Integrating 16S RRNA gene sequencing and metabolomics to evaluate the association between gut microbiota and serum metabolites in patients with myositis.

AIMS: Gut microbiota and metabolites have a profound impact on the maintenance of body health. In this study, we assessed the association between gut microbiota and serum metabolite changes in myositis using 16S rRNA gene sequencing and metabolomics to provide new ideas for screening and treating myositis. METHODS AND RESULTS: Blood and faecal samples were collected from 20 myositis patients and 20 healthy control subjects. Then, 16S rRNA gene sequencing, enzyme-linked immunosorbent assays and untargeted metabolomics study were performed to evaluate the relationship between gut microbiota and serum metabolites in patients with myositis. Compared to healthy control subjects, the blood samples from the patients with myositis had elevated levels of interleukin-4 (IL-4), tumour necrosis factor-α (TNF-α), and malondialdehyde (MDA) and decreased superoxide dismutase (SOD) levels. The increase in Bacteroidota (including Bacteroides and Parabacteroides, but not Prevotella) and the decrease in Firmicutes in the patients were accompanied by functional changes in amino acid and lipid metabolism. The gut microbiota (Bacteroides and Parabacteroides) were negatively correlated with the differential serum metabolites (glutamate and taurine). The differential serum metabolites (glutamate, pyrrolidonecarboxylic acid, and taurine) were also correlated with inflammatory factors (IL-4 and TNF-α) and oxidative stress indexes (MDA and SOD). CONCLUSION: Dysbiosis of gut microbiota in patients with myositis was accompanied by changes in inflammatory factors, oxidative stress indexes, and small molecule metabolites in serum. SIGNIFICANCE AND IMPACT OF STUDY: Blood and faecal biomarkers could be used for screening myositis.

High Basal Levels of γH2AX in Human Induced Pluripotent Stem Cells Are Linked to Replication-Associated DNA Damage and Repair.

Human induced pluripotent stem cells (iPSCs) have great potential as source cells for therapeutic uses. However, reports indicate that iPSCs carry genetic abnormalities, which may impede their medical use. Little is known about mechanisms contributing to intrinsic DNA damage in iPSCs that could lead to genomic instability. In this report, we investigated the level of DNA damage in human iPSC lines compared with their founder fibroblast line and derived mesenchymal stromal cell (MSC) lines using the phosphorylated histone variant, γH2AX, as a marker of DNA damage. We show that human iPSCs have elevated basal levels of γH2AX, which correlate with markers of DNA replication: 5-ethynyl-2'-deoxyuridine and the single-stranded binding protein, replication protein A. γH2AX foci in iPSCs also colocalize to BRCA1 and RAD51, proteins in the homologous repair pathway, implying γH2AX in iPSCs marks sites of double strand breaks. Our study demonstrates an association between increased basal levels of γH2AX and the rapid replication of iPSCs. Stem Cells 2018;36:1501-1513.

Ultrasmall Nanodots with Dual Anti-Ferropototic Effect for Acute Kidney Injury Therapy.

Ferroptosis is known to mediate the pathogenesis of chemotherapeutic drug-induced acute kidney injury (AKI); however, leveraging the benefits of ferroptosis-based treatments for nephroprotection remains challenging. Here, ultrasmall nanodots, denoted as FerroD, comprising the amphiphilic conjugate (tetraphenylethylene-L-serine-deferoxamine, TPE-lys-Ser-DFO (TSD)) and entrapped ferrostatin-1 are designed. After being internalized through kidney injury molecule-1-mediated endocytosis, FerroD can simultaneously remove the overloaded iron ions and eliminate the overproduction of lipid peroxides by the coordination-disassembly mechanisms, which collectively confer prominent inhibition efficiency of ferroptosis. In cisplatin (CDDP)-induced AKI mice, FerroD equipped with dual anti-ferroptotic ability can provide long-term nephroprotective effects. This study may shed new light on the design and clinical translation of therapeutics targeting ferroptosis for various ferroptosis-related kidney diseases.

The proportion of C1q-high and ISG15-high monocytes in the skin of patients with Behçet disease.

Behçet disease (BD) is a chronic systemic vasculitis that is clinically characterized by recurrent oral ulcers, genital ulcers, uveitis, and skin lesions. Here, we conducted bulk RNA-seq of skin samples from 4 BD patients and 4 normal controls (NCs). A total of 260 differentially expressed genes (DEGs), including 99 upregulated and 161 downregulated genes, were detected in the skin lesions of BD patients compared to NCs. These DEGs were mainly enriched in the following biological processes: the activation and migration of immune cells, the release of proinflammatory factors, and the IFN-γ signaling pathway. The top upregulated DEGs were CXCL10, CXCL9, FCGR3A, GBP5, GBP4, LILRB2, ADIPOQ, PLIN1, SLC43A2, and MYO1G. Using the deconvolution method CIBERSORT, we analyzed the immune cells subtypes in the skin of BD by integrating the single cell RNA-seq data from PBMC (GSE198616) and bulk RNA-seq data of skin. There was a higher proportion of C1q+ and ISG15 + monocyte subtypes in skin of BD. IHC staining of CD14 and CD16 showed that the monocyte number increased in the skin of BD. IF staining confirmed there was a higher proportion of the C1Q + Mono and ISG15 + Mono subsets in the skin of BD patients. Moreover, we analyzed the average expression level of the top upregulated genes in immune cell types found in PBMC from BD patients and NCs. Almost all the top upregulated genes expressed in monocytes. CXCL10 was specifically expressed in ISG15 + monocyte, and GBP5, GBP4 and IFI44L were expressed more strongly in ISG15 + monocytes. LILRB2 was expressed more strongly in CD16+ monocytes and C1Q + monocytes. In conclusion, our study identified that the IFN-γ pathway was activated in skin of BD and the proportion of C1q+ and ISG15 + monocyte subtype increased in the skin of BD.

Targeting Lymph Nodes for Systemic Immunosuppression Using Cell-Free-DNA-Scavenging And cGAS-Inhibiting Nanomedicine-In-Hydrogel for Rheumatoid Arthritis Immunotherapy.

Rheumatoid arthritis (RA) is a systemic autoimmune disease with pathogenic inflammation caused partly by excessive cell-free DNA (cfDNA). Specifically, cfDNA is internalized into immune cells, such as macrophages in lymphoid tissues and joints, and activates pattern recognition receptors, including cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS), resulting in overly strong proinflammation. Here, nanomedicine-in-hydrogel (NiH) is reported that co-delivers cGAS inhibitor RU.521 (RU) and cfDNA-scavenging cationic nanoparticles (cNPs) to draining lymph nodes (LNs) for systemic immunosuppression in RA therapy. Upon subcutaneous injection, NiH prolongs LN retention of RU and cNPs, which pharmacologically inhibit cGAS and scavenged cfDNA, respectively, to inhibit proinflammation. NiH elicits systemic immunosuppression, repolarizes macrophages, increases fractions of immunosuppressive cells, and decreases fractions of CD4+ T cells and T helper 17 cells. Such skewed immune milieu allows NiH to significantly inhibit RA progression in collagen-induced arthritis mice. These studies underscore the great potential of NiH for RA immunotherapy.

Ferroptosis MRI for early detection of anticancer drug-induced acute cardiac/kidney injuries.

Ferroptosis has been realized in anticancer drug-induced acute cardiac/kidney injuries (ACI/AKI); however, molecular imaging approach to detect ferroptosis in ACI/AKI is a challenge. We report an artemisinin-based probe (Art-Gd) for contrast-enhanced magnetic resonance imaging of ferroptosis (feMRI) by exploiting the redox-active Fe(II) as a vivid chemical target. In vivo, the Art-Gd probe showed great feasibility in early diagnosis of anticancer drug-induced ACI/AKI, which was at least 24 and 48 hours earlier than the standard clinical assays for assessing ACI and AKI, respectively. Furthermore, the feMRI was able to provide imaging evidence for the different mechanisms of action of ferroptosis-targeted agents, either by blocking lipid peroxidation or depleting iron ions. This study presents a feMRI strategy with simple chemistry and robust efficacy for early evaluation of anticancer drug-induced ACI/AKI, which may shed light on the theranostics of a variety of ferroptosis-related diseases.

Derived myeloid lineage induced pluripotent stem as a platform to study human C-C chemokine receptor type 5Δ32 homozygotes.

The C-C chemokine receptor type 5 (CCR5) expressed on immune cells supports inflammatory responses by directing cells to the inflammation site. CCR5 is also a major coreceptor for macrophage tropic human immunodeficiency viruses (R5-HIV-1) and its variants can confer protection from HIV infection, making it an ideal candidate to target for therapy. We developed a stepwise protocol that differentiates induced pluripotent stem cells (iPSCs) from individuals homozygous for the CCR5Δ32 variant and healthy volunteers into myeloid lineage induced monocytes (iMono) and macrophages (iMac). By characterizing iMono and iMac against their primary counterparts, we demonstrated that CCR5Δ32 homozygous cells are endowed with similar pluripotent potential for self-renewal and differentiation as iPSC lines generated from non-variant individuals while also showing resistance to HIV infection. In conclusion, these cells are a platform to investigate CCR5 pathophysiology in HIV-positive and negative individuals and to help develop novel therapies.

Human induced pluripotent stem cells generated from STING-associated vasculopathy with onset in infancy (SAVI) patients with a heterozygous mutation in the STING gene.

We have successfully created induced pluripotent stem cells (iPSC) from patients carrying a heterozygous mutation in the gene encoding STING. The gain-of-function mutation leads to constitutive activation of STING which leads to the development of the disease STING-associated vasculopathy with onset in infancy (SAVI). The iPSC lines derived from the SAVI patitents are shown to be morphologically and phenotypically normal and have the potential to self renew and differentiate into the three germ layers. These iPSC provide a powerful tools to investigate the role of STING in the regulation of immune responses and vascular renegeration.

Human induced pluripotent stem cells generated from a patient with a homozygous L272P mutation in the OTULIN gene (NIHTVBi014-A).

We have successfully generated induced pluripotent stem cells (iPSC) from dermal fibroblasts of a patient with a homozygous p.Leu272Pro mutation in the gene encoding the linear deubiquitinase OTULIN. Biallelic loss of function mutations in this gene are responsible for the OTULIN deficiency termed Otulipenia or OTULIN-related autoinflammatory syndrome (ORAS). The iPSC carrying homozygous L272P OTULIN gene mutations are phenotypically normal and they have capacity to differentiate toward the three germ layers. These iPSC have great potential to study the role of linear ubiquitination in the regulation of immune responses and other cellular pathways.

Generation of human induced pluripotent stem cells (NIHTVBi004-A, NIHTVBi005-A, NIHTVBi006-A, NIHTVBi007-A, NIHTVBi008-A) from 5 CADASIL patients with NOTCH3 mutation.

We have successfully generated induced pluripotent stem cell (iPSC) lines derived from peripheral blood mononuclear cells of five patients with Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). These cells carry the genetic NOTCH3 mutation present in their parental cells. These iPSC cells exhibited normal karyotype and phenotype, which were sustained through propagation. Furthermore, these iPSCs displayed the capacity of differentiating toward the three germ layers in vitro.

STAT3 modulates reprogramming efficiency of human somatic cells; insights from autosomal dominant Hyper IgE syndrome caused by STAT3 mutations.

Human induced pluripotent stem cell (iPSC) technology has opened exciting opportunities for stem-cell-based therapy. However, its wide adoption is precluded by several challenges including low reprogramming efficiency and potential for malignant transformation. Better understanding of the molecular mechanisms of the changes that cells undergo during reprograming is needed to improve iPSCs generation efficiency and to increase confidence for their clinical use safety. Here, we find that dominant negative mutations in STAT3 in patients with autosomal-dominant hyper IgE (Job's) syndrome (AD-HIES) result in greatly reduced reprograming efficiency of primary skin fibroblasts derived from skin biopsies. Analysis of normal skin fibroblasts revealed upregulation and phosphorylation of endogenous signal transducer and activator of transcription 3 (STAT3) and its binding to the NANOG promoter following transduction with OKSM factors. This coincided with upregulation of NANOG and appearance of cells expressing pluripotency markers. Upregulation of NANOG and number of pluripotent cells were greatly reduced throughout the reprograming process of AD-HIES fibroblasts that was restored by over-expression of functional STAT3. NANOGP8, the human-specific NANOG retrogene that is often expressed in human cancers, was also induced during reprogramming, to very low but detectable levels, in a STAT3-dependent manner. Our study revealed the critical role of endogenous STAT3 in facilitating reprogramming of human somatic cells.

Clinical Features and Cytokine Profile in Myositis Patients with Anti-EJ Autoantibodies Detected by a Novel Immunoprecipitation Assay.

OBJECTIVE: This study aimed to clarify the clinical features, the serum level of autoantibodies, and cytokine of myositis patients with anti-EJ antibody, which targets glycyl tRNA-synthetase (GlyRS). METHODS: Sera of 236 Chinese patients with myositis were screened for anti-EJ by a novel immunoprecipitation assay of flag-tagged GlyRS. Anti-EJ positive patients are evaluated for the clinical features and cytokine profile. RESULTS: The sera from 4 of 236 adult myositis patients were found to carry the anti-EJ using established novel immunoprecipitation assay and immunoblotting. The prevalence of anti-EJ in our cohorts is about 1.7%. The decline of anti-EJ level was detected in two patients during disease remission. Interstitial lung disease and muscle weakness, but not skin involvement, are common clinical features of anti-EJ positive patients. Moreover, using a cytokine profile analyses, we found that the serum levels of IP-10, IL-6, MCP-1, and VEGF were significantly elevated in patients with anti-EJ and gradually decreased during disease remission of two patients, whereas IL-8 level was obviously reduced in these patients. CONCLUSION: The novel immunoprecipitation assay is suitable to detect and monitor the levels of anti-EJ autoantibody. The serum levels of anti-EJ, IP-10, IL-6, MCP-1, and VEGF may be related to disease activity in myositis patients with anti-EJ antibodies.

Generation of human induced pluripotent stem cells from individuals with a homozygous CCR5Δ32 mutation.

Chemokine receptor 5 (CCR5) is the primary coreceptor for HIV entry into macrophages. Individuals with a homozygous deletion of 32 bp in the CCR5 gene (CCR5Δ32) are highly resistant to HIV infection (Samson et al., 1996). Allogeneic stem cell transplantation from a healthy donor with the homozygous CCR5Δ32 variant to an HIV positive individual has demonstrated efficient long-term control of HIV. We identified three individuals with this homozygous CCR5Δ32 variant, and successfully generated induced pluripotent stem cell (iPSC) lines from their dermal fibroblasts. The iPSCs lines carrying homozygous CCR5Δ32 variant displayed phenotypically normal and the potential to differentiation toward the three germ layers.

Generation of human induced pluripotent stem cell lines (NIHTVBi011-A, NIHTVBi012-A, NIHTVBi013-A) from autosomal dominant Hyper IgE syndrome (AD-HIES) patients carrying STAT3 mutation.

Autosomal dominant Hyper IgE syndrome (AD-HIES), a rare immune deficiency affecting fewer than one per million people, is caused by heterozygous deleterious mutations in STAT3. STAT3 signaling plays crucial roles in basic cellular functions affecting broad aspects of cellular homeostasis. Accordingly, in addition to immunological deficits, patients experience severe multisystem non-immunological features. Human induced pluripotent stem cells (hiPSC) are well established as in vivo disease models for various human pathologies. We describe the generation of iPSC from three AD-HIES patients. These iPSCs express pluripotency markers, differentiate into three germ layers, have normal karyotype and similar genome identity to parental cells.

Increased activity of TNAP compensates for reduced adenosine production and promotes ectopic calcification in the genetic disease ACDC.

ACDC (arterial calcification due to deficiency of CD73) is an autosomal recessive disease resulting from loss-of-function mutations in NT5E, which encodes CD73, a 5'-ectonucleotidase that converts extracellular adenosine monophosphate to adenosine. ACDC patients display progressive calcification of lower extremity arteries, causing limb ischemia. Tissue-nonspecific alkaline phosphatase (TNAP), which converts pyrophosphate (PPi) to inorganic phosphate (Pi), and extracellular purine metabolism play important roles in other inherited forms of vascular calcification. Compared to cells from healthy subjects, induced pluripotent stem cell-derived mesenchymal stromal cells (iMSCs) from ACDC patients displayed accelerated calcification and increased TNAP activity when cultured under conditions that promote osteogenesis. TNAP activity generated adenosine in iMSCs derived from ACDC patients but not in iMSCs from control subjects, which have CD73. In response to osteogenic stimulation, ACDC patient-derived iMSCs had decreased amounts of the TNAP substrate PPi, an inhibitor of extracellular matrix calcification, and exhibited increased activation of AKT, mechanistic target of rapamycin (mTOR), and the 70-kDa ribosomal protein S6 kinase (p70S6K), a pathway that promotes calcification. In vivo, teratomas derived from ACDC patient cells showed extensive calcification and increased TNAP activity. Treating mice bearing these teratomas with an A2b adenosine receptor agonist, the mTOR inhibitor rapamycin, or the bisphosphonate etidronate reduced calcification. These results show that an increase of TNAP activity in ACDC contributes to ectopic calcification by disrupting the extracellular balance of PPi and Pi and identify potential therapeutic targets for ACDC.

Low tumor blood flow assessed with perfusion CT correlates with lymphatic involvement in patients with stage T1b non-small cell lung cancer.

BACKGROUND: To investigate the correlation of computed tomography (CT) perfusion parameters and lymphatic involvement in patients with stage T1b non-small cell lung cancer (NSCLC). METHODS: Forty-six patients (30 men and 16 women; age range, 36-73 years; mean age, 57 years), with stage T1b non-small cell lung cancer, underwent perfusion CT before surgery. The correlations between CT perfusion parameters (blood flow, blood volume, peak enhancement intensity), tumor angiogenesis (microvessel density and maturity of microvessels of surgical specimens) and lymphatic involvement were retrospectively investigated. Receiver operator curve (ROC) analysis was used to identify the parameter threshold at which tumors had or did not have lymph node metastasis, and the corresponding sensitivity and specificity were calculated. RESULTS: A significant tendency for tumors with low blood flow and high density of immature microvessels to show lymphatic involvement was found (all P < 0.001). High correlation (r =-0.769, P < 0.001) was observed between tumor blood flow and immature microvessels. The area under ROC curves (AUC) for blood flow to detect lymph node metastasis was 0.866 (95% confidence interval, 0.766-0.966). For blood flow, the sensitivity, specificity, and accuracy of predicting lymph node metastasis were 88.9, 64.3, and 73.9% respectively, if the cutoff point was set at 43.05 mL/100 g/minute. CONCLUSIONS: Blood flow may be useful to predict lymphatic involvement before surgery in stage T1b NSCLC.

[Correlation between podoplanin-positive lymphatic microvessel density and CT characteristics of non-small cell lung cancer].

BACKGROUND AND OBJECTIVE: It has been proven that ymphatic microvessel density (LMVD) was closely correlated with the lymphatic metastasis of non-small cell lung cancer (NSCLC). The aim of the present study is to explore the relationship between podoplanin-LMVD and multi-slice spiral computed tomography (MSCT) characteristics of NSCLC. METHODS: MSCT scanning was performed on 34 cases of NSCLC (squamous carcinoma, 15 cases; adenocarcinoma, 15 cases; and adenosquamous carcinoma, 4 cases) prior to operation. Clinical pathology results, including lymph node metastasis, were obtained. CT characteristics, such as shape of the edge, internal structure, and adjacent structures, were described. LMVD in the central and peripheral areas examined respectively using SP immunohistochemical technique were analyzed. RESULTS: Lymph node metastasis was found to be associated with LMVD in the peripheral areas. LMVD in the peripheral areas of the resected lesions, the MSCT findings of which included spinous process, pleural indentation, and carcinomatous lymphangitis, was higher than that of the lesions without these MSCT characteristics (P<0.05). CONCLUSION: MSCT findings of spinous process, pleural indentation, or carcinomatous lymphangitis of NSCLC may suggest a higher level of tumor lymphangiogenesis with a higher risk of lymph node metastasis.

Lymphatic microvessel density combined with CT used in the diagnosis of mediastinal and hilar lymph node metastasis of non-small cell lung cancer.

BACKGROUND AND AIMS: Lymphatic microvessel density (LMVD) has been demonstrated to correlate with tumor metastasis. The purpose of this study is to determine whether the criteria combining LMVD with computed tomography (CT) could improve the diagnostic accuracy of lymph node (LN) metastasis in non-small cell lung cancer (NSCLC). METHODS: Ninety four patients with NSCLC who had chest CT scans preoperatively and LMVD tested by immunohistochemistry postoperatively were randomized into two groups: the training set (n = 66) and the test set (n = 28). Cut-off point of LMVD was selected to separate the LN metastasis-predictive positive and negative groups. On the basis of LMVD levels, chest CTs of the training set were re-analyzed and hypothetical criteria for LN metastasis diagnosis were established. Diagnostic characteristics for LN metastasis were tested by using the combined criteria in the test set as compared to those of CT alone. RESULTS: There was a significantly positive correlation between LMVD and LN metastasis (p <0.01). For sensitivity, specificity, positive predictive value (PPV) and negative predictive values (NPV), accuracy was 67, 81, 75, 81 and 79% for the combined criteria, respectively. Diagnostic efficacy of the combined criteria was significantly higher than that of CT only (p <0.05). CONCLUSIONS: Diagnosis of LN metastasis using a combination of LMVD and CT is superior to the CT-only diagnosis. In future clinical trials, it is necessary to evaluate the efficacy of adjuvant therapy for the selection of patients according to the combined criteria.