Structural and biological characterization of pAC65, a macrocyclic peptide that blocks PD-L1 with equivalent potency to the FDA-approved antibodies.

Recent advances in immuno-oncology have opened up new and impressive treatment options for cancer. Notwithstanding, overcoming the limitations of the current FDA-approved therapies with monoclonal antibodies (mAbs) that block the PD-1/PD-L1 pathway continues to lead to the testing of multiple approaches and optimizations. Recently, a series of macrocyclic peptides have been developed that exhibit binding strengths to PD-L1 ranging from sub-micromolar to micromolar. In this study, we present the most potent non-antibody-based PD-1/PD-L1 interaction inhibitor reported to date. The structural and biological characterization of this macrocyclic PD-L1 targeting peptide provides the rationale for inhibition of both PD-1/PD-L1 and CD80/PD-L1 complexes. The IC50 and EC50 values obtained in PD-L1 binding assays indicate that the pAC65 peptide has potency equivalent to the current FDA-approved mAbs and may have similar activity to the BMS986189 peptide, which entered the clinical trial and has favorable safety and pharmacokinetic data. The data presented here delineate the generation of similar peptides with improved biological activities and applications not only in the field of cancer immunotherapy but also in other disorders related to the immune system.

Monocyte subpopulations display disease-specific miRNA signatures depending on the subform of Spondyloarthropathy.

Spondyloarthropathies (SpA) are a family of rheumatic disorders that could be divided into axial (axSpA) and peripheral (perSpA) sub-forms depending on the disease clinical presentation. The chronic inflammation is believed to be driven by innate immune cells such as monocytes, rather than self-reactive cells of adaptive immune system. The aim of the study was to investigate the micro-RNA (miRNA) profiles in monocyte subpopulations (classical, intermediate and non-classical subpopulations) acquired from SpA patients or healthy individuals in search for prospective disease specific and/or disease subtype differentiating miRNA markers. Several SpA-specific and axSpA/perSpA differentiating miRNAs have been identified that appear to be characteristic for specific monocyte subpopulation. For classical monocytes, upregulation of miR-567 and miR-943 was found to be SpA-specific, whereas downregulation of miR-1262 could serve as axSpA-differentiating, and the expression pattern of miR-23a, miR-34c, mi-591 and miR-630 as perSpA-differentiating markers. For intermediate monocytes, expression levels of miR-103, miR-125b, miR-140, miR-374, miR-376c and miR-1249 could be used to distinguish SpA patients from healthy donors, whereas the expression pattern of miR-155 was identified as characteristic for perSpA. For non-classical monocytes, differential expression of miR-195 was recognized as general SpA indicator, while upregulation of miR-454 and miR-487b could serve as axSpA-differentiating, and miR-1291 as perSpA-differentiating markers. Our data indicate for the first time that in different SpA subtypes, monocyte subpopulations bear disease-specific miRNA signatures that could be relevant for SpA diagnosis/differentiation process and may help to understand SpA etiopathology in the context of already known functions of monocyte subpopulations.

Improving the Effect of Cancer Cells Irradiation with X-rays and High-Energy Protons Using Bimetallic Palladium-Platinum Nanoparticles with Various Nanostructures.

Nano-sized radiosensitizers can be used to increase the effectiveness of radiation-based anticancer therapies. In this study, bimetallic, ~30 nm palladium-platinum nanoparticles (PdPt NPs) with different nanostructures (random nano-alloy NPs and ordered core-shell NPs) were prepared. Scanning transmission electron microscopy (STEM), selected area electron diffraction (SAED), energy-dispersive X-ray spectroscopy (EDS), zeta potential measurements, and nanoparticle tracking analysis (NTA) were used to provide the physicochemical characteristics of PdPt NPs. Then, PdPt NPs were added to the cultures of colon cancer cells and normal colon epithelium cells in individually established non-toxic concentrations and irradiated with the non-harmful dose of X-rays/protons. Cell viability before and after PdPt NPs-(non) assisted X-ray/proton irradiation was evaluated by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Flow cytometry was used to assess cell apoptosis. The results showed that PdPt NPs significantly enhanced the effect of irradiation on cancer cells. It was noticed that nano-alloy PdPt NPs possess better radiosensitizing properties compared to PtPd core-shell NPs, and the combined effect against cancer cells was c.a. 10% stronger for X-ray than for proton irradiation. Thus, the radio-enhancing features of differently structured PdPt NPs indicate their potential application for the improvement of the effectiveness of radiation-based anticancer therapies.

Supportive, Delegated, and Common Dyadic Coping Mediates the Association between Adult Attachment Representation and Relationship Satisfaction: A Dyadic Approach.

The aim of this study was to examine intrapersonal (actor) and interpersonal (partner) associations between attachment, assessed by the Adult Attachment Interview, and satisfaction with the relationship, as well as to establish the possibility of the mediatory effect of supportive, delegated, and common dyadic coping on the aforementioned associations. A dyadic approach has been introduced, using the actor-partner interdependence mediation model and data from 114 heterosexual couples, aged 26 to 60. It has been shown that one's own secure attachment can be perceived as the predictor of one's own relationship satisfaction in women and men and the predictor of a partner's relationship satisfaction in men. The findings support the partially mediating role of dyadic coping in the association between attachment and relationship satisfaction and are a significant contribution to the issue of dyadic coping in general. Adults' secure representations of their childhood experiences may be effective in using their partners as a secure base and also in serving as a secure base themselves, but it is not the sole influence on the quality of the couple's experience together. The we-ness phenomenon and resulting clinical implications were discussed.

Inflammasome function in monocyte subsets and a risk of late-onset sepsis in preterm very low birth weight neonates.

BACKGROUND: Immature immune systems predispose very low birth weight (VLBW) neonates to systemic infections in early life. Defective inflammasome function may increase a neonate's susceptibility to late-onset sepsis (LOS). METHODS: Blood samples were taken on the 5th day of life (DOL) for all VLBW neonates (non-LOS and before-LOS groups; N.=76), and within 24 hours of sepsis onset (LOS group; N.=39). Monocyte (MO) subsets and intracellular interleukin-1ß (IL-1ß) expression were analyzed using flow cytometry. Inflammasome function, defined as level of IL-1ß and interleukin-18 (IL-18) was measured with enzyme-linked immunosorbent assay. IRA B cells were reported as a fraction of all B cells. RESULTS: Stimulation of classical MO in non-LOS cells demonstrated a higher expression of intracellular IL-1ß in comparison to MO from before LOS group. Serum from the LOS group revealed a higher level of IL-18. Stimulation of mononuclear cultures from samples taken during LOS resulted in significantly increased supernatant level of IL-1ß and IL-18 in comparison to samples taken on 5th DOL. No changes in the levels of IRA B cells were detected with the onset of sepsis. CONCLUSIONS: We did not observe a difference in the functioning of the inflammasome within monocytes taken on 5th DOL from premature VLBW neonates. Furthermore, there was no observable change in the IRA B cells of the septic and non-septic groups. The decreased expression of intracellular IL-1ß within classical MO of the before-LOS group may be an independent risk factor for LOS development.

Gold-Decorated Platinum and Palladium Nanoparticles as Modern Nanocomplexes to Improve the Effectiveness of Simulated Anticancer Proton Therapy.

Noble metal nanoparticles, such as gold (Au NPs), platinum (Pt NPs), or palladium (Pd NPs), due to their highly developed surface, stability, and radiosensitizing properties, can be applied to support proton therapy (PT) of cancer. In this paper, we investigated the potential of bimetallic, c.a. 30 nm PtAu and PdAu nanocomplexes, synthesized by the green chemistry method and not used previously as radiosensitizers, to enhance the effect of colorectal cancer PT in vitro. The obtained nanomaterials were characterized by scanning transmission electron microscopy (STEM), selected area electron diffraction (SAED), energy-dispersive X-ray spectroscopy (EDS), UV-Vis spectroscopy, and zeta potential measurements. The effect of PtAu and PdAu NPs in PT was investigated on colon cancer cell lines (SW480, SW620, and HCT116), as well as normal colon epithelium cell line (FHC). These cells were cultured with both types of NPs and then irradiated by proton beam with a total dose of 15 Gy. The results of the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) test showed that the NPs-assisted PT resulted in a better anticancer effect than PT used alone; however, there was no significant difference in the radiosensitizing properties between tested nanocomplexes. The MTS results were further verified by defining the cell death as apoptosis (Annexin V binding assay). Furthermore, the data showed that such a treatment was more selective for cancer cells, as normal cell viability was only slightly affected.

Terphenyl-Based Small-Molecule Inhibitors of Programmed Cell Death-1/Programmed Death-Ligand 1 Protein-Protein Interaction.

We describe a new class of potent PD-L1/PD-1 inhibitors based on a terphenyl scaffold that is derived from the rigidified biphenyl-inspired structure. Using in silico docking, we designed and then experimentally demonstrated the effectiveness of the terphenyl-based scaffolds in inhibiting PD-1/PD-L1 complex formation using various biophysical and biochemical techniques. We also present a high-resolution structure of the complex of PD-L1 with one of our most potent inhibitors to identify key PD-L1/inhibitor interactions at the molecular level. In addition, we show the efficacy of our most potent inhibitors in activating the antitumor response using primary human immune cells from healthy donors.

Expression of VEGFA-mRNA in classical and MSX2-mRNA in non-classical monocytes in patients with spondyloarthritis is associated with peripheral arthritis.

Spondyloarthritis (SpA) is characterized by chronic inflammation and structural damage involving spine and peripheral joints. Monocytes, as part of innate immune system, following migration into affected tissue, may play a role in the pathogenesis of SpA. Here, potential associations between osteogenesis-linked gene expression profile in particular monocyte subpopulations and clinical signs of SpA were investigated. The 20 patients with axial and 16 with peripheral SpA were enrolled in the study. Monocyte subpopulations (classical-CD14++CD16-, intermediate-CD14++CD16+ and non-classical-CD14+CD16++) were isolated from blood using flow cytometry and gene expression analysis was performed using real-time PCR method and TaqMan Array, Human Osteogenesis, Fast 96-well plates. Next, the characteristic clinical features shared by axial and peripheral SpA were analyzed in the context of the expression of selected genes in the three subpopulations of monocytes. We demonstrated that expression of VEGFA in classical and MSX2 in non-classical monocytes were associated with the number of swollen and painful peripheral joints of SpA patients. We conclude that monocytes may contribute to the development of peripheral arthritis in SpA patients. This might be possible through subpopulation specific effects, linking number of inflamed joints with expression of VEGFA in classical monocytes and MSX2 in non-classical monocytes.

Down-Regulation of Dkk-1 in Platelets of Patients With Axial Spondyloarthritis.

OBJECTIVE: Axial spondyloarthritis (SpA) is a chronic autoinflammatory disease with new bone formation, which is controlled by Wnt/ß-catenin signaling. Dkk-1 is an inhibitor of the Wnt pathway, and in humans, platelets represent a major source of Dkk-1. This study was undertaken to investigate whether levels of Dkk-1 in serum and platelet expression of DKK1 messenger RNA (mRNA) and Dkk-1 protein are affected in patients with axial SpA compared to healthy controls. METHODS: Forty-one patients with axial SpA and 35 healthy controls were enrolled in the study. Total serum Dkk-1 levels in all patients and healthy controls were measured by quantitative enzyme-linked immunosorbent assay. Platelet DKK1 mRNA was analyzed by quantitative reverse transcriptase-polymerase chain reaction in 20 patients with axial SpA and 20 controls, and Dkk-1 protein levels were measured by immunoblotting in 20 patients with axial SpA and 18 controls. RESULTS: We found a lower concentration of Dkk-1 in the serum from patients with axial SpA compared to the serum from healthy controls (P < 0.0001). Furthermore, the expression of Dkk-1 was significantly reduced both at the transcriptional level (P < 0.04) and at the protein level (P < 0.007) in platelets isolated from the blood of patients with axial SpA. CONCLUSION: Our preliminary observations suggest that dysfunction of the megakaryocyte/platelet axis might be responsible for reduced serum Dkk-1 levels in patients with axial SpA. Dkk-1 is down-regulated in the platelets of patients with axial SpA, a mechanism that might play a role in new bone formation.

Fancy-Shaped Gold-Platinum Nanocauliflowers for Improved Proton Irradiation Effect on Colon Cancer Cells.

Enhancing the effectiveness of colorectal cancer treatment is highly desirable. Radiation-based anticancer therapy-such as proton therapy (PT)-can be used to shrink tumors before subsequent surgical intervention; therefore, improving the effectiveness of this treatment is crucial. The addition of noble metal nanoparticles (NPs), acting as radiosensitizers, increases the PT therapeutic effect. Thus, in this paper, the effect of novel, gold-platinum nanocauliflowers (AuPt NCs) on PT efficiency is determined. For this purpose, crystalline, 66-nm fancy shaped, bimetallic AuPt NCs were synthesized using green chemistry method. Then, physicochemical characterization of the obtained AuPt NCs by transmission electron microscopy (TEM), selected area electron diffraction (SAED), energy dispersive X-ray spectroscopy (EDS), and UV-Vis spectra measurements was carried out. Fully characterized AuPt NCs were placed into a cell culture of colon cancer cell lines (HCT116, SW480, and SW620) and a normal colon cell line (FHC) and subsequently subjected to proton irradiation with a total dose of 15 Gy. The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) test, performed after 18-h incubation of the irradiated cell culture with AuPt NCs, showed a significant reduction in cancer cell viability compared to normal cells. Thus, the radio-enhancing features of AuPt NCs indicate their potential application for the improvement in effectiveness of anticancer proton therapy.

Di-bromo-Based Small-Molecule Inhibitors of the PD-1/PD-L1 Immune Checkpoint.

Immune checkpoint blockade is one of the most promising strategies of cancer immunotherapy. However, unlike classical targeted therapies, it is currently solely based on expensive monoclonal antibodies, which often inflict immune-related adverse events. Herein, we propose a novel small-molecule inhibitor targeted at the most clinically relevant immune checkpoint, PD-1/PD-L1. The compound is capable of disrupting the PD-1/PD-L1 complex by antagonizing PD-L1 and, therefore, restores activation of T cells similarly to the antibodies, while being cheap in production and possibly nonimmunogenic. The final compound is significantly smaller than others reported in the literature while being nontoxic to cells even at high concentrations. The scaffold was designed using a structure-activity relationship screening cascade based on a new antagonist-induced dissociation NMR assay, called the weak-AIDA-NMR. Weak-AIDA-NMR finds true inhibitors, as opposed to only binders to the target protein, in early steps of lead compound development, and this process makes it less time and cost consuming.

Similarities in the General Chemical Composition of Colon Cancer Cells and Their Microvesicles Investigated by Spectroscopic Methods-Potential Clinical Relevance.

Colon cancer constitutes 33% of all cancer cases in humans and the majority of patients with metastatic colon cancer still have poor prognosis. An important role in cancer development is the communication between cancer and normal cells. This may occur, among others, through extracellular vesicles (including microvesicles) (MVs), which are being released by both types of cells. MVs may regulate a diverse range of biological processes and are considered as useful cancer biomarkers. Herein, we show that similarity in the general chemical composition between colon cancer cells and their corresponding tumor-derived microvesicles (TMVs) does exist. These results have been confirmed by spectroscopic methods for four colon cancer cell lines: HCT116, LoVo, SW480, and SW620 differing in their aggressiveness/metastatic potential. Our results show that Raman and Fourier Transform InfraRed (FTIR) analysis of the cell lines and their corresponding TMVs did not differ significantly in the characterization of their chemical composition. However, hierarchical cluster analysis of the data obtained by both of the methods revealed that only Raman spectroscopy provides results that are in line with the molecular classification of colon cancer, thus having potential clinical relevance.

Transition Metal Containing Particulate Matter Promotes Th1 and Th17 Inflammatory Response by Monocyte Activation in Organic and Inorganic Compounds Dependent Manner.

In recent years, a significant increase in the frequency of disorders caused by air pollutants has been observed. Here we asked whether transition metal-containing particulate matter (TMCPM), a component of air pollution, has an effect on the activity of human CD4+ T cell subsets (Th1, Th2, Th17, and Treg). Peripheral blood mononuclear cells (PBMC) from healthy donors were cultured with or without NIST (SRM 1648a-standard urban particulate matter purchased from the National Institute for Standards and Technology) and LAP (SRM 1648a particulate matter treated within 120 min with cold oxygen plasma) preparations of TMCPM, differing in organic compounds content. Data show that TMCPM treatment increased the level of CD4+ cells positive for IFN-γ and IL-17A, specific for Th1 and Th17 cells, respectively. Moreover, a substantial decrease in frequency of Foxp3 positive CD4+ cells was observed in parallel. This effect was more pronounced for NIST particles, containing more organic components, including endotoxin (LPS - lipopolysaccharide) and required the presence of monocytes. Inactivation of LPS by treatment of TMCPM with polymyxin B reduced the inflammatory response of monocytes and Th subsets but did not abolish this activity, suggesting a role of their inorganic components. In conclusion, treatment of human PBMC with TMCPM skews the balance of Th1/Th2 and Treg/Th17 cells, promoting polarization of CD4+ T cells into Th1 and Th17 subsets. This phenomenon requires activation of monocytes and depends on the organic and inorganic fractions, including endotoxin content in TMCPM, as significantly higher inflammatory response was observed for the NIST comparing to LAP. This observation may shed a new light on the role of TMCPM in development and exacerbation of allergies, inflammatory, and autoimmune disorders.

Fe3O4@SiO2@Au nanoparticles for MRI-guided chemo/NIR photothermal therapy of cancer cells.

Novel functionalized (biofunctionalization followed by cisplatin immobilization) Fe3O4@SiO2@Au nanoparticles (NPs) were designed. The encapsulation of Fe3O4 cores inside continuous SiO2 shells preserves their initial structure and strong magnetic properties, while the shell surface can be decorated by small Au NPs, and then cisplatin (cPt) can be successfully immobilized on their surface. The fabricated NPs exhibit very strong T 2 contrasting properties for magnetic resonance imaging (MRI). The functionalized Fe3O4@SiO2@Au NPs are tested for a potential application in photothermal cancer therapy, which is simulated by irradiation of two colon cancer cell lines (SW480 and SW620) with a laser (λ = 808 nm, W = 100 mW cm-2). It is found that the functionalized NPs possess low toxicity towards cancer cells (∼10-15%), which however could be drastically increased by laser irradiation, leading to a mortality of the cells of ∼43-50%. This increase of the cytotoxic properties of the Fe3O4@SiO2@Au NPs, due to the synergic effect between the presence of cPt plus Au NPs and laser irradiation, makes these NPs perspective agents for potential (MRI)-guided stimulated chemo-photothermal treatment of cancer.

Autologous tumor­derived microvesicles influence gene expression profiles and enhance protumorigenic chemotactic potential, signal transduction and cellular respiration in gastric cancer cells.

Tumor­derived microvesicles (TMVs) interact with a variety of different cell types within the immune system, including lymphocytes, monocytes, dendritic cells and tumor cells that they have originated from. In the present study, the effects of autologous­TMVs (auto­TMVs) on gene expression, chemotaxis, intercellular signaling and cellular metabolism were examined in cells of the gastric cancer (GC) cell line 1415 (GC1415). The effects of auto­TMVs on mRNA gene expression in GC1415 cells were assessed using pathway­focused PCR arrays. A chemotaxis assay was performed using the HoloMonitor M4 System. Signaling pathways were evaluated using western blot analysis, and cellular respiration was measured using the Seahorse XF Cell Mito Stress Test. Exposure of the GC1415 cells to auto­TMVs led to the overexpression (75 genes) and underexpression (96 genes) of genes that are associated with signal transduction, metabolism, chemotaxis, angiogenesis and metastasis. The auto­TMVs were indicated to induce chemotaxis and activate the PI3K/AKT signaling pathway in GC1415 cells. However, the MAPK/ERK signaling pathway was not indicated to be activated. Furthermore, studies on cellular respiration in GC1415 cells exposed to auto­TMVs demonstrated a metabolic shift to glycolysis. The results of the current study thus indicate that auto­TMVs may exert an effect on tumor cell function.

Control of Arms of Au Stars Size and its Dependent Cytotoxicity and Photosensitizer Effects in Photothermal Anticancer Therapy.

Gold nanostars (AuS NPs) are a very attractive nanomaterial, which is characterized by high effective transduction of the electromagnetic radiation into heat energy. Therefore, AuS NPs can be used as photosensitizers in photothermal therapy (PTT). However, understanding the photothermal conversion efficiency in nanostars is very important to select the most appropriate shape and size of AuS NPs. Therefore, in this article, the synthesis of AuS NPs with different lengths of star arms for potential application in PTT was investigated. Moreover, the formation mechanism of these AuS NPs depending on the reducer concentration is proposed. Transmission electron microscopy (TEM) with selected area diffraction (SEAD) and X-ray diffraction (X-Ray) showed that all the obtained AuS NPs are crystalline and have cores with similar values of the diagonal (parameter d), from 140 nm to 146 nm. However, the widths of the star arm edges (parameter c) and the lengths of the arms (parameter a) vary between 3.75 nm and 193 nm for AuS1 NPs to 6.25 nm and 356 nm for AuS4 NPs. Ultraviolet-visible (UV-Vis) spectra revealed that, with increasing edge widths and lengths of the star arms, the surface plasmon resonance (SPR) peak is shifted to the higher wavelengths, from 640 nm for AuS1 NPs to 770 nm for AuS4 NPs. Moreover, the increase of temperature in the AuS NPs solutions as well as the values of calculated photothermal efficiency grew with the elongation of the star arms. The potential application of AuS NPs in the PTT showed that the highest decrease of viability, around 75%, of cells cultured with AuS NPs and irradiated by lasers was noticed for AuS4 NPs with the longest arms, while the smallest changes were visible for gold nanostars with the shortest arms. The present study shows that photothermal properties of AuS NPs depend on edge widths and lengths of the star arms and the values of photothermal efficiency are higher with the increase of the arm lengths, which is correlated with the reducer concentration.

CA-170 - A Potent Small-Molecule PD-L1 Inhibitor or Not?

CA-170 is currently the only small-molecule modulator in clinical trials targeting PD-L1 and VISTA proteins - important negative checkpoint regulators of immune activation. The reported therapeutic results to some extent mimic those of FDA-approved monoclonal antibodies overcoming the limitations of the high production costs and adverse effects of the latter. However, no conclusive biophysical evidence proving the binding to hPD-L1 has ever been presented. Using well-known in vitro methods: NMR binding assay, HTRF and cell-based activation assays, we clearly show that there is no direct binding between CA-170 and PD-L1. To strengthen our reasoning, we performed control experiments on AUNP-12 - a 29-mer peptide, which is a precursor of CA-170. Positive controls consisted of the well-documented small-molecule PD-L1 inhibitors: BMS-1166 and peptide-57.

Sera of patients with axial spondyloarthritis (axSpA) enhance osteoclastogenic potential of monocytes isolated from healthy individuals.

BACKGROUND: Axial spondyloarthritis (axSpA) is characterized by significant bone loss caused by dysregulation of physiological bone turnover, possibly resulting from intensified differentiation of osteoclasts. The aim of this study was to reevaluate the levels of osteoclastogenesis-mediating factors: soluble RANKL, M-CSF, OPG and other cytokines in sera of untreated, with sDMARDs and/or bDMARDs, axSpA patients and to test whether these sera influence differentiation of healthy monocytes towards osteoclast lineage. METHODS: Bone remodeling molecules (RANKL, M-CSF, OPG, IL-6, OSM, IL-17A, TGFß, and TNFα) were evaluated in 27 patients with axSpA and 23 age and sex-matched controls. Disease activity (BASDAI, ASDAS) and inflammatory markers (ESR, CRP) were assessed. Monocytes obtained from healthy individuals were cultured in vitro in presence of sera from 11 randomly chosen axSpA patients and 10 controls, with addition of exogenous M-CSF and/or RANKL or without. Osteoclastic differentiation was assessed analyzing osteoclast markers (cathepsin K and RANK at mRNA level) and with osteoclast-specific staining. RESULTS: axSpA patients' sera levels of soluble RANKL were significantly lower and M-CSF, IL-6, OSM, IL-17A and TNFα significantly higher in comparison to controls, whereas of OPG and TGFß were comparable in both groups. Numbers of generated in vitro osteoclasts and cathepsin K mRNA levels did not differ between cultures supplemented with sera of healthy and axSpA patients, both in the absence and presence of M-CSF. Instead, addition of exogenous RANKL boosted osteoclastogenesis, which was significantly higher in cultures with axSpA sera. Furthermore, sera from axSpA patients induced substantially higher levels of RANK mRNA, independently of M-CSF and RANKL stimulation. CONCLUSION: We show that, paradoxically, serum levels of soluble RANKL observed in axSpA are in fact significantly lower in comparison to healthy blood donors. Our results indicate that sera of axSpA patients - in contrary to healthy subjects - contain circulating, soluble factors (presumably IL-6, OSM, IL-17A, TNFα and others) able to stimulate healthy monocytes responsiveness to even relative low RANKL serum levels, by inducing high RANK mRNA expression and - as a net effect - boosting their osteoclastogenic potential. We suggest also that locally produced RANKL in axSpA may induce overactive osteoclasts from their precursors.

Secretion of tumoricidal human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by recombinant Lactococcus lactis: optimization of in vitro synthesis conditions.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively eliminates tumor cells. However, the short biological half-life of this molecule limits its potential use in the clinic. Our aim was to construct a recombinant strain of nonpathogenic Lactococcus lactis bacteria as a vector for effective and prolonged human TRAIL production. Herein, we examined the expression and secretion conditions leading to the production of biologically active protein in vitro. RESULTS: The human soluble TRAIL-cDNA (hsTRAIL-cDNA) with optimized codons was designed to fit the codon usage pattern (codon bias) of the L. lactis host. This cDNA construct was synthesized and cloned in lactococcal plasmid secretion vector pNZ8124 under the control of the nisin-induced PnisA promoter. The pNZ8124-hsTRAIL plasmid vector was transformed into the L. lactis NZ9000 host strain cells by electroporation. Secretion of the protein occurred at the neutral pH during induction, with optimized concentration of the inducer and presence of serine proteases inhibitor. Using Western blotting and amino acid sequencing method we found that TRAIL was secreted in two forms, as visualized by the presence of two distinct molecular size bands, both deprived of the usp45 protein, the bacterial signal peptide. By the use of MTS assay we were able to prove that hsTRAIL present in supernatant from L. lactis (hsTRAIL+) broth culture was cytotoxic to human HCT116 colon cancer cells but not to normal human fibroblasts. Flow cytometry analysis revealed TRAIL-induced apoptosis of cancer cells. CONCLUSIONS: We designed recombinant L. lactis bacteria, which efficiently produce biologically active, anti-tumorigenic human TRAIL in vitro. Further studies in tumor-bearing NOD-SCID mice will reveal whether the TRAIL-secreting L. lactis bacteria can be used as a safe carrier of this protein, capable of inducing effective elimination of human colon cancer cells in vivo.

Characterization of human gastric adenocarcinoma cell lines established from peritoneal ascites.

The three cell lines, designated as gastric cancer (GC)1401, GC1415 and GC1436 were derived from peritoneal effusions from patients with gastric adenocarcinoma. Cell lines were established in tissue culture and in immunodeficient, non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. All cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. These cell lines were grown as an adherent monolayer with doubling time ranging between 25 h (GC1436 cell line) and 30-34 h (GC1401 and GC1415, respectively). All cells showed morphological features of epithelial-like cells, forming sheets of polygonal cells. Chromosomal analysis showed that the modal numbers ranged from 52 (GC1401), 51-56 (GC1415) and 106 (GC1436). High heterogeneity, resulting from several structural and numerical chromosomal abnormalities were evident in all cell lines. The surface marker expression suggested a tumor origin of the cells, and indicated the intestinal phenotype of a GC (CD10+, MUC1). All three cell lines were tumorigenic but not metastatic, in vivo, in NOD/SCID mice. The lack of metastatic potential was suggested by the lack of aldehyde dehydrogenase 1A1 activity. In conclusion, these newly established GC cell lines widen the feasibility of the functional studies on biology of GC as well as drug testing for potential therapeutic purposes.