Ascorbic acid induced TET2 enzyme activation enhances cancer immunotherapy efficacy in renal cell carcinoma

Exploring the regulatory mechanism of PD-L1 in renal cancer is one of the key strategies to improve the response of renal cancer patients to checkpoint blockade therapy. In this study, the synergistic effect of ascorbic acid (vitamin C) supplementation and the impact of TET2 depletion on anti-PD-L1 therapy were determined in xenograft model experiments. Lymphocyte infiltration and chemokine expression were determined using flow cytometry and qRT-PCR. To determine the downstream targets of TET2, we performed hMeDip-seq and RNA-seq analyses. The molecular mechanism was further confirmed by hMeDip-qPCR, MeDip-qPCR, bisulfite sequencing, Western blotting, qRT-PCR and xenograft model experiments in vitro and in vivo. The present study demonstrated that ascorbic acid enhanced the efficacy of immunotherapy and that the loss of TET2 function enabled renal cancer cells to evade antitumor immunity. Ascorbic acid treatment significantly increased the intratumoral infiltration of T cells and the expression of cytokines and chemokines, while the loss of TET2 impaired the infiltration of T cells and the expression of cytokines and chemokines. TET2 was recruited to IRF1 by IFN-γ-STAT1 signaling, thereby maintaining IRF1 demethylation and ultimately inducing PD-L1 expression. These results suggest a new strategy of stimulating TET activity to improve immunotherapy for renal cell carcinoma.     

New sense organ in larval hookworms.

A new ciliary sense organ is described which is bilaterally arranged, one in each lateral cord. It is just posterior to the nerve ring and adjacent to the excretory duct. The cilium is closely associated with another structure which is itself connected to the cuticle beneath the lateral alae. A nervous circuit is proposed which links this receptor via the hemizonid to the nerve ring. The receptor and associated structures are in the infective larvae of Necator americanus and Ancylostoma tubaeforme.     

新型声学造影剂在常规诊断超声频率下对在 体肾脏及离体培养细胞影响的研究

目的：评价新型超声造影剂在常规诊断超声频率下对组织细胞结构的影响。资料和方法：体外培养的人近曲小管上皮细胞分为造影剂无超声照射组,单纯超声照射组,MI 1．5,造影剂加超声照射组。MI 0．28,造影剂加超声照射组,MI 1．5。超声照射时间为10分钟。实验完后用2.5％的戊二醛固定4小时,送电镜室做扫描电镜。正常免肾经耳缘静脉按0．02ml／kg团注脂氟显（新桥医院超声科实验室自制）,用6MHz的超声频率照射,MI 0．28。照射10分钟。活体取肾皮质。放入3．0％的戊二醛溶液中固定,送电镜室做投射电镜。结果：离体培养的人近端小管上皮细胞单纯超声照射、单纯加造影剂、造影剂加超声照射MI 0．28和MI 1．5时扫描电镜观察细胞形态无改变,细胞表面未见异常。透射电镜观察免肾小囊腔和肾小管上皮细胞超微结构未见特异性政变。结论：新型脂质体声学造影剂在诊断剂量超声照射下不会对组织细胞结构产生影响。新型脂质体声学造影剂在诊断剂量下是安全的。     

Activity estimation and biokinetic analysis of 99mTc-DMSA in renal infant patients using a gamma camera

Biokinetic data from the administration of radiopharmaceuticals is essential in nuclear medicine dosimetry. It has particular significance in children, as their metabolism is very different from adults. Biokinetic models for paediatric patients could therefore need to be adapted to better reflect their absorption, retention and excretion functions, when compared to adults. Obtaining quality in vivo infant or paediatric biokinetic data is then essential to improve the available reference models, which in turn can lead to the optimization of paediatric procedures and protocols in clinical practice.This study analyses the biokinetic behaviour of ^99mTc-dimercaptosuccinic acid (DMSA), in 8 infants aged 4 months to 2 years old, through an imaging study using a gamma camera, and compares the obtained values with those obtained with the reference ICRP biokinetic model. The in vivo data was treated using an adapted methodology from the MIRD 16 pamphlet. Activity curves for the liver, the kidney and the whole body, were built, and new effective absorption, retention and excretion half-lives were estimated, and compared with the reference biokinetic parameters of ICRP 128. The obtained residence time in the kidneys of 2.56 h, has a deviation of 30.8% to the ICRP 128 value of 3.70 h. The obtained maximum uptake in the kidneys was of 0.22/A₀, which compares to the value of 0.31/A₀ for ICRP.The obtained biokinetic parameters were used to estimate the absorbed dose. The obtained dose values are smaller than the reference ICRP 128 ones by 32.1% in the kidneys, and 18.4% in the liver.     

Reduced L-selectin (CD62LLow) expression identifies tumor-specific type 1 T cells from lymph nodes draining an autologous tumor cell vaccine

Reduced expression of CD62L can identify tumor-specific T cells in lymph nodes draining murine tumors. Here, we examined whether this strategy could isolate tumor-specific T cells from vaccinated patients. Tumor vaccine-draining lymph node (TVDLN) T cells of seven patients were separated into populations with reduced (CD62LLow) or high levels of CD62L (CD62LHigh). Effector T cells generated from CD62LLow cells maintained or enriched the autologous tumor-specific type 1 cytokine response compared to unseparated TVDLN T cells in four of four patients showing tumor-specific cytokine secretion. Interestingly, effector T cells generated from CD62LLow or CD62LHigh TVDLN were polarized towards a dominant type 1 or type 2 cytokine profile, respectively. For CD62LLow T cells the type 1 cytokine profile appeared determined prior to culture. Since a tumor-specific type 1 cytokine profile appears critical for mediating anti-tumor activity in vivo, this approach might be used to isolate T cells for adoptive immunotherapy.     

T cell epitope definition by differential mass spectrometry: identification of a novel, immunogenic HLA-B8 ligand directly from renal cancer tissue

In this study, we describe a differential mass spectrometric technique for the immuno-proteomic analysis of the major histocompatibility complex (MHC) peptides of a renal cell carcinoma (RCC) biopsy compared with the healthy kidney tissue of the same patient after nephrectomy. Using a stable isotope labeling approach, we could directly compare and relatively quantify 43 MHC-peptide pairs, most of which were present in similar proportions on both normal kidney and tumor. Significantly, two dominant peptides of monoisotopic masses ([M+H](+)) 973.43 u and 967.59 u, respectively, were found exclusively in the tumor sample. One of these was identified as originating from heme oxygenase-1 (HO-1), a protein involved in induction of apoptosis resistance, immuno-suppression and neoangiogenesis and reported to be up-regulated in various cancer types. Moreover, the corresponding synthetic HO-1-derived peptide was shown to be immunogenic in vitro by generation of CD8+ T cell lines with peptide-specific cytolytic activity. Thus, this peptide is an example of a differentially identified T cell epitope that could be considered as a target for immunotherapy.     

Membrane Tension Modulates the Effects of Apical Cholesterol on the Renal Epithelial Sodium Channel

We used patch-clamp techniques and A6 distal nephron cells as a model to determine how cholesterol regulates the renal epithelial sodium channel (ENaC). We found that luminal methyl-β-cyclodextrin (mβCD, a cholesterol scavenger) did not acutely affect ENaC activity at a previously used concentration of 10 mm but significantly decreased ENaC activity both when the cell membrane was stretched and at a higher concentration of 50 mm. Luminal cholesterol had no effect on ENaC activity at a concentration of 50 μg/ml but significantly increased ENaC activity both when the cell membrane was stretched and at a higher concentration of 200 μg/ml. Confocal microscopy data indicate that membrane tension facilitates both mβCD extraction of cholesterol and A6 cell uptake of exogenous cholesterol. Together with previous findings that cholesterol in the apical membrane is tightly packed with sphingolipids and that stretch can affect lipid distribution, our data suggest that membrane tension modulates the effects of mβCD and cholesterol on ENaC activity, probably by facilitating both extraction and enrichment of apical cholesterol.     

Role of structural organization in the urine concentrating mechanism of an avian kidney

The organization of tubules and blood vessels in the quail medullary cone is highly structured. This structural organization may result in preferential interactions among tubules and vessels, interactions that may enhance urine concentrating capability. In this study, we formulate a model framework for the urine concentrating mechanism of the quail kidney. The model simulates preferential interactions among renal tubules by representing two concentric cores and by specifying the fractions of tubules assigned to each of the concentric cores. The model equations are based on standard expressions for transmural transport and on solute and water conservation. Model results suggest that the preferential interactions among tubules enhance the urine concentration capacity of short medullary cones by reducing the diluting effect of the descending limbs on the region of the interstitium where the collecting ducts are located; however, the effects on longer cones are unclear.     

Characterization of renal interstitial fibroblast-specific protein 1/S100A4-positive cells in healthy and inflamed rodent kidneys

Fibrosis is considered as a central factor in the loss of renal function in chronic kidney diseases. The origin of fibroblasts and myofibroblasts that accumulate in the interstitium of the diseased kidney is still a matter of debate. It has been shown that accumulation of myofibroblasts in inflamed and fibrotic kidneys is associated with upregulation of fibroblast-specific protein 1 (FSP1, S100A4), not only in the renal interstitium but also in the injured renal epithelia. The tubular expression of FSP1 has been taken as evidence of myofibroblast formation by epithelial–mesenchymal transition (EMT). The identity of FSP1/S100A4 cells has not been defined in detail. We originally intended to use FSP1/S100A4 as a marker of putative EMT in a model of distal tubular injury. However, since the immunoreactivity of FSP1 did not seem to fit with the distribution and shape of fibroblasts or myofibroblasts, we undertook the characterization of FSP1/S100A4-expressing cells in the interstitium of rodent kidneys. We performed immunolabeling for FSP1/S100A4 on thin cryostat sections of perfusion-fixed rat and mouse kidneys with peritubular inflammation, induced by thiazides and glomerulonephritis, respectively, in combination with ecto-5-nucleotidase (5NT), recognizing local cortical peritubular fibroblasts, with CD45, MHC class II, CD3, CD4 and Thy 1, recognizing mononuclear cells, with alpha smooth muscle actin (SMA), as marker for myofibroblasts, and vimentin for intracellular intermediate filaments in cells of mesenchymal origin. In the healthy interstitium of rodents the rare FSP1/S100A4+ cells consistently co-expressed CD45 or lymphocyte surface molecules. Around the injured distal tubules of rats treated for 3–4 days with thiazides, FSP1+/S100A4+, 5NT+, SMA+, CD45+ and MHC class II+ cells accumulated. FSP1+/S100A4+ cells consistently co-expressed CD45. In the inflamed regions, SMA was co-expressed by 5NT+ cells. In glomerulonephritic mice, FSP1+/S100A4+ cells co-expressed Thy 1, CD4 or CD3. Thus, in the inflamed interstitium around distal tubules of rats and of glomerulonephritic mice, the majority of FSP1+ cells express markers of mononuclear cells. Consequently, the usefulness of FSP1/S100A4 as a tool for detection of (myo)fibroblasts in inflamed kidneys and of EMT in vivo is put into question. In the given rat model the consistent co-expression of SMA and 5NT suggests that myofibroblasts originate from resident peritubular fibroblasts.Ivan Hegyi and Michel Le Hir contributed equally to the study     

Anti-tumor effects of fusion vaccine prepared by renal cell carcinoma 786-O cell line and peripheral blood dendritic cells of healthy volunteers in vitro and in human immune reconstituted SCID mice

Dendritic cells (DC), as professional antigen presenting cells, play the central role in the process of body initiating the anti-tumor immunity, and the study on DC anti-tumor vaccine has become heated in recent years. In this study, we used polyethylene glycol (PEG) to induce renal cell carcinoma (RCC) 786-O cell line fused with peripheral blood DC of healthy volunteers, and discuss the biological characteristics of fusion vaccine and its anti-tumor effects in vitro and in human immune reconstituted SCID mice model of RCC. The study found that PEG could effectively induce cell fusion, and the expressions of CD86 and HLA-DR in fusion vaccine group were significantly up-regulated compared with the DC control group; the secretion of IL-12 was much higher and longer than that of the control; the functions of dendritic cell-tumor fusion vaccine to stimulate the proliferation of allogenic T lymphocytes and to kill RCC786-O cells in vitro were significantly higher than those of the control group, and after the killing, apoptosis body was observed in the target cells; after the injection of fusion vaccine into human immune reconstituted SCID mice model of RCC786-O via vena caudalis, the volume of mice tumor was reduced significantly, proliferation index of tumor cells decreased obviously compared with that of the control group, and more hemorrhage and putrescence focuses presented, accompanying large quantity of lymphocytes soakage. The results of this experimental study shows that fusion vaccine of RCC786-O cell line and DC can significantly stimulate the proliferation of allogenic T cells and specifically inhibit and kill RCC cells in vitro and in vivo, which makes the DC-RCC786-O fusion vaccine a possible new way of effective RCC immunotherapy.