency. The three tested variants with substitutions of glycine to methionine in position 3 displayed reduced efficacy and are, therefore, considered partial agonists. The two most promising activating candidates were further investigated for functional selectivity between the G-protein and the β-arrestin pathway, as well as for activation of mTOR. No difference was detected in the respective read-outs, compared to wild-type DynB. Our data indicate that the assessment of affinity to KOPr alone is not sufficient to predict either potency or efficacy of peptidergic agonists on KOPr. Further assessment of downstream pathways is required to allow more reliable predictions of in vivo effects.

Effect of Hypoxia on Siglec-7 and Siglec-9 Receptors and Sialoglycan Ligands and Impact of Their Targeting on NK Cell Cytotoxicity

Background/Objectives: Tumor microenvironmental hypoxia is an established hallmark of solid tumors. It significantly contributes to tumor aggressiveness and therapy resistance and has been reported to affect the balance of activating/inhibitory surface receptors’ expression and activity on NK cells. In the current study, we investigated the impact of hypoxia on the surface expression of Siglec-7 and Siglec-9 (Sig-7/9) and their ligands in NK cells and tumor target cells. The functional consequence of Siglec blockage using nanoparticles specifically designed to target and block Sig-7/9 receptors on NK cell cytotoxicity was elucidated. Methods: CD56⁺ CD3− NK cells were isolated from PBMCs along with an NK-92 clone and used as effector cells, while MCF-7 and K562 served as target cells. All cells were incubated under normoxic or hypoxic conditions for 24 h. To assess Siglec-7 and Siglec-9 receptor expression, U937, NK-92, and primary NK cells were stained with PE-labeled antibodies against CD328 Siglec-7/9. Interactions between Siglec-7/9 and their sialylated ligands, along with their functional impact on NK cell activity, were evaluated using polymeric nanoparticles coated with a sialic acid mimetic. Immunological synapse formation and live-cell imaging were performed with a ZEISS LSM 800 with Airyscan at 10x magnification for 24 h. Results: Our data indicate that hypoxia had no effect on the expression of Siglec-7/9 receptors by NK cells. In contrast, hypoxic stress resulted in an increase in Siglec-7 sialoglycan ligand expression by a sub-population of NK target cells. Using polymeric nanoparticles coated with a sialic acid mimetic that binds both Siglec-7 and -9 (Sig-7/9 NP), we demonstrated that incubation of these nanoparticles with NK cells resulted in increased immunological synapse formation, granzyme B accumulation, and killing of NK target cells. These studies indicate that hypoxic stress may have an impact on NK cell-based therapies and highlight the need to consider the hypoxic microenvironment for tumor-specific glycosylation. Conclusions: Our findings point to the role of Siglec–sialylated glycan interactions in hypoxic stress-induced NK cell dysfunction and recommend the potential integration of the manipulation of this axis through the targeting of Siglecs in future cancer immunotherapy strategies.

Cellular Profile of Subfornical Organ Insulin Receptors in Mice.

Brain insulin receptor signaling is strongly implicated in cardiovascular and metabolic physiological regulation. In particular, we recently demonstrated that insulin receptors within the subfornical organ (SFO) play a tonic role in cardiovascular and metabolic regulation in mice. The SFO is a forebrain sensory circumventricular organ that regulates cardiometabolic homeostasis due to its direct exposure to the circulation and thus its ability to sense circulating factors, such as insulin. Previous work has demonstrated broad distribution of insulin receptor-expressing cells throughout the entire SFO, indirectly indicating insulin receptor expression in multiple cell types. Based on this, we sought to determine the cellular phenotypes that express insulin receptors within the SFO by combining immunohistochemistry with genetically modified reporter mouse models. Interestingly, SFO neurons, including both excitatory and inhibitory types, were the dominant cell site for insulin receptor expression, although a weak degree of insulin receptor expression was also detected in astrocytes. Moreover, SFO angiotensin type 1a receptor neurons also expressed insulin receptors. Collectively, these anatomical findings indicate the existence of potentially complex cellular networks within the SFO through which insulin signaling can influence physiology and further point to the SFO as a possible brain site for crosstalk between angiotensin-II and insulin.

Investigating the IgM and IgG B Cell Receptor Repertoires and Expression of Ultralong Complementarity Determining Region 3 in Colostrum and Blood from Holstein-Friesian Cows at Calving.

In cattle, colostral maternal immunoglobulins and lymphocytes transfer across the neonate's intestinal epithelium to provide protection against pathogens. This study aimed to compare repertoires of B cell populations in blood and colostrum in cows for the first time, with an emphasis on ultralong complementarity determining region 3 (CDR3, ≥40 amino acids). Blood mononuclear cells (BMCs, n= 7) and colostral cells (n = 7) were isolated from Holstein-Friesian dairy cows. Magnetic-activated cell sorting was used to capture IgM and IgG B cells from BMCs. Colostral cells were harvested by centrifugation. RNA was extracted and cDNA was produced; IgM and IgG transcripts were amplified using polymerase chain reactions. Amplicons were sequenced using the Nanopore Native barcoding kit 24 V14 and MinION with R10.4 flow cells. In colostrum, there was a significantly greater percentage of IgM B cells with ultralong CDR3s (8.09% ± 1.73 standard error of the mean) compared to blood (4.22% ± 0.70, p = 0.05). There was a significantly greater percentage of IgG B cells in colostrum with ultralong CDR3s (12.98% ± 1.98) compared to blood (6.61% ± 1.11, p = 0.05). A higher percentage of IgM and IgG B cells with ultralong CDR3s in colostrum may be indicative of a potential role in protecting the neonate.

B-343 Development of CD40 and HER2 Bispecific Antibodies for Enhanced Efficacy against HER2-Positive Tumors

Abstract Background CD40, a member of the tumor necrosis factor superfamily, is primarily expressed on antigen-presenting cells. This receptor plays a crucial role in facilitating the interaction between dendritic cells, activating antigen-specific T cells. Agonist antibodies targeting the CD40 receptor have the potential to mimic the natural CD40 ligand, fostering dendritic cell maturation and stimulating immunity against “cold” tumors. Although this approach has shown promise in preclinical studies, translating it to clinical success has been challenging. Only modest anti-tumor activity was observed in cancer patients, and none of the developed CD40 monoclonal antibodies (mAbs) progressed beyond early clinical trials, highlighting a need for improved anti-tumor efficacy. Methods In this study, we successfully developed an anti-human CD40 agonist rabbit monoclonal antibody using Yurogen's proprietary SMAB(TM) platform. The most potent CD40 mAb clone was combined with trastuzumab (an anti-HER2 mAb) to create a therapeutic bispecific antibody. Our goal is to leverage immune cell activation through HER2-targeted cancer therapy to achieve enhanced anti-tumor activity. Dozens of diverse CD40xHER2 bispecific antibody designs (both symmetry and asymmetry) were explored, with careful evaluation of expression yield, purity, and binding profiles for each target respectively. Results A few bispecific antibody designs demonstrated robust binding to both HER2 and CD40 antigen proteins, as well as receptor-expressing cell lines. Notably, two developed bispecific antibodies exhibited potent killing of HER2-positive cancer cells with the assistance of innate immune cells. Conclusions In sum, this study provides novel insights into designing bispecific antibodies against HER2 and CD40, offering a promising avenue for advancing cancer immunotherapy.

Single-hit genome editing optimized for maturation in B cells redirects their specificity toward tumor antigens.

T-cell-based adoptive immunotherapy is a new pillar of cancer care. Tumor-redirected B cells could also contribute to therapy if their manipulation to rewire immunoglobulin (Ig) genes is mastered. We designed a single-chain Ig-encoding cassette ("scFull-Ig") that redirects antigen specificity when inserted at a single position of the IgH locus. This design, which places combined IgH and IgL variable genes downstream of a pVH promoter, nevertheless preserves all Ig functional domains and the intrinsic mechanisms that regulate expression from the IgM B cell receptor (BCR) expression to Ig secretion, somatic hypermutation and class switching. This single-locus editing provides an efficient and safe strategy to both disrupt endogenous Ig expression and encode a new Ig paratope. As a proof of concept, the functionality of scFull BCR and/or secreted Ig was validated against two different classical human tumor antigens, HER2 and hCD20. Once validated in cell lines, the strategy was extended to primary B cells, confirming the successful engineering of BCR and Ig expression and the ability of scFull-Ig to undergo further class switching. These results further pave the way for future B cell-based adoptive immunotherapy and strategies to express a therapeutic mAb with a variety of switched H-chains that provide complementary functions.

Selective Transfection of a Transferrin Receptor-Expressing Cell Line with DNA-Lipid Nanoparticles.

Despite considerable progress in using lipid nanoparticle (LNP) vehicles for gene delivery, achieving selective transfection of specific cell types remains a significant challenge, hindering the advancement of new gene or gene-editing therapies. Although LNPs have been equipped with ligands aimed at targeting specific cellular receptors, achieving complete selectivity continues to be elusive. The exact reasons for this limited selectivity are not fully understood, as cell targeting involves a complex interplay of various cellular factors. Assessing how much ligand/receptor binding contributes to selectivity is challenging due to these additional influencing factors. Nonetheless, such data are important for developing new nanocarriers and setting realistic expectations for selectivity. Here, we have quantified the selective, targeted transfection using two uniquely engineered cell lines that eliminate unpredictable and interfering cellular influences. We have compared the targeted transfection of Chinese ovary hamster (CHO) cells engineered to express the human transferrin receptor 1 (hTfR1), CHO-TRVb-hTfR1, with CHO cells that completely lack any transferrin receptor, CHO-TRVb-neo cells (negative control). Thus, the two cell lines differ only in the presence/absence of hTfR1. The transfection was performed with pDNA-encapsulating LNPs equipped with the DT7 peptide ligand that specifically binds to hTfR1 and enables targeted transfection. The LNP's pDNA encoded for the monomeric GreenLantern (mGL) reporter protein, whose fluorescence was used to quantify transfection. We report a novel LNP composition designed to achieve an optimal particle size and ζ-potential, efficient pDNA encapsulation, hTfR1-targeting capability, and sufficient polyethylene glycol sheltering to minimize random cell targeting. The transfection efficiency was quantified in both cell lines separately through flow cytometry based on the expression of the fluorescent gene product. Our results demonstrated an LNP dose-dependent mGL expression, with a 5-fold preference for the CHO-TRVb-hTfR1 when compared to CHO-TRVb-neo. In another experiment, when both cell lines were mixed at a 1:1 ratio, the DT7-decorated LNP achieved a 3-fold higher transfection of the CHO-TRVb-hTfR1 over the CHO-TRVb-neo cells. Based on the low-level transfection of the CHO-TRVb-neo cells in both experiments, our results suggest that 17-25% of the transfection occurred in a nonspecific manner. The observed transfection selectivity for the CHO-TRVb-hTfR1 cells was based entirely on the hTfR1/DT7 interaction. This work showed that the platform of two engineered cell lines which differ only in the hTfR1 can greatly facilitate the development of LNPs with hTfR1-targeting ligands.

Developing New Peptides and Peptide-Drug Conjugates for Targeting the FGFR2 Receptor-Expressing Tumor Cells and 3D Spheroids.

In this work, we utilized a biomimetic approach for targeting KATO (III) tumor cells and 3D tumoroids. Specifically, the binding interactions of the bioactive short peptide sequences ACSAG (A-pep) and LPHVLTPEAGAT (L-pep) with the fibroblast growth factor receptor (FGFR2) kinase domain was investigated for the first time. Both peptides have been shown to be derived from natural resources previously. We then created a new fusion trimer peptide ACSAG-LPHVLTPEAGAT-GASCA (Trimer-pep) and investigated its binding interactions with the FGFR2 kinase domain in order to target the fibroblast growth factor receptor 2 (FGFR2), which is many overexpressed in tumor cells. Molecular docking and molecular dynamics simulation studies revealed critical interactions with the activation loop, hinge and glycine-rich loop regions of the FGFR2 kinase domain. To develop these peptides for drug delivery, DOX (Doxorubicin) conjugates of the peptides were created. Furthermore, the binding of the peptides with the kinase domain was further confirmed through surface plasmon resonance studies. Cell studies with gastric cancer cells (KATO III) revealed that the conjugates and the peptides induced higher cytotoxicity in the tumor cells compared to normal cells. Following confirmation of cytotoxicity against tumor cells, the ability of the conjugates and the peptides to penetrate 3D spheroids was investigated by evaluating their permeation in co-cultured spheroids grown with KATO (III) and colon tumor-associated fibroblasts (CAFs). Results demonstrated that Trimer-pep conjugated with DOX showed the highest permeation, while the ACSAG conjugate also demonstrated reasonable permeation of the drug. These results indicate that these peptides may be further explored and potentially utilized to create drug conjugates for targeting tumor cells expressing FGFR2 for developing therapeutics.

Synthesis and Characterization of Folic Acid-Conjugated Terbium Complexes as Luminescent Probes for Targeting Folate Receptor-Expressing Cells.

Several conjugates between folic acid and a series of kinetically stable lanthanide complexes have been synthesized, using amide coupling and azide-alkyne cycloaddition methodologies to link the metal-binding domain to folate through a variety of spacer groups. While all these complexes exhibit affinity for the folate receptor, it is clear that the point of attachment to folate is essential, with linkage through the γ-carboxylic acid giving rise to significantly enhanced receptor affinity. All the conjugates studied show affinities consistent with displacing biological circulating folate derivatives, 5-methyltetrahydrofolate, from folate receptors. All the complexes exhibit luminescence with a short-lived component arising from ligand fluorescence overlaid on a much longer lived terbium-centered component. These can be separated using time-gating methods. From the results obtained, the most promising approach to achieve sensitized luminescence in these systems requires incorporating a sensitizing chromophore close to the lanthanide.

Enhancing the cytotoxicity of immunotoxins by facilitating their dissociation from target receptors under the reducing conditions of the endocytic pathway

Immunotoxins (ITs) are recombinant chimeric proteins that combine a protein toxin with a targeting moiety to facilitate the selective delivery of the toxin to cancer cells. Here, we present a novel strategy to enhance the cytosolic access of ITs by promoting their dissociation from target receptors under the reducing conditions of the endocytic pathway. We engineered monobodySS, a human fibronectin type III domain-based monobody with disulfide bond (SS)-containing paratopes, targeting receptors such as EGFR, EpCAM, Her2, and FAP. MonobodySS exhibited SS-dependent target receptor binding with a significant reduction in binding under reducing conditions. We then created monobodySS-based ITs carrying a 25 kDa fragment of Pseudomonas exotoxin A (PE25), termed monobodySS-PE25. These ITs showed dose-dependent cytotoxicity against target receptor-expressing cancer cells and a wider therapeutic window due to higher efficacy at lower doses compared to controls with SS reduction inhibited. ERSS/28-PE25, with a KD of 28 nM for EGFR, demonstrated superior tumor-killing potency compared to ER/21-PE25, which lacks an SS bond, at equivalent and lower doses. In vivo, ERSS/28-PE25 outperformed ER/21-PE25 in suppressing tumor growth in EGFR-overexpressing xenograft mouse models. This study presents a strategy for developing solid tumor-targeting ITs using SS-containing paratopes to enhance cytosolic delivery and antitumor efficacy.

Local synthesis of estradiol in the rostral ventromedial medulla protects against widespread muscle pain in male mice.

Animal studies consistently demonstrate that testosterone is protective against pain in multiple models, including an animal model of activity-induced muscle pain. In this model, females develop widespread muscle hyperalgesia, and reducing testosterone levels in males results in widespread muscle hyperalgesia. Widespread pain is believed to be mediated by changes in the central nervous system, including the rostral ventromedial medulla (RVM). The enzyme that converts testosterone to estradiol, aromatase, is highly expressed in the RVM. Therefore, we hypothesized that testosterone is converted by aromatase to estradiol locally in the RVM to prevent development of widespread muscle hyperalgesia in male mice. This was tested through pharmacological inhibition of estrogen receptors (ER), aromatase, or ER-α in the RVM which resulted in contralateral hyperalgesia in male mice (C57BL/6J). ER inhibition in the RVM had no effect on hyperalgesia in female mice. As prior studies show modulation of estradiol signaling alters GABA receptor and transporter expression, we examined if removal of testosterone in males would decrease mRNA expression of GABA receptor subunits and vesicular GABA transporter (VGAT). However, there were no differences in mRNA expression of GABA receptor subunits of VGAT between gonadectomized and sham control males. Lastly, we used RNAscope to determine expression of ER-α in the RVM and show expression in inhibitory (VGAT+), serotonergic (tryptophan hydroxylase 2+), and μ-opioid receptor expressing (MOR+) cells. In conclusion, testosterone protects males from development of widespread hyperalgesia through aromatization to estradiol and activation of ER-α which is widely expressed in multiple cell types in the RVM.Significance Statement This study's findings reveal, for the first time, that testosterone protects males from development of widespread muscle hyperalgesia through aromatization to estradiol and activation of ER-α within the RVM. The findings also show, for the first time, ER-α expression in the RVM in male mice, and the distribution patterns of this expression among multiple cell types. Thus, together our data suggest a unique endogenous mechanism in the RVM for protection against widespread pain in males only.

Therapeutic effect of F127-folate@PLGA/CHL/IR780 nanoparticles on folate receptor-expressing cancer cells.

Theragnostic platforms, which integrate therapeutic and diagnostic capabilities, have gained significant interest in drug research because of to their potential advantages. This study reports the development of a novel multifunctional nanoparticle carrier system based on poly(ᴅ,ʟ-lactic-co-glycolic acid) (PLGA) for the targeted delivery of the chemotherapeutic agent chlorambucil (CHL) and the imaging agent IR780. The approach in this study incorporates Pluronic F127-folate onto the PLGA nanoparticles, which enables targeted delivery to folate receptor-expressing cancer cells. The F127-folate@PLGA/CHL/IR780 nanoparticles were formulated using a nanoprecipitation technique, resulting in small size, high homogeneity, and negative surface charge. Importantly, the folate-targeted nanoparticles demonstrated enhanced uptake and cytotoxicity in folate receptor-positive cancer cell lines (MCF-7 and HepG-2) compared to folate receptor-negative cells (HEK 293). Additionally, the F127-folate@PLGA/CHL/IR780 nanoparticles exhibited a lower IC50 value against cancer cells than non-targeted F127@PLGA/CHL/IR780 nanoparticles. These findings suggest that the developed F127-folate@PLGA/CHL/IR780 nanoparticles hold promise as a theragnostic system for targeted cancer therapy and diagnosis, leveraging the advantages of PLGA, folate targeting, and the integration of therapeutic and imaging agents.

Lysosomal-Associated Protein Transmembrane 5, Tubular Senescence, and Progression of CKD.

Tubular senescence is a major determinant of chronic kidney disease (CKD) and identification of potential therapeutic targets involved in senescent tubular epithelial cells has clinical importance. Lysosomal-associated protein transmembrane 5 (LAPTM5) is a key molecule related to T and B cell receptor expression and inflammation. However, the expression pattern of LAPTM5 in the kidney and the contribution of LAPTM5 to the development of CKD keep unknown. LAPTM5-/- mice and tubule specific-LAPTM5 knockout mice were used to examine the role of LAPTM5 in tubular senescence by establishing different experimental mouse CKD models. LAPTM5 expression was significantly induced in the kidney, especially in proximal tubules and distal convoluted tubules, from mice with aristolochic acid nephropathy, bilateral ischemia/reperfusion injury (IRI)-induced CKD or unilateral ureter obstruction (UUO). Tubule-specific deletion of LAPTM5 inhibited senescence of tubular epithelial cells and alleviated tubulointerstitial fibrosis in aged mice. Moreover, LAPTM5 deficiency ameliorated kidney injury and tubular senescence in mice with CKD. Mechanistically, LAPTM5 inhibited ubiquitination of NICD1 by mediating WWP2 lysosomal degradation, then leading to cellular senescence in tubular epithelial cells. Notably, we also observed a higher expression of LAPTM5 in tubules from individuals with CKD and the level of LAPTM5 was correlated with kidney fibrosis and tubular senescence in people with CKD. LAPTM5 contributed to tubular senescence by regulating WWP2/NICD1 signaling pathway and exacerbated kidney injury during the progression of CKD.

Descending facilitation from rostral ventromedial medulla mu opioid receptor-expressing neurons is necessary for maintenance of sensory and affective dimensions of chronic neuropathic pain.

Pharmacological ablation of rostral ventromedial medulla (RVM) mu opioid receptor-expressing cells before peripheral nerve injury prevents the development of neuropathic pain. However, whether these neurons are required for the expression of established neuropathic pain is not known. Male Oprm1Cre heterozygous (MORCre) or wild-type (MORWT) mice received AAV8-hSyn-DIO-hM4D(Gi)-mCherry in the RVM. After partial sciatic nerve ligation (PSNL), we evaluated pain behaviors and descending control of nociception in response to acute or sustained chemogenetic inhibition of RVM-MOR cells expressing hM4D(Gi). A single systemic administration of hM4D(Gi) agonist clozapine-N-oxide (CNO) reversibly inhibited hind paw tactile allodynia and produced conditioned place preference only in MORCre mice with PSNL. Intrathecal CNO also reversibly inhibited PSNL-induced hind paw allodynia, suggesting that the spinal projections from these RVM-MOR cells are critical for manifestation of pain behaviors. Consistent with enhanced descending facilitation from RVM-MOR cells, MORCre-hM4D(Gi) mice with PSNL showed diminished descending control of nociception that was restored by systemic CNO. Sustained CNO in drinking water before PSNL prevented expression of chronic pain without affecting acute surgical pain; however, relief of chronic pain required sustained CNO treatment. Thus, in male mice, activity of spinally projecting RVM-MOR cells is required (1) for expression and manifestation of both sensory and affective dimensions of established neuropathic pain and (2) to promote descending facilitation that overcomes apparently intact descending inhibition to maintain chronic pain. Enhanced descending facilitation likely regulates the output signal from the spinal cord to the brain to shape the pain experience and may provide a mechanism for nonopioid management of pain.

The dual amylin and calcitonin receptor agonist KBP-336 elicits a unique combination of weight loss, antinociception and bone protection – a novel disease-modifying osteoarthritis drug

BackgroundDespite the extensive research to provide a disease-modifying osteoarthritis drug (DMOAD), there is still no approved DMOAD. Dual amylin and calcitonin receptor agonists (DACRA) can provide metabolic benefits along with antinociceptive and potential structural preserving effects. In these studies, we tested a DACRA named KBP-336 on a metabolic model of OA in meniscectomised (MNX) rats.MethodsWe evaluated KBP-336’s effect on pain-like symptoms in Sprague Dawley (SD) rats on high-fat diet (HFD) that underwent meniscectomy using the von Frey test to measure the 50% paw withdrawal threshold (PWT) and analyzed using one-way ANOVA. Short in vivo studies and in vitro cell receptor expression systems were used to illustrate receptor pharmacology.ResultsAfter 30 weeks on HFD, including an 8-week treatment, female MNX animals receiving KBP-336 4.5 nmol/Kg/72 h had lower body weight and smaller adipose tissues than their vehicle-treated counterparts. After 20 weeks on HFD, including an 8-week treatment, male rats receiving KBP-336 had lower body weight than the vehicle group. In both the female and male rats, the MNX groups on KBP-336 treatment had a higher PWT than the vehicle-treated MNX group. Aiming to identify the receptor influencing pain alleviation, KBP-336 was compared to the long-acting human calcitonin (hCTA). Single-dose studies on 12-week-old male rats showed that hCTA lowers CTX-I without affecting food intake, confirming its calcitonin receptor selectivity. On the metabolic OA model with 18 weeks of HFD, including 6-week treatment, hCTA at 100 nmol/Kg/24 h and KBP-336 at 0.5, 1.5, and 4.5 nmol/Kg/72 h produced significantly higher PWT in MNX animals compared to MNX animals on vehicle treatment. hCTA and KBP-336 at 0.5 nmol/Kg did not affect body weight and fat tissues.ConclusionOverall, KBP-336 improved the pain observed in the metabolic OA model. Calcitonin receptor activation proved to be essential in this antinociceptive effect.

Release of CM-12 from A2-type casein by the cleavage of Ser-Leu-Xaa at the C-terminus using Aspergillus oryzae alkaline protease.

Aspergillus oryzae protease can release the opioid peptide β-casomorphin-10 (CM-10, YPFPGPIPNS, 60-69) from A2-type casein. However, not only is the yield of the active peptide low, but the key enzyme involved in processing has yet to be identified. A significant amount of the opioid peptide 60YPFPGPIPNSLP71 (CM-12) was produced from the A2-type casein peptide 53AQTQSLVYPFPGPIPNSLPQNIPPLTQTPV82 when the active protease in A. oryzae protease extract was fractionated with DEAE-Sepharose. The fractionated enzyme produced CM-12 from bovine A2-type casein but not from bovine A1 casein. A major protein of 34 kDa was purified and identified as an alkaline protease (Alp). Motif prediction of the Alp cleavage site using Multiple EM for Motif Elicitation analysis revealed preferable cleavage at the C-terminal end of Ser-Leu-Xaa for the release of CM-12. A2-type casein hydrolysate by Alp exhibited similar levels of opioid activity to that of synthetic CM-12 in cAMP-Glo assays with μ-opioid receptor-expressing HEK293 cells. These results suggest that CM-12 is a major opioid peptide in the casein hydrolysate. Our findings showed that Alp fractionated from A. oryzae protease extract produced the opioid peptide CM-12 from A2-type casein as a result of preferential cleavage at the C-terminal end of Ser-Leu-Xaa and the removal of coexisting enzymes. Moreover, docking predictions suggested a stable interaction between CM-12 and the 3D structure of Alp. Casein hydrolysate with Alp-containing CM-12 has the potential for use as a bioactive peptide material with opioid activity. © 2024 Society of Chemical Industry.