Exosomes have been implicated in numerous biological processes, and they may serve as important disease markers. Surface proteins on exosomes carry information about their tissues of origin. Because ...of the heterogeneity of exosomes it is desirable to investigate them individually, but this has so far remained impractical. Here, we demonstrate a proximity-dependent barcoding assay to profile surface proteins of individual exosomes using antibody-DNA conjugates and next-generation sequencing. We first validate the method using artificial streptavidin-oligonucleotide complexes, followed by analysis of the variable composition of surface proteins on individual exosomes, derived from human body fluids or cell culture media. Exosomes from different sources are characterized by the presence of specific combinations of surface proteins and their abundance, allowing exosomes to be separately quantified in mixed samples to serve as markers for tissue-specific engagement in disease.
Summary Background Hepatocellular carcinoma (HCC) is prevalent worldwide and improvements in timely and effective diagnosis are needed. We assessed whether measurement of Dickkopf-1 (DKK1) in serum ...could improve diagnostic accuracy for HCC. Methods We analysed data for patients with HCC, chronic hepatitis B virus (HBV) infection, liver cirrhosis, and healthy controls, recruited from two Chinese centres between December, 2008, and July, 2009. A validation cohort matched for age and sex was recruited from another centre in China between February, 2009, and June, 2011. DKK1 was measured in serum by ELISA by independent researchers who had no access to patients' clinical information. We used receiver operating characteristics (ROC) to calculate diagnostic accuracy. Findings We assessed serum DKK1 in 831 participants: 424 with HCC, 98 with chronic HBV infection, 96 with cirrhosis, and 213 healthy controls. The validation cohort comprised 453 participants: 209 with HCC, 73 with chronic HBV infection, 72 with cirrhosis, and 99 healthy controls. Levels of DKK1 in serum were significantly higher in patients with HCC than in all controls. ROC curves showed the optimum diagnostic cutoff was 2·153 ng/mL (area under curve AUC 0·848 95% CI 0·820–0·875, sensitivity 69·1%, and specificity 90·6% in the test cohort; 0·862 0·825–0·899, 71·3%, and 87·2% in the validation cohort). Similar results were noted for early-stage HCC (0·865 0·835–0·895, 70·9%, and 90·5% in the test cohort; 0·896 0·846–0·947, 73·8%, and 87·2% in the validation cohort). Furthermore, DKK1 maintained diagnostic accuracy for patients with HCC who were α-fetoprotein (AFP) negative (0·841 0·801–0·882, 70·4%, and 90·0% in the test cohort; 0·869 0·815–0·923, 66·7%, and 87·2% in the validation cohort), including for patients with early-stage HCC (0·870 0·829–0·911, 73·1%, and 90·0% in the test cohort; 0·893 0·804–0·983, 72·2%, and 87·2% in the validation cohort), compared with all controls. Raised concentrations of DKK1 in serum could differentiate HCC from chronic HBV infection and cirrhosis (0·834 0·798–0·871, 69·1%, and 84·7% in the test cohort; 0·873 0·832–0·913, 71·3%, and 90·6% in the validation cohort). Moreover, measurement of DKK1 and AFP together improved diagnostic accuracy for HCC versus all controls compared with either test alone (0·889 0·866–0·913, 73·3%, and 93·4% in the test cohort; 0·888 0·856–0·920, 78·5%, and 87·2% in the validation cohort). Interpretation DKK1 could complement measurement of AFP in the diagnosis of HCC and improve identification of patients with AFP-negative HCC and distinguish HCC from non-malignant chronic liver diseases. Funding National Key Basic Research Programme of China, National Key Sci-Tech Special Projects of Infectious Diseases, National Natural Science Foundation of China, Research Fund for the Doctoral Programme of Higher Education of China.
An important motivation for the construction of biobanks is to discover biomarkers that identify diseases at early, potentially curable stages. This will require biobanks from large numbers of ...individuals, preferably sampled repeatedly, where the samples are collected and stored under conditions that preserve potential biomarkers. Dried blood samples are attractive for biobanking because of the ease and low cost of collection and storage. Here we have investigated their suitability for protein measurements. Ninety-two proteins with relevance for oncology were analyzed using multiplex proximity extension assays (PEA) in dried blood spots collected on paper and stored for up to 30 years at either +4 °C or −24 °C.
Our main findings were that (1) the act of drying only slightly influenced detection of blood proteins (average correlation of 0.970), and in a reproducible manner (correlation of 0.999), (2) detection of some proteins was not significantly affected by storage over the full range of three decades (34 and 76% of the analyzed proteins at +4 °C and −24 °C, respectively), whereas levels of others decreased slowly during storage with half-lives in the range of 10 to 50 years, and (3) detectability of proteins was less affected in dried samples stored at −24 °C compared with at +4 °C, as the median protein abundance had decreased to 80 and 93% of starting levels after 10 years of storage at +4 °C or −24 °C, respectively. The results of our study are encouraging as they suggest an inexpensive means to collect large numbers of blood samples, even by the donors themselves, and to transport, and store biobanked samples as spots of whole blood dried on paper. Combined with emerging means to measure hundreds or thousands of protein, such biobanks could prove of great medical value by greatly enhancing discovery as well as routine analysis of blood biomarkers.
The neurotransmitter γ-aminobutyric acid (GABA) is an extracellular signaling molecule in the brain and in pancreatic islets. Here, we demonstrate that GABA regulates cytokine secretion from human ...peripheral blood mononuclear cells (PBMCs) and CD4+ T cells. In anti-CD3 stimulated PBMCs, GABA (100nM) inhibited release of 47 cytokines in cells from patients with type 1 diabetes (T1D), but only 16 cytokines in cells from nondiabetic (ND) individuals. CD4+ T cells from ND individuals were grouped into responder or non-responder T cells according to effects of GABA (100nM, 500nM) on the cell proliferation. In the responder T cells, GABA decreased proliferation, and inhibited secretion of 37 cytokines in a concentration-dependent manner. In the non-responder T cells, GABA modulated release of 8 cytokines. GABA concentrations in plasma from T1D patients and ND individuals were correlated with 10 cytokines where 7 were increased in plasma of T1D patients. GABA inhibited secretion of 5 of these cytokines from both T1D PBMCs and ND responder T cells. The results identify GABA as a potent regulator of both Th1- and Th2-type cytokine secretion from human PBMCs and CD4+ T cells where GABA generally decreases the secretion.
•GABA regulates cytokine secretion in anti-CD3-stimulated peripheral blood mononuclear cells (PBMCs) and CD4+ T cells.•GABA inhibits secretion of 47 cytokines in PBMCs from type 1 diabetes patients.•GABA regulates secretion of pro- and anti-inflammatory cytokines in a concentration-dependent manner.
GABA is a signal molecule in the brain, blood and pancreatic islets where it is secreted by the insulin-producing β cells. GABA has many roles in human islets including optimizing function and survival of β cells. Bhandage et al. now show that GABA is a potent regulator of secretion of both pro- and anti-inflammatory cytokines in stimulated immune cells. In type 1 diabetes the β-cell mass is diminished and thus the protective effect of GABA in the islets although not in blood. Targeting GABA signaling in diabetes mellitus is likely to be a part of the solution when curing diabetes.
To report how the Chinese mainland battled its first omicron wave, which happened in Tianjin, a metropolis with 14 million residents. We also sought to better understand how clinical features ...affected the timing of viral clearance.
A retrospective study of the omicron wave in Tianjin between 8 January 2022 and 3 March 2022.
Except for the first cases on 8 January, all the omicron cases were identified through PCR mass testing in the residential communities. Residential quarantine and serial PCR mass testing were dynamically adjusted according to the trends of new cases.
All the 417 consecutive PCR-positive cases identified through mass screening of the entire city's 14 million residents. 45.3% of the cases were male, and the median age was 37 (range 0.3-90). 389 (93%) cases had complete data for analysing the correlation between clinical features and the timing of viral clearance.
Time to viral clearance.
Tianjin initiated the 'dynamic zero-COVID' policy very early, that is, when daily new case number was ≈0.4 cases per 1 000 000 residents. Daily new cases dropped to <5 after 3 February, and the number of affected residential subdivisions dropped to ≤2 after 13 February. 64% (267/417) of the cases had no or mild symptoms. The median interval from hospital admission to viral clearance was 10 days (range 3-28). An exploratory analysis identified a feature cluster associated with earlier viral clearance, with HRs of 3.56 (95% CI 1.66 to 7.63) and 3.15 (95% CI 1.68 to 5.91) in the training and validation sets, respectively.
The 'dynamic zero-COVID' policy can suppress an omicron wave within a month. It might be possible to predict in advance which cases will require shorter periods of isolation based on their clinical features.
The timely diagnosis and effective treatment are essential for improving the survival and prognosis of hepatocellular carcinoma (HCC) patients. Alpha-fetoprotein (AFP) is the most widely used ...biomarker for diagnosis of HCC, but the low sensitivity and specificity limits its clinical application. In this study, we evaluated the diagnostic capability of the combination of AFP with two novel potential biomarkers, dickkopf-1 (DKK1) and osteopontin (OPN), for HCC in 390 participants including 89 patients with HCC, 36 patients with liver cirrhosis, 65 patients with chronic hepatitis B, and 200 health controls. We found the combination of all three markers as a panel showed a better diagnostic performance than that of AFP alone, with increased AUC 0.948 (95 % CI 0.921–0.968) vs. 0.831 (95 % CI 0.790–0.867) and sensitivity (88.76 vs. 71.91 %). Moreover, this combination showed a great improvement in diagnosing early-stage HCC patients. In conclusion, the combined use of AFP, DKK1, and OPN as a biomarker panel could enhance the diagnostic ability for HCC.
Hepatocellular carcinoma (HCC) is one of the most common cancers, and its incidence is increasing worldwide. Neuropeptide Y (NPY) broadly expressed in the central and peripheral nervous system. It ...participates in multiple physiological and pathological processes through specific receptors. Evidences are accumulating that NPY is involved in development and progression in neuro- or endocrine-related cancers. However, little is known about the potential roles and underlying mechanisms of NPY receptors in HCC. In this study, we analyzed the expression of NPY receptors by real-time polymerase chain reaction, Western blot, and immunohistochemical staining. Correlation between NPY1R levels and clinicopathological characteristics, and survival of HCC patients were explored, respectively. Cell proliferation was researched by CCK-8 in vitro, and tumor growth was studied by nude mice xenografts in vivo. We found that mRNA and protein level of NPY receptor Y1 subtype (NPY1R) significantly decreased in HCC tissues. Low expression of NPY1R closely correlated with poor prognosis in HCC patients. Proliferation of HCC cells was significantly inhibited by recombinant NPY protein in vitro. This inhibitory effect could be blocked by selected NPY1R antagonist BIBP3226. Furthermore, overexpression of NPY1R could significantly inhibit HCC cell proliferation. Knockdown of NPY1R promoted cell multiplication in vitro and increased tumorigenicity and tumor growth in vivo. NPY1R was found to participate in the inhibition of cell proliferation via inactivating mitogen-activated protein kinase signal pathway in HCC cells. Collectively, NPY1R plays an inhibitory role in tumor growth and may be a promising therapeutic target for HCC.
Abstract Background Hepatitis B virus X protein is a key regulator of hepatocarcinogenesis. The pregnane X receptor is a xenobiotic nuclear receptor that plays a role in the regulation of ...drug-metabolizing enzymes including the cytochrome P450 3A4, an enzyme important for the bioactivation of the liver carcinogen aflatoxin B1. Aims To identify novel host factor that interacts with hepatitis B virus X protein and the functional interaction between hepatitis B virus X protein and pregnane X receptor in hepatocarcinogenesis. Methods Co-immunoprecipitation, glutathione S -transferase pull-down, and chromatin immunoprecipitation were utilized to assess the interaction between hepatitis B virus X protein and pregnane X receptor. The functional relevance of hepatitis B virus X protein–pregnane X receptor interaction was investigated in cell cultures and hepatocellular carcinoma samples. Results We observed that hepatitis B virus X protein and pregnane X receptor co-localize in hepatic cells. Pregnane X receptor interacted with hepatitis B virus X protein via the ligand-binding domain of pregnane X receptor. Functionally, hepatitis B virus X protein increased the transcriptional activity of pregnane X receptor. Pregnane X receptor was able to recruit hepatitis B virus X protein to the CYP3A4 gene promoter. In clinic samples, the expression of pregnane X receptor was high in hepatitis B virus-associated liver cirrhosis and stage I hepatocellular carcinoma, but low in state II and stage III hepatocellular carcinoma. Conclusion We revealed a novel function of hepatitis B virus X protein in co-activating pregnane X receptor. The increased expression of pregnane X receptor and its target gene CYP3A4 are potential biomarkers for the early stage of hepatitis B virus-associated hepatocarcinogenesis.
ETV6::RUNX1 is the most common fusion gene in childhood acute lymphoblastic leukaemia (ALL) and is associated with favorable outcomes, especially in low‐risk children. However, as many as 10% of ...children relapse within 3 years, and such early relapses have poor survival. Identifying children at risk for early relapse is an important challenge. We interrogated data from 87 children with low‐risk ETV6::RUNX1‐positive B‐cell ALL and with available preserved bone marrow samples (discovery cohort). We profiled somatic point mutations in a panel of 559 genes and genome‐wide transcriptome and single‐nucleotide variants. We found high TIMD4 expression (> 85th‐percentile value) at diagnosis was the most important independent prognostic factor of early relapse (hazard ratio HR = 5.07 1.76, 14.62; p = 0.03). In an independent validation cohort of low‐risk ETV6::RUNX1‐positive B‐cell ALL (N = 68) high TIMD4 expression at diagnosis had an HR = 4.78 1.07, 21.36 (p = 0.04) for early relapse. In another validation cohort including 78 children with low‐risk ETV6::RUNX1‐negative B‐cell ALL, high TIMD4 expression at diagnosis had an HR = 3.93 1.31, 11.79 (p = 0.01). Our results suggest high TIMD4 expression at diagnosis in low‐risk B‐cell ALL in children might be associated with high risk for early relapse.
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