Co-Analysis of Serum and Urine Differentially Expressed Proteins in Mucopolysaccharidosis Type I.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease caused by the deficiency of the enzyme α-l-iduronidase (IDUA), typically leading to devastating secondary pathophysiological cascades. Due to the irreversible nature of the disease's progression, early diagnosis and interventional treatment has become particularly crucial. Considering the fact that serum and urine are the most commonly used specimens in clinical practice for detection, we conducted an analysis to identify the differential protein profile in the serum and urine of MPS I patients using the tandem mass tag (TMT) technique. A total of 182 differentially expressed proteins (DEPs) were detected in serum, among which 9 showed significant differences as confirmed by parallel reaction monitoring (PRM) analysis. The proteins APOA1 and LGFBP3 were downregulated in serum, while the expression levels of ALDOB, CD163, CRTAC1, DPP4, LAMP2, SHBG, and SPP2 exhibited an increase. In further exploratory studies of urinary proteomics, 32 identified DEPs were consistent with the discovered findings in serum tests, specifically displaying a high diagnostic area under the curve (AUC) value. Thus, our study demonstrates the value of serum-urine integrated proteomic analysis in evaluating the clinical course of MPS I and other potential metabolic disorders, shedding light on the importance of early detection and intervention in these conditions.

ITIH4, as an inflammation biomarker, mainly increases in bacterial bloodstream infection.

Bloodstream infection (BSI) is usually accompanied with the changes of varieties of inflammation proteins. In our previous study, we identified that inter-α-trypsin inhibitor heavy chain H4 (ITIH4) was highly expressed in the infection arms than the normal control arm. However, the correlated verification and mechanism remain obscure. Escherichia coli infected mice model and clinical serum samples were used to validate the concentration of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), as well as ITIH4, in ELISA method. Cytokines (IL-6, TNF-α, IL-10 and lipopolysaccharide (LPS)) were used to stimulate the HepG2 cell model to explore which cytokines influence the expression of ITIH4. JAK/STAT inhibitor was treated before IL-6 and LPS stimulation. Westernblot, as well as real-time PCR were performed to detect the expression of ITIH4 in liver tissue from protein and transcription levels. Immunohistochemistry analysis was used to observe the expression of ITIH4 in mice liver tissue. In mice model, IL-6, TNF-α, as well as IL-10 increased in the infection arms than the normal control arm. ITIH4 in serum and liver tissue of mice model increased from 1 h to 128 h, which were remarkably different from that of the normal control arm. Besides, ITIH4 increased in the bacterial infection arm greatly than the fungemia arm, mycoplasma pneumoniae (MP) arm and febrile arm in clinical serum samples. Furthermore, using the HepG2 cell line, we demonstrated that ITIH4 was up-regulated at both protein and mRNA levels upon dose- and time- response treatments with IL-6, as well as LPS. Moreover, IL-6 or LPS mediated induction of ITIH4 expression could be significantly decreased by treatment with an JAK/STAT inhibitor in protein or mRNA level. No changes were observed after TNF-α or IL-10 stimulation. ITIH4 might be a critical inflammatory biomarker which correlated with the development of BSI, especially with bacterial bloodstream infection. It is expected that this study would provide some insights into potential functional mechanisms underlying BSI.

Evaluation of an Element-Tagged Duplex Immunoassay Coupled with Inductively Coupled Plasma Mass Spectrometry Detection: A Further Study for the Application of the New Assay in Clinical Laboratory.

BACKGROUND: Element-tagged immunoassay coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection has the potential to revolutionize immunoassay analysis for multiplex detection. However, a further study referring to the standard evaluation and clinical sample verification is needed to ensure its reliability for simultaneous analysis in clinical laboratories. METHODS: Carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) were chosen for the duplex immunoassay. The performance of the assay was evaluated according to guidelines from the Clinical and Laboratory Standards Institute (CLSI). Moreover, reference intervals (RIs) of CEA and AFP were established. At last, 329 clinical samples were analyzed by the proposed method and results were compared with those obtained with electrochemiluminescent immunoassay (ECLIA) method. RESULTS: The measurement range of the assay was 2-940 ng/mL for CEA and 1.5-1000 ng/mL for AFP, with a detection limit of 0.94 ng/mL and 0.34 ng/mL, respectively. The inter-assay and intra-assay imprecision were all less than 6.58% and 10.62%, respectively. The RI of CEA and AFP was 0-3.84 ng/mL and 0-9.94 ng/mL, respectively. Regarding to clinical sample detection, no significant difference was observed between the proposed duplex assay and the ECLIA method. CONCLUSIONS: The ICP-MS-based duplex immunoassay was successfully developed and the analytical performance fully proved clinical applicability. Well, this could be different with other analytes.

A preliminary study for the establishment of a reference interval for vitamin B12 in China after performance verification of a second-generation ECLIA kit.

BACKGROUND: The second-generation electrochemiluminescence immunoassay (ECLIA) kit of vitamin B12 is widely used in clinical laboratories, and the establishment of a reference interval (RI) is essential to provide the basis for clinical monitoring. The purpose of this study was to establish a laboratory RI for vitamin B12 in China and at the same time verify the method performance of the second-generation kit. METHODS: The verification of the method performance was conducted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Based on these guidelines, a total of 580 serum samples were collected, and 391 serum samples were used for the establishment of the RI according to CLSI guidelines. The subjects were grouped by sex and age. The age groups were as follows: 21-40, 41-60, and 61-80 years. The RI was defined by nonparametric 2.5th and 97.5th percentile intervals. RESULTS: The performance of the second-generation kit of vitamin B12 from the Roche Cobas E602 system was in compliance with laboratory requirements. Serum vitamin B12 levels conformed to a non-Gaussian distribution. Harris-Boyd's test did not indicate partitioning for different age and gender group. Besides, there was no significant difference between different age groups (P = .07) and gender groups (P = .2002). The RI for healthy Chinese adults (aged 21-80 years) calculated by the nonparametric method was 250.8-957.1 pg/mL. CONCLUSIONS: The reference range of vitamin B12 was established, which provided a theoretical basis for the clinical application and monitoring of vitamin B12 detection.

Development and evaluation of an element-tagged immunoassay coupled with inductively coupled plasma mass spectrometry detection: can we apply the new assay in the clinical laboratory?

Introduction Element-tagged immunoassay coupled with inductively coupled plasma-mass spectrometry (ICP-MS) detection has the potential to revolutionize immunoassay analysis in clinical detection; however, a systematic evaluation with the standard guidelines of the assay is needed to ensure its performance meets the requirements of the clinical laboratory. Methods Carcinoembryonic antigen (CEA) was chosen for analysis using the proposed method. A systematic evaluation of the proposed assay was carried out according to the Clinical and Laboratory Standards Institute (CLSI). The 469 clinical samples were analyzed using the new method and compared with the electrochemiluminescent immunoassay (ECLIA) method. Results The measurement range of the assay was 1-900 ng/mL, with a detection limit of 0.83 ng/mL. The inter-assay and intra-assay imprecision were 4.67% and 5.38% with high concentration samples, and 9.27% and 17.64% with low concentration samples, respectively. The cross-reactivity (%) for different antigens was less than 0.05%, and the recovery was between 94% and 108%. Percentage deviation of all the dilutions was less than 12.5% during linearity estimation. The interference bias caused by different substances was less than 10%. The reference interval of the assay was 0-4.442 ng/mL. Comparison with the commercial ECLIA method for clinical sample detection, the proposed method showed a correlation of 0.9878 and no significant differences between the methods were observed (p = 0.6666). Conclusions The ICP-MS based immunoassay was successfully developed, and the analytical performance of the assay met the requirements of the CLSI, which fully proved the clinical transferability and application of the new method.

The Impact of Sharing Primer, the Quantity of the Internal Control Gene and the Primer Dimer on Reaction System in Duplex PCR.

BACKGROUND: In duplex real time quantitative PCR (qPCR), there are several factors affecting the sensitivity of duplex qPCR, such as sharing primer, quantity of the internal control (IC) gene, and the primer dimer (PD). The aim of the study is to find out the relationship of interference among templates with different primer pairs, the internal control gene, and the PD in duplex PCR. METHODS: We designed and synthesized plasmids with partial same sequence and different types of primers include central-homo primer pair, ordinary primer pair, and complementary primer pair. Then we compared the amplification efficiencies of different kinds of primer pairs. Besides, we adjusted the amount of IC plasmid and IC primer to find out the key factor that influences the sensitivity of the target template. RESULTS: The concentration ratios of two plasmids showed interference in sharing the universal primer pair, sharing one forward primer, and sharing no primer were 50:1, 200:1 and 500:1, respectively. The amplification efficiency of the ordinary primer pair was higher than that of the universal primer pair for the plasmid. Sensitivity of the duplex qPCR remained unchanged when the amount of PDs increased, but it declined rapidly when the quantity of the IC increased. CONCLUSIONS: IC is the major factor influencing the sensitivity of the duplex qPCR and it would be better to use one universal primer for IC and target template to achieve minimum interference.

The clinical value of IL-3, IL-4, IL-12p70, IL17A, IFN-γ, MIP-1ß, NLR, P-selectin, and TNF-α in differentiating bloodstream infections caused by gram-negative, gram-positive bacteria and fungi in hospitalized patients: An Observational Study.

Early differential diagnosis of bloodstream infections (BSIs) caused by different sources and species of bacteria in hospitalized patients is crucial for the timely targeted interventions including appropriate use of antibiotics. The aim of this study was to identify 9 biomarkers for the early differentiation of gram-negative-bloodstream infection (GN-BSI), gram-positive (GP)-BSI, and fungal-BSI.A prospective study was conducted for a total of 390 inpatients who underwent blood culture in the Chinese PLA General Hospital from September 2015 to March 2018. Patients with positive culture of a single pathogen were divided into GN-BSI, GP-BSI, and Fungal-BSI groups, and a culture-negative disease control group was also established. The serum levels of macrophage inflammatory protein 1ß (MIP-1ß), tumor necrosis factor α (TNF-α), interleukin (IL)-3, interferon (IFN)-γ, IL-17A, IL-4, IL-12p70, and P-selectin were detected and the NLR was calculated from routine blood test. Receiver-operating characteristic analysis was used to determine the efficacy of various indicators in the differential diagnosis of BSIs. Prediction and validation experiments on clinical patient samples (263 cases) were also performed.The level of IL-3 in the GP-BSI group was significantly higher than those in the other 3 groups. The level of IFN-γ in the fungal-BSI group was significantly higher than those in the other 3 groups. NLR, MIP-1ß, TNF-α, IL-17A, and IL3 exhibited some efficacy when distinguishing between GN-BSI and GP-BSI and NLR had the largest area under curve (AUC) (0.728), followed by MIP-1ß with an AUC of 0.679. IFN-γ and IL-3 exhibited some value in differential diagnosis between GN-BSI and Fungal-BSI. IL-3, MIP-1ß, TNF-α, IFN-γ, NLR, IL-17A, and IL-4 exhibited some value in distinguishing fungal-BSI and GP-BSI, with IL-3 had the largest AUC (0.722), followed by MIP-1ß with an AUC of 0.703.NLR and MIP-1ß may be valuable in differentiating GN-BSI from GP-BSI in hospitalized patients. IFN-γ and IL-3 may be helpful in differential diagnosis GN-BSI and fungal-BSI. IL-3 and MIP-1ß exhibited some diagnostic efficacy in distinguishing fungal-BSI and GP-BSI. Additionally, IL-3 with high serum level may be a marker for GP-BSI and IFN-γ with high serum level may be a valuable marker for the prediction of Fungal-BSI. The utility of these biomarkers to predict BSIs owing to different pathogens in hospitalized patients needs to be assessed in further studies.

Proteomic approaches in the discovery of potential urinary biomarkers of mucopolysaccharidosis type II.

Mucopolysaccharindosis type II (MPS II) is a rare lysosomal storage disorder caused by deficient or absent activity of the iduronate-2-sulfatase (IDS) enzyme, which leads to pathological accumulation of the glycosaminoglycans(GAGs). The absence of early diagnosis can result in irreversible developmental, neurological, and physiological damage. The lack of clear understanding of the etiology of physiological dysfunction in MPS II has been a major obstacle to the development of new treatment. Therefore, a reliable biomarker for early diagnosis and exploration of pathogenic mechanism are of great importance. Proteomics provides powerful tool for protein expression alterations and study of complicated pathological process. This study was performed to identify the differential protein profile in urine of MPS II patients using two-dimensional gel electrophoresis(2D-PAGE)combining with MALDI-TOF/TOF and a total of 15 differentially expressed proteins were identified. Content of alpha1-antitrypsin, Gm2 activator and lipocalin-type prostaglandin D synthase was measured by ELISA method. The value of urinary α1-AT/Cr in MPS II group was 0.79 ±â€¯0.10 mg/mmol, significantly higher than 0.42 ±â€¯0.05 mg/mmol in healthy control group; whereas the value of GM2A/Cr and L-PGDS/Cr in MPS II group was 1.30 ±â€¯0.12 µg/mmol and 9.86 ±â€¯1.16 ng/mmol respectively, which was significantly lower than 2.19 ±â€¯0.19 µg/mmol and 13.98 ±â€¯1.48 ng/mmol in healthy control group. The proteins can be considered as accessory diagnostic biomarkers for MPS II. This approach helped to discover early diagnostic markers and provided a better understanding of the pathogenic mechanism of MPS II.

Primary Tr1 cells from metastatic melanoma eliminate tumor-promoting macrophages through granzyme B- and perforin-dependent mechanisms.

In malignant melanoma, tumor-associated macrophages play multiple roles in promoting tumor growth, such as inducing the transformation of melanocytes under ultraviolet irradiation, increasing angiogenesis in melanomas, and suppressing antitumor immunity. Because granzyme B- and perforin-expressing Tr1 cells could specifically eliminate antigen-presenting cells of myeloid origin, we examined whether Tr1 cells in melanoma could eliminate tumor-promoting macrophages and how the interaction between Tr1 cells and macrophages could affect the growth of melanoma cells. Tr1 cells were characterized by high interleukin 10 secretion and low Foxp3 expression and were enriched in the CD4+CD49b+LAG-3+ T-cell fraction. Macrophages derived from peripheral blood monocytes in the presence of modified melanoma-conditioned media demonstrated tumor-promoting capacity, exemplified by improving the proliferation of cocultured A375 malignant melanoma cells. But when primary Tr1 cells were present in the macrophage-A375 coculture, the growth of A375 cells was abrogated. The conventional CD25+ Treg cells, however, were unable to inhibit macrophage-mediated increase in tumor cell growth. Further analyses showed that Tr1 cells did not directly eliminate A375 cells, but mediated the killing of tumor-promoting macrophages through the secretion of granzyme B and perforin. The tumor-infiltrating interleukin 10+Foxp3-CD4+ T cells expressed very low levels of granzyme B and perforin, possibly suggested the downregulation of Tr1 cytotoxic capacity in melanoma tumors. Together, these data demonstrated an antitumor function of Tr1 cells through the elimination of tumor-promoting macrophages, which was not shared by conventional Tregs.

CD4+ T cell-mediated cytotoxicity eliminates primary tumor cells in metastatic melanoma through high MHC class II expression and can be enhanced by inhibitory receptor blockade.

Metastatic melanoma is a rapidly progressing disease with high mortality rate and limited treatment options. Immunotherapy based on tumor-targeting cytotoxic T cell responses represents a promising strategy. To assist in its development, we examined the possibility and efficacy of using CD4+ cytotoxic T cells. The regulatory mechanisms controlling CD4+ T cell-mediated cytotoxicity were also investigated. We found that naturally occurring granzyme B and perforin-expressing CD4+ cytotoxic T cells can be recovered from metastatic melanoma patients at significantly elevated frequencies compared to those from healthy controls. These CD4+ cytotoxic T cells were also capable of killing autologous tumor cells harvested from metastatic melanoma, independent of CD8+ T cells or any other cell types. However, several restricting factors were observed. First, the cytolytic activity by CD4+ T cells required high MHC class II expression on melanoma cells, which was not satisfied in a subset of melanomas. Second, the granzyme B and perforin release by activated CD4+ cytotoxic T cells was reduced after coculturing with autologous melanoma cells, characterized by low LAMP-1 expression and low granzyme B and perforin secretion in the supernatant. This suggested that inhibitory mechanisms were present to suppress CD4+ cytotoxic T cells. Indeed, blockade of PD-1 and CTLA-4 had increased the cytolytic activity of CD4+ T cells but was only effective in MHC class II high but not MHC class II low melanomas. Together, our study showed that CD4+ T cell-mediated cytotoxicity could eliminate primary melanoma cells but the efficacy depended on MHC class II expression.