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Background: A high-throughput method using inductively coupled plasma mass spectrometry (ICP–MS) was developed and validated for the quantitative analysis of antimony in human plasma and peripheral blood mononuclear cells from patients with cutaneous leishmaniasis undergoing treatment with meglumine antimoniate. Materials & methods: Antimony was digested in clinical samples with 1% tetramethylammonium hydroxide/1% EDTA and indium was used as internal standard. Accuracy, precision and stability were evaluated. Conclusion: Taking the lower limit of quantitation to be the lowest validation concentration with precision and accuracy within 20%, the current assay was successfully validated from 25 to 10000 ng/ml for antimony in human plasma and peripheral blood mononuclear cells. This protocol will serve as a baseline for future analytical designs, aiming to provide a reference method to allow inter-study comparisons.Lay abstractCutaneous leishmaniasis is a disease caused by single-cell parasites in the genus Leishmania which results in painful skin ulcers and is spread by insect bites. Drugs containing antimony are the mainstay therapy for cutaneous leishmaniasis, but if and how the amount of these compounds in the cells can affect the success of the treatment, remains unknown. Validated methods to reliably measure these amounts in human cells are limited. Here we have developed a validated method that allows quantifying antimony in human plasma and peripheral blood cells from patients undergoing antileishmanial treatment. This protocol will serve as a baseline for future studies aiming to understand how antimonials work to treat leishmaniasis infections and how this therapy can be improved.

AimA high-throughput method using inductively coupled plasma mass spectrometry (ICP-MS) was developed and validated for the quantitative analysis of antimony in human plasma and peripheral blood mononuclear cells (PBMCs) from patients with cutaneous leishmaniasis undergoing treatment with meglumine antimoniate.MethodsFor this study, antimony was digested in clinical samples with 1% TMAH / 1% EDTA and indium was used as internal standard. Calibration curves for antimony, over the range of 25 to 10000 ng/mL were fitted to a linear model using a weighting of 1/concentration2. Accuracy, precision and stability were evaluated.ResultsTaking the lower limit of quantitation (LLOQ) to be the lowest validation concentration with precision and accuracy within 20% (25% at the LLOQ), the current assay was successfully validated from 25 to 10000 ng/mL for antimony in human plasma and PBMCs. Dilution studies demonstrated that concentrations up to 100000 ng/mL of antimony in plasma were reliably analyzed when diluted into the calibration range.ConclusionThis protocol will serve as a baseline for future analytical designs, aiming to provide a reference method to allow inter-study comparisons.What is already known about this subjectAntimonial drugs are the mainstay treatment for cutaneous leishmaniasis of which systemically administered pentavalent antimonials (SbV) are widely used, however the pharmacokinetics (PK) of these drugs at the site of action is unknown.A wide range of analytical strategies have been used to quantify antimony in biological samples and atomic absorption spectroscopy is the most employed technique, however, no standardized methods for determination of intracellular concentrations of antimony were available.Relationships between plasma and intracellular drug concentrations remain unknown for most antiparasitic drugs, and PK studies rely on plasma drug concentrations assuming these act as surrogates of intracellular concentrations.What this study addsWe have developed and validated a reproducible and accurate ICP-MS method for the quantification of total antimony in human plasma and peripheral blood mononuclear cells (PBMCs) in accordance with the European Bioanalysis Forum (EBF) recommendations.This method was successfully used to compare pharmacokinetic curves of antimony in plasma and intracellular compartments, in samples collected from patients undergoing treatment for cutaneous leishmaniasis with meglumine antimoniate.

BACKGROUND Tuberculosis (TB) kills more people than any other infection, and new diagnostic tests to identify active cases are required. We aimed to discover and verify novel markers for TB in nondepleted plasma. METHODS We applied an optimized quantitative proteomics discovery methodology based on multidimensional and orthogonal liquid chromatographic separation combined with high-resolution mass spectrometry to study nondepleted plasma of 11 patients with active TB compared with 10 healthy controls. Prioritized candidates were verified in independent UK (n = 118) and South African cohorts (n = 203). RESULTS We generated the most comprehensive TB plasma proteome to date, profiling 5022 proteins spanning 11 orders-of-magnitude concentration range with diverse biochemical and molecular properties. We analyzed the predominantly low–molecular weight subproteome, identifying 46 proteins with significantly increased and 90 with decreased abundance (peptide FDR ≤ 1%, q ≤ 0.05). Verification was performed for novel candidate biomarkers (CFHR5, ILF2) in 2 independent cohorts. Receiver operating characteristics analyses using a 5-protein panel (CFHR5, LRG1, CRP, LBP, and SAA1) exhibited discriminatory power in distinguishing TB from other respiratory diseases (AUC = 0.81). CONCLUSION We report the most comprehensive TB plasma proteome to date, identifying novel markers with verification in 2 independent cohorts, leading to a 5-protein biosignature with potential to improve TB diagnosis. With further development, these biomarkers have potential as a diagnostic triage test. FUNDING Colciencias, Medical Research Council, Innovate UK, NIHR, Academy of Medical Sciences, Program for Advanced Research Capacities for AIDS, Wellcome Centre for Infectious Diseases Research., Analysis of a comprehensive tuberculosis plasma proteome identified a 5-protein biosignature with potential to improve active tuberculosis diagnosis.

Previously, we developed a 3-dimensional cell culture model of human tuberculosis (TB) and demonstrated its potential to interrogate the host-pathogen interaction (Tezera et al., 2017a). Here, we use the model to investigate mechanisms whereby immune checkpoint therapy for cancer paradoxically activates TB infection. In patients, PD-1 is expressed in Mycobacterium tuberculosis (Mtb)-infected lung tissue but is absent in areas of immunopathology. In the microsphere model, PD-1 ligands are up-regulated by infection, and the PD-1/PD-L1 axis is further induced by hypoxia. Inhibition of PD-1 signalling increases Mtb growth, and augments cytokine secretion. TNF-α is responsible for accelerated Mtb growth, and TNF-α neutralisation reverses augmented Mtb growth caused by anti-PD-1 treatment. In human TB, pulmonary TNF-α immunoreactivity is increased and circulating PD-1 expression negatively correlates with sputum TNF-α concentrations. Together, our findings demonstrate that PD-1 regulates the immune response in TB, and inhibition of PD-1 accelerates Mtb growth via excessive TNF-α secretion.

Epidemiological data suggest that pre-eclampsia (PE) is associated with an increased risk of post-delivery metabolic dysregulation. The aim of the present case–control observational study was to examine the global plasma proteomic profile 1 year postpartum in women who developed PE during pregnancy (n = 5) compared to controls (n = 5), in order to identify a novel predictive marker linking PE with long-term metabolic imbalance. Key findings were verified with enzyme-linked immunosorbent assay (ELISA) in a separate cohort (n = 17 women with PE and n = 43 controls). One hundred and seventy-two proteins were differentially expressed in the PE vs. control groups. Gene ontology analysis showed that Inflammatory|Immune responses, Blood coagulation and Metabolism were significantly enriched terms. CD14, mapping to the inflammatory response protein network, was selected for verification based on bibliographic evidence. ELISA measurements showed CD14 to be significantly increased 1 year postpartum in women with PE during pregnancy compared to controls [PE group (median ± SD): 296.5 ± 113.6; control group (median ± SD): 128.9 ± 98.5; Mann–Whitney U test p = 0.0078]. Overall, the identified proteins could provide insight into the long-term disease risk among women with PE during pregnancy and highlight the need for their postpartum monitoring. CD14 could be examined in larger cohorts as a predictive marker of insulin resistance and type II diabetes mellitus among women with PE.

Background: Polycystic ovary syndrome (PCOS) is a common endocrine disorder associated with insulin resistance, even in the absence of overweight/obesity. The aim of the present study is to examine the global serum proteomic profile of adolescent, normal-weight females with PCOS in order to gain novel insight in the association of this endocrine disorder with insulin physiology and to identify novel circulating markers that can guide intervention protocols. Methods: Non-depleted serum from normal-weight (BMI: 18–23 kg m −2 ), adolescent females (13–21 years old) with PCOS (n = 20) is compared to BMI- and age-matched healthy controls (n = 20) using our 3D quantitative proteomics methodology. Serum samples from study participants are randomly pooled to form four biological replicates of females with PCOS and four of healthy controls (n = 5 per sample pool). Results: One-hundred and twenty-six proteins are differentially expressed in females with PCOS compared to controls. Gene ontology analysis shows significant enrichment for terms related to inflammatory immune response, metabolism and insulin-like growth factor receptor signaling pathway. Circulating levels of IGF-1 and -2 and IGFBP-2, -3, and -4 are found to be lower in females with PCOS compared to healthy controls. Conclusions: The present serum proteomics study provides insight into the pro-inflammatory status and insulin dysregulation in young females with PCOS and identifies potential serological markers that can guide early intervention protocols.

Background: Endometriosis affects about 4% of women in the reproductive age and is associated with subfertility. The aim of the present study was to examine the quantitative proteomic profile of eutopic endometrium and serum from women with endometriosis compared to controls in order to identify candidate disease-specific serological markers.Methods: Eutopic endometrium and serum from patients with endometriosis (n=8 for tissue and n=4 for serum) was respectively compared to endometrium and serum from females without endometriosis (n=8 for tissue and n=4 for serum) using a shotgun quantitative proteomics method. All study participants were at the proliferative phase of their menstrual cycle.Results: At the tissue and serum level, 1,214 and 404 proteins were differentially expressed (DEPs) in eutopic endometrium and serum respectively of women with endometriosis vs. control. Gene ontology analysis showed that terms related to immune response | inflammation, cell adhesion | migration and blood coagulation were significantly enriched in the DEPs of eutopic endometrium as well as serum. Twenty-one DEPs had the same trend of differential expression in both matrices and can be further examined as potential disease- and tissue-specific serological markers of endometriosis.Conclusions: The present in-depth proteomic profiling of eutopic endometrium and serum from women with endometriosis identified promising serological markers that can be further validated in larger cohorts for the minimally invasive diagnosis of endometriosis.

Objective Subjects with low vitamin D levels are at risk of cardiometabolic disease. The aim of this study was to identify novel serological markers linking vitamin D status with cardiometabolic profile in non-diabetic adults with obesity. Methods For the discovery phase, we used quantitative serum proteomics in sex-matched, age-matched and BMI-matched subjects with obesity [BMI: 25–35 kg/m2] and low [25(OH)D 50 nmol/L] (n = 16). For the validation phase, we performed ELISA in a larger cohort with similar characteristics (n = 179). Results We identified 423 and 549 differentially expressed proteins in the high vs. low vitamin D groups of the male and female cohorts, respectively. The small molecule biochemistry protein networks and the glycolysis|gluconeogenesis pathway were significantly enriched in the DEPs of both sexes. As surrogate markers to these processes, the insulin-like growth factor binding protein -2 (IGFBP-2) was upregulated in males, whereas IGFBP-3 was upregulated in females from the high Vitamin D status. This sex-specific trend was confirmed using Luminex ELISA to an independent but clinically analogous cohort of males (n = 84, p = 0.002) and females (n = 95, p = 0.03). Conclusions The high Vitamin D status correlated with the serological upregulation of IGFBP-2 in males and IGFBP-3 in females with obesity and may constitute surrogate markers of risk reduction of cardiometabolic disease.

Background Early-onset breast cancer (EOBC) affects about one in 300 women aged 40 years or younger and is associated with worse outcomes than later onset breast cancer. This study explored novel serum proteins as surrogate markers of prognosis in patients with EOBC. Methods Serum samples from EOBC patients (stages 1–3) were analysed using agnostic high-precision quantitative proteomics. Patients received anthracycline-based chemotherapy. The discovery cohort (n = 399) either had more than 5-year disease-free survival (DFS) (good outcome group, n = 203) or DFS of less than 2 years (poor outcome group, n = 196). Expressed proteins were assessed for differential expression between the two groups. Bioinformatics pathway and network analysis in combination with literature research were used to determine clinically relevant proteins. ELISA analysis against an independent sample set from the Prospective study of Outcomes in Sporadic versus Hereditary breast cancer (POSH) cohort (n = 181) was used to validate expression levels of the selected target. Linear and generalized linear modelling was applied to determine the effect of target markers, body mass index (BMI), lymph node involvement (LN), oestrogen receptor (ER), progesterone receptor and human epidermal growth factor receptor 2 status on patients’ outcome. Results A total of 5346 unique proteins were analysed (peptide FDR p ≤ 0.05). Of these, 812 were differentially expressed in the good vs poor outcome groups and showed significant enrichment for the insulin signalling (p = 0.01) and the glycolysis/gluconeogenesis (p = 0.01) pathways. These proteins further correlated with interaction networks involving glucose and fatty acid metabolism. A consistent nodal protein to these metabolic networks was resistin (upregulated in the good outcome group, p = 0.009). ELISA validation demonstrated resistin to be upregulated in the good outcome group (p = 0.04), irrespective of BMI and ER status. LN involvement was the only covariate with a significant association with resistin measurements (p = 0.004). An ancillary in-silico observation was the induction of the inflammatory response, leucocyte infiltration, lymphocyte migration and recruitment of phagocytes (p < 0.0001, z-score > 2). Survival analysis showed that resistin overexpression was associated with improved DFS. Conclusions Higher circulating resistin correlated with node-negative patients and longer DFS independent of BMI and ER status in women with EOBC. Overexpression of serum resistin in EOBC may be a surrogate indicator of improved prognosis. Electronic supplementary material The online version of this article (10.1186/s13058-018-0938-6) contains supplementary material, which is available to authorized users.

The insulin-like growth factor (IGF) family is organized in a complex regulatory network at the cellular and sub-cellular levels. In the human the IGF system has a key physiological role in the development of the organism and maintenance of normal cellular function during foetal and postnatal life. The IGF system consists of three ligands, IGF-I, IGF-II and Insulin; three cell membrane receptors, IGF-I receptor (IGF-IR), insulin receptor (IR), and IGF-II receptor (IGF-IIR); six high-affinity IGF binding proteins, IGFBP-1 through -6, their specific proteases (IGFBP proteases) and membrane receptors (IGFBP-R) (Fig. 1).

gene or in ‘small for gestational age’ infants are associated with increased risk of developing a metabolic syndrome. Additionally, recent studies also imply the use of the IGF-1R as a target tool to study insulin resistance and Type 2 diabetes. Our lab has used the Igf1r mice to unravel the mechanism of disrupted glucose metabolism caused by IGF-1R haploinsufficiency. The results demonstrate a consistent reduction in the IGF-1 signaling in the muscle tissue of Igf1r mice. The aged male Igf1r mice, in particular, exhibit glucose and insulin intolerance, fasting hyperinsulinemia, reduced pGSK3 levels and an increased expression of lipogenic enzymes, a signature of metabolic disorder. This study proposes a new mechanism whereby reduced IGF-1 signaling results in hyperglycemia and hyperinsulinemia, a secondary consequence of which is ectopic fat accretion in the muscle and liver. An intense metabolic profiling of this model will be imminent in the understanding of molecular mechanisms favoring insulin resistance, thereby leading to the development of novel therapies for Type 2 diabetes.