Renal Cell Carcinoma-Infiltrating CD3low Vγ9Vδ1 T Cells Represent Potentially Novel Anti-Tumor Immune Players

Due to the highly immunogenic nature of renal cell carcinoma (RCC), the tumor microenvironment (TME) is enriched with various innate and adaptive immune subsets. In particular, gamma-delta (γδ) T cells can act as potent attractive mediators of adoptive cell transfer immunotherapy because of their unique properties such as non-reliance on major histocompatibility complex expression, their ability to infiltrate human tumors and recognize tumor antigens, relative insensitivity to immune checkpoint molecules, and broad tumor cytotoxicity. Therefore, it is now critical to better characterize human γδ T-cell subsets and their mechanisms in RCCs, especially the stage of differentiation. In this study, we aimed to identify γδ T cells that might have adaptive responses against RCC progression. We characterized γδ T cells in peripheral blood and tumor-infiltrating lymphocytes (TILs) in freshly resected tumor specimens from 20 RCC patients. Furthermore, we performed a gene set enrichment analysis on RNA-sequencing data from The Cancer Genome Atlas (TCGA) derived from normal kidneys and RCC tumors to ascertain the association between γδ T-cell infiltration and anti-cancer immune activity. Notably, RCC-infiltrating CD3^low Vγ9Vδ1 T cells with a terminally differentiated effector memory phenotype with up-regulated activation/exhaustion molecules were newly detected as predominant TILs, and the cytotoxic activity of these cells against RCC was confirmed in vitro. In an additional analysis of the TCGA RCC dataset, γδ T-cell enrichment scores correlated strongly with those for CTLs, Th1 cells, “exhausted” T cells, and M1 macrophages, suggesting active involvement of γδ T cells in anti-tumor rather than pro-tumor activity, and Vδ1 cells were more abundant than Vδ2 or Vδ3 cells in RCC tumor samples. Thus, we posit that Vγ9Vδ1 T cells may represent an excellent candidate for adoptive immunotherapy in RCC patients with a high risk of relapse after surgery.     

Critical considerations for the development of potency tests for therapeutic applications of mesenchymal stromal cell-derived small extracellular vesicles

Mesenchymal stromal/stem cells (MSCs) have been widely tested against many diseases, with more than 1000 registered clinical trials worldwide. Despite many setbacks, MSCs have been approved for the treatment of graft-versus-host disease and Crohn disease. However, it is increasingly clear that MSCs exert their therapeutic functions in a paracrine manner through the secretion of small extracellular vesicles (sEVs) of 50–200 nm in diameter. Unlike living cells that can persist long-term, sEVs are non-living and non-replicative and have a transient presence in the body. Their small size also renders sEV preparations highly amenable to sterilization by filtration. Together, acellular MSC-sEV preparations are potentially safer and easier to translate into the clinic than cellular MSC products. Nevertheless, there are inherent challenges in the development of MSC-sEV drug products. MSC-sEVs are products of living cells, and living cells are sensitive to changes in the external microenvironment. Consequently, quality control metrics to measure key identity and potency features of MSC-sEV preparations have to be specified during development of MSC-sEV therapeutics. The authors have previously described quantifiable assays to define the identity of MSC-sEVs. Here the authors discuss requirements for prospective potency assays to predict the therapeutic effectiveness of the drug substance in accordance with International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines. Although potency assays should ideally reflect the mechanism of action (MoA), this is challenging because the MoA for the reported efficacy of MSC-sEV preparations against multiple diseases of diverse underlying pathology is likely to be complex and different for each disease and difficult to fully elucidate. Nevertheless, robust potency assays could be developed by identifying the EV attribute most relevant to the intended biological activity in EV-mediated therapy and quantifying the EV attribute. Specifically, the authors highlight challenges and mitigation measures to enhance the manufacture of consistent and reproducibly potent sEV preparations, to identify and select the appropriate EV attribute for potency assays despite a complex “work-in-progress” MoA and to develop assays likely to be compliant with regulatory guidance for assay validation.     

A Hierarchically Organized Photoelectrode Architecture for Highly Efficient CdS/CdSe‐Sensitized Solar Cells

A form of photoelectrode architecture suitable for inorganic semiconductor solar cells is reported. The developed architecture consists of hierarchically organized TiO₂ nanostructures with several tens of nanometer‐sized particles that have a large surface area and open channels with several hundred‐nanometer‐gaps perpendicular to the substrate. These are tailored by controlling the kinetic energy of the ablated species during pulsed laser deposition (PLD). To fabricate the solar cells, CdS and CdSe inorganic sensitizers are assembled onto the architecture by successive ionic layer adsorption and reaction and polysulfide solution is used as an electrolyte with lead sulfide counter‐electrodes. The inorganic semiconductor solar cells using the developed architecture (PLD‐TiO₂) show high energy conversion efficiencies of 5.57% compared to a conventional mesoporous TiO₂ film(NP‐TiO₂) (3.84%) with an optical mask at 1 sun of illumination. The improved cell performance of PLD‐TiO₂ is attributed to greater light‐harvesting ability, which results in the enhancement of the J_sc value. PLD‐TiO₂ absorbs more CdS/CdSe because of its larger surface area and excellent adhesion properties with fluorine‐doped tin oxide (FTO) substrates. Additionally, due to its unique channel‐shaped architecture, PLD‐TiO₂ has a longer electron lifetime compared to NP‐TiO₂.     

A hydrophilic and hydrophobic organic solvent mixture enhances enzyme stability in organic media

Binary mixtures of hydrophilic and hydrophobic solvents were assessed for their ability to balance enzyme activity with the conservation of enzyme stability in organic media. Acetone, dioxane and dodecane were chosen as model organic solvents, and subtilisin Carlsberg and horseradish peroxidase (HRP) were chosen as model enzymes. Residual enzyme activities were measured to monitor enzyme stability, and the fluorescence intensity of HRP was monitored to investigate structural changes due to the presence of an organic solvent. Enzyme stability increased with the increasing hydrophobicity of the solvent mixture used, and a solvent mixture with a high log P value (~ >4) was capable of conserving enzyme stability. Enzyme stability in organic media can be conserved therefore with a mixture of hydrophilic and hydrophobic solvents: this approach might be used as a general and practical strategy for optimizing enzyme activity and stability for industrial applications.     

Tumorigenesis of Papillary Thyroid Cancer Is Not BRAF-Dependent in Patients with Acromegaly

Introduction

Several studies have reported a high frequency of papillary thyroid cancer (PTC) in patients with acromegaly. The aim of this study was to determine the prevalence and predictors of thyroid cancer in patients with acromegaly and to investigate the frequency of the BRAF ^V600E mutation in PTC patients with and without acromegaly.

Materials and Methods

We conducted a retrospective study of 60 patients with acromegaly. Thyroid ultrasonography (US) and US-guided fine needle aspiration were performed on nodules with sonographic features of malignancy. We selected 16 patients with non-acromegalic PTC as a control group. The BRAF ^V600E mutation was analyzed in paraffin-embedded surgical specimens of PTC by real-time polymerase chain reaction, and tumor specimens from patients with PTC were stained immunohistochemically with an antibody against insulin-like growth factor-1 receptor β (IGF-1Rβ).

Results

Thyroid cancer was found in 15 (25.0%) patients. No differences in age, sex, initial growth hormone (GH) and IGF-1 percentage of the upper limit of normal values or treatment modalities were observed between patients with and without PTC. Acromegaly was active in 12 of 15 patients at the time of PTC diagnosis; uncontrolled acromegaly had a significantly higher frequency in the PTC group (60%) than in the non-PTC group (28.9%) (p = 0.030). The BRAF ^V600E mutation was present in only 9.1% (1/11) of PTC patients with acromegaly, although 62.5% (10/16) of control patients with PTC had the mutation (p = 0.007). IGF-1Rβ immunostaining showed moderate-to-strong staining in all malignant PTC cells in patients with and without acromegaly. Significantly less staining for IGF-1Rβ was observed in normal adjacent thyroid tissues of PTC patients with acromegaly compared with those without (p = 0.014).

Conclusion

The prevalence of PTC in acromegalic patients was high (25%). An uncontrolled hyperactive GH-IGF-1 axis may play a dominant role in the development of PTC rather than the BRAF ^V600E mutation in patients with acromegaly.     

Functional Phenotype of Synovial Monocytes Modulating Inflammatory T-Cell Responses in Rheumatoid Arthritis (RA)

Monocytes function as crucial innate effectors in the pathogenesis of chronic inflammatory diseases, including autoimmunity, as well as in the inflammatory response against infectious pathogens. Human monocytes are heterogeneous and can be classified into three distinct subsets based on CD14 and CD16 expression. Although accumulating evidence suggests distinct functions of monocyte subsets in inflammatory conditions, their pathogenic roles in autoimmune diseases remain unclear. Thus, we investigated the phenotypic and functional characteristics of monocytes derived from synovial fluid and peripheral blood in RA patients in order to explore the pathogenic roles of these cells. In RA patients, CD14⁺CD16⁺, but not CD14^dimCD16⁺, monocytes are predominantly expanded in synovial fluid and, to a lesser degree, in peripheral blood. Expression of co-signaling molecules of the B7 family, specifically CD80 and CD276, was markedly elevated on synovial monocytes, while peripheral monocytes of RA and healthy controls did not express these molecules without stimulation. To explore how synovial monocytes might gain these unique properties in the inflammatory milieu of the synovial fluid, peripheral monocytes were exposed to various stimuli. CD16 expression on CD14⁺ monocytes was clearly induced by TGF-β, although co-treatment with IL-1β, TNF-α, or IL-6 did not result in any additive effects. In contrast, TLR stimulation with LPS or zymosan significantly downregulated CD16 expression such that the CD14⁺CD16⁺ monocyte subset could not be identified. Furthermore, treatment of monocytes with IFN-γ resulted in the induction of CD80 and HLA-DR expression even in the presence of TGF-β. An in vitro assay clearly showed that synovial monocytes possess the unique capability to promote Th1 as well as Th17 responses of autologous peripheral CD4 memory T cells. Our findings suggest that the cytokine milieu of the synovial fluid shapes the unique features of synovial monocytes as well as their cardinal role in shaping inflammatory T-cell responses in RA.     

Metabolic Syndrome and Chronic Kidney Disease in an Adult Korean Population: Results from the Korean National Health Screening

Background

This study was aimed to examine the prevalence of metabolic syndrome (MS) and chronic kidney disease (CKD), and the association between MS and its components with CKD in Korea.

Methods

We excluded diabetes to appreciate the real impact of MS and performed a cross-sectional study using the general health screening data of 10,253,085 (48.86±13.83 years, men 56.18%) participants (age, ≥20 years) from the Korean National Health Screening 2011. CKD was defined as dipstick proteinuria ≥1 or an estimated glomerular filtration rate (eGFR) <60 ml/min/1.73 m².

Results

The prevalence of CKD was 6.15% (men, 5.37%; women, 7.15%). Further, 22.25% study population had MS (abdominal obesity, 27.98%; hypertriglyceridemia, 30.09%; low high-density cholesterol levels, 19.74%; high blood pressure, 43.45%; and high fasting glucose levels, 30.44%). Multivariate-adjusted analysis indicated that proteinuria risk increased in participants with MS (odds ratio [OR] 1.884, 95% confidence interval [CI] 1.867–1.902, P<0.001). The presence of MS was associated with eGFR<60 mL/min/1.73 m² (OR 1.364, 95% CI 1.355–1.373, P<0.001). MS individual components were also associated with an increased CKD risk. The strength of association between MS and the development of CKD increase as the number of components increased from 1 to 5. In sub-analysis by men and women, MS and its each components were a significant determinant for CKD.

Conclusions

MS and its individual components can predict the risk of prevalent CKD for men and women.     

Induction of the Differentiation of HL-60 Promyelocytic Leukemia Cells by l-Ascorbic Acid

The present study was undertaken to examine the effect of l-ascorbic acid (LAA) on the growth of HL-60 promyelocytic leukemia cells, besides induction of apoptosis. LAA (≥10^-4?M) was found to markedly inhibit the proliferation of HL-60 in liquid culture and clonogenicity in semisolid culture. Moreover, LAA-treated HL-60 showed activity to produce chemiluminescence and expressed CD 66b cell surface antigens, indicating that LAA induces the differentiation of HL-60 mainly into granulocytes. The results are supported by morphological changes of LAA-treated HL-60 into segmented neutrophils. Therefore, the inhibitory effect of LAA on the growth of HL-60 cells seems to arise from the induction of differentiation. To assess the potential role of LAA, cells were exposed to oxygen radical scavengers in the absence or presence of LAA. Catalase abolished and superoxide dismutase promoted LAA-induced differentiation of HL-60. Thus, H²O² produced as a result of LAA treatment seems to play a major role in induction of HL-60 differentiation.     

The protein kinase 2 inhibitor CX-4945 regulates osteoclast and osteoblast differentiation In vitro

Drug repositioning can identify new therapeutic applications for existing drugs, thus mitigating high R&D costs. The Protein kinase 2 (CK2) inhibitor CX-4945 regulates human cancer cell survival and angiogenesis. Here we found that CX-4945 significantly inhibited the RANKL-induced osteoclast differentiation, but enhanced the BMP2-induced osteoblast differentiation in a cell culture model. CX-4945 inhibited the RANKL-induced activation of TRAP and NFATc1 expression accompanied with suppression of Akt phosphorylation, but, in contrast, it enhanced the BMP2-mediated ALP induction and MAPK ERK1/2 phosphorylation. CX-4945 is thus a novel drug candidate for bone-related disorders such as osteoporosis.     

Eisenstasin,new antistasin family inhibitor from the earthworm

A couple of new antistasin family serine protease inhibitors have been isolated from the non-hematophagous earthworm, Eisenia andrei. These novel inhibitors have been designated as eisenstasin I and II. Similar to other antistasin family inhibitors, eisenstasin I and II feature 3 and 4 internal repeats, respectively, of a 24–29 amino acid sequence, both of which exhibit a conserved pattern of 6-cysteine/2-glycine at an identical position between the third and fourth cysteine residues. This suggests that the eisenstasins isolated from the earthworm are members of the antistasin family. The eisenstasins are 82% similar with regard to amino acid sequences and exhibit over 70% similarity with the antistasins from the earthworm Lumbricus rubellus, while also displaying less than 40% sequence similarity with the leech antistasins. Earthworm eisenstasins are basic proteins, primarily due to the frequent occurrence of arginine residues in their structure, especially at the C-terminal region. As arginine is a key residue for the substrate specificity of some serine proteases including FXa, it is thought that these multiple arginine residues may play a role in the inhibitory characteristics of the eisenstasins. Considering the structure and number of the internal repeats derived from a variety of animal species, the deletion as well as the duplication of all or part of an internal repeat may be implicated in the evolution of the structure and function of the antistasin family inhibitors.