A multiparameter panel method for outcome prediction following aneurysmal subarachnoid hemorrhage

Purpose: Accurate early anticipation of long-term irreversible brain damage during the acute phase of patients with aneurysmal subarachnoid hemorrhage (aSAH) remains difficult. Using a combination of clinical scores together with brain injury-related biomarkers (H-FABP, NDKA, UFD1 and S100β), this study aimed at developing a multiparameter prognostic panel to facilitate early outcome prediction following aSAH. Methods: Blood samples of 141 aSAH patients from two separated cohorts (sets of 28 and 113 patients) were prospectively enrolled and analyzed with 14months of delay. Patients were admitted within 48h following aSAH onset. A venous blood sample was withdrawn within 12h after admission. H-FABP, NDKA, UFD1, S100β and troponin I levels were determined using classical immunoassays. The World Federation of Neurological Surgeons (WFNS) at admission and the Glasgow Outcome Score (GOS) at 6months were evaluated. Results: In the two cohorts, blood concentration of H-FABP, S100β and troponin I at admission significantly predicted unfavorable outcome (GOS 1-2-3). A multivariate analysis identified a six-parameter panel, including WFNS, H-FABP, S100β, troponin I, NDKA and UFD-1; when at least three of these parameters were simultaneously above cutoff values, prediction of unfavorable outcome reached around 70% sensitivity in both cohorts for 100% specificity. Conclusion: The use of this panel, including four brain injury-related proteins, one cardiac marker and a clinical score, could be a valuable tool to identify aSAH patients at risk of poor outcom

Integrative Multi-omics Analysis to Characterize Human Brain Ischemia

Stroke is a major cause of death and disability. A better comprehension of stroke pathophysiology is fundamental to reduce its dramatic outcome. The use of high-throughput unbiased omics approaches and the integration of these data might deepen the knowledge of stroke at the molecular level, depicting the interaction between different molecular units. We aimed to identify protein and gene expression changes in the human brain after ischemia through an integrative approach to join the information of both omics analyses. The translational potential of our results was explored in a pilot study with blood samples from ischemic stroke patients. Proteomics and transcriptomics discovery studies were performed in human brain samples from six deceased stroke patients, comparing the infarct core with the corresponding contralateral brain region, unveiling 128 proteins and 2716 genes significantly dysregulated after stroke. Integrative bioinformatics analyses joining both datasets exposed canonical pathways altered in the ischemic area, highlighting the most influential molecules. Among the molecules with the highest fold-change, 28 genes and 9 proteins were selected to be validated in five independent human brain samples using orthogonal techniques. Our results were confirmed for NCDN, RAB3C, ST4A1, DNM1L, A1AG1, A1AT, JAM3, VTDB, ANXA1, ANXA2, and IL8. Finally, circulating levels of the validated proteins were explored in ischemic stroke patients. Fluctuations of A1AG1 and A1AT, both up-regulated in the ischemic brain, were detected in blood along the first week after onset. In summary, our results expand the knowledge of ischemic stroke pathology, revealing key molecules to be further explored as biomarkers and/or therapeutic targets

Integrative Multi-omics Analysis to Characterize Human Brain Ischemia

Stroke is a major cause of death and disability. A better comprehension of stroke pathophysiology is fundamental to reduce its dramatic outcome. The use of high-throughput unbiased omics approaches and the integration of these data might deepen the knowledge of stroke at the molecular level, depicting the interaction between different molecular units. We aimed to identify protein and gene expression changes in the human brain after ischemia through an integrative approach to join the information of both omics analyses. The translational potential of our results was explored in a pilot study with blood samples from ischemic stroke patients. Proteomics and transcriptomics discovery studies were performed in human brain samples from six deceased stroke patients, comparing the infarct core with the corresponding contralateral brain region, unveiling 128 proteins and 2716 genes significantly dysregulated after stroke. Integrative bioinformatics analyses joining both datasets exposed canonical pathways altered in the ischemic area, highlighting the most influential molecules. Among the molecules with the highest fold-change, 28 genes and 9 proteins were selected to be validated in five independent human brain samples using orthogonal techniques. Our results were confirmed for NCDN, RAB3C, ST4A1, DNM1L, A1AG1, A1AT, JAM3, VTDB, ANXA1, ANXA2, and IL8. Finally, circulating levels of the validated proteins were explored in ischemic stroke patients. Fluctuations of A1AG1 and A1AT, both up-regulated in the ischemic brain, were detected in blood along the first week after onset. In summary, our results expand the knowledge of ischemic stroke pathology, revealing key molecules to be further explored as biomarkers and/or therapeutic targets. Graphical abstract: [Figure not available: see fulltext.].This work has been funded by Instituto de Salud Carlos III (PI15/00354, PI18/00804), MINECO (MTM2015-64465-C2-1R) and GRBIO (2014-SGR-464) and co-financed by the European Regional Development Fund (FEDER). Neurovascular Research Laboratory takes part in the Spanish stroke research network INVICTUS + (RD16/0019/0021). L.R is supported by a pre-doctoral fellowship from the Instituto de Salud Carlos III (IFI17/00012).Peer reviewe

Neopterin is a cerebrospinal fluid marker for treatment outcome evaluation in patients affected by Trypanosoma brucei gambiense sleeping sickness.

BACKGROUND: Post-therapeutic follow-up is essential to confirm cure and to detect early treatment failures in patients affected by sleeping sickness (HAT). Current methods, based on finding of parasites in blood and cerebrospinal fluid (CSF) and counting of white blood cells (WBC) in CSF, are imperfect. New markers for treatment outcome evaluation are needed. We hypothesized that alternative CSF markers, able to diagnose the meningo-encephalitic stage of the disease, could also be useful for the evaluation of treatment outcome. METHODOLOGY/PRINCIPAL FINDINGS: Cerebrospinal fluid from patients affected by Trypanosoma brucei gambiense HAT and followed for two years after treatment was investigated. The population comprised stage 2 (S2) patients either cured or experiencing treatment failure during the follow-up. IgM, neopterin, B2MG, MMP-9, ICAM-1, VCAM-1, CXCL10 and CXCL13 were first screened on a small number of HAT patients (n = 97). Neopterin and CXCL13 showed the highest accuracy in discriminating between S2 cured and S2 relapsed patients (AUC 99% and 94%, respectively). When verified on a larger cohort (n = 242), neopterin resulted to be the most efficient predictor of outcome. High levels of this molecule before treatment were already associated with an increased risk of treatment failure. At six months after treatment, neopterin discriminated between cured and relapsed S2 patients with 87% specificity and 92% sensitivity, showing a higher accuracy than white blood cell numbers. CONCLUSIONS/SIGNIFICANCE: In the present study, neopterin was highlighted as a useful marker for the evaluation of the post-therapeutic outcome in patients suffering from sleeping sickness. Detectable levels of this marker in the CSF have the potential to shorten the follow-up for HAT patients to six months after the end of the treatment

Cerebrospinal fluid neopterin as marker of the meningo-encephalitic stage of Trypanosoma brucei gambiense sleeping sickness.

BACKGROUND: Sleeping sickness, or human African trypanosomiasis (HAT), is a protozoan disease that affects rural communities in sub-Saharan Africa. Determination of the disease stage, essential for correct treatment, represents a key issue in the management of patients. In the present study we evaluated the potential of CXCL10, CXCL13, ICAM-1, VCAM-1, MMP-9, B2MG, neopterin and IgM to complement current methods for staging Trypanosoma brucei gambiense patients. METHODS AND FINDINGS: Five hundred and twelve T. b. gambiense HAT patients originated from Angola, Chad and the Democratic Republic of the Congo (D.R.C.). Their classification as stage 2 (S2) was based on the number of white blood cells (WBC) (>5/µL) or presence of parasites in the cerebrospinal fluid (CSF). The CSF concentration of the eight markers was first measured on a training cohort encompassing 100 patients (44 S1 and 56 S2). IgM and neopterin were the best in discriminating between the two stages of disease with 86.4% and 84.1% specificity respectively, at 100% sensitivity. When a validation cohort (412 patients) was tested, neopterin (14.3 nmol/L) correctly classified 88% of S1 and S2 patients, confirming its high staging power. On this second cohort, neopterin also predicted both the presence of parasites, and of neurological signs, with the same ability as IgM and WBC, the current reference for staging. CONCLUSIONS: This study has demonstrated that neopterin is an excellent biomarker for staging T. b. gambiense HAT patients. A rapid diagnostic test for detecting this metabolite in CSF could help in more accurate stage determination

A divergent cyclin/cyclin-dependent kinase complex controls the atypical replication of a malaria parasite during gametogony and transmission

Cell cycle transitions are generally triggered by variation in the activity of cyclin-dependent kinases (CDKs) bound to cyclins. Malaria-causing parasites have a life cycle with unique cell-division cycles, and a repertoire of divergent CDKs and cyclins of poorly understood function and interdependency. We show that Plasmodium berghei CDK-related kinase 5 (CRK5), is a critical regulator of atypical mitosis in the gametogony and is required for mosquito transmission. It phosphorylates canonical CDK motifs of components in the pre-replicative complex and is essential for DNA replication. During a replicative cycle, CRK5 stably interacts with a single Plasmodium-specific cyclin (SOC2), although we obtained no evidence of SOC2 cycling by transcription, translation or degradation. Our results provide evidence that during Plasmodium male gametogony, this divergent cyclin/CDK pair fills the functional space of other eukaryotic cell-cycle kinases controlling DNA replication

Ca2+ signals critical for egress and gametogenesis in malaria parasites depend on a multipass membrane protein that interacts with PKG.

Calcium signaling regulated by the cGMP-dependent protein kinase (PKG) controls key life cycle transitions in the malaria parasite. However, how calcium is mobilized from intracellular stores in the absence of canonical calcium channels in Plasmodium is unknown. Here, we identify a multipass membrane protein, ICM1, with homology to transporters and calcium channels that is tightly associated with PKG in both asexual blood stages and transmission stages. Phosphoproteomic analyses reveal multiple ICM1 phosphorylation events dependent on PKG activity. Stage-specific depletion of Plasmodium berghei ICM1 prevents gametogenesis due to a block in intracellular calcium mobilization, while conditional loss of Plasmodium falciparum ICM1 is detrimental for the parasite resulting in severely reduced calcium mobilization, defective egress, and lack of invasion. Our findings suggest that ICM1 is a key missing link in transducing PKG-dependent signals and provide previously unknown insights into atypical calcium homeostasis in malaria parasites essential for pathology and disease transmission

A Combined CXCL10, CXCL8 and H-FABP Panel for the Staging of Human African Trypanosomiasis Patients

The actual serological and parasitological tests used for the diagnosis of human African trypanosomiasis (HAT), also known as sleeping sickness, are not sensitive and specific enough. The card agglutination test for trypanosomiasis (CATT) assay, widely used for the diagnosis, is restricted to the gambiense form of the disease, and parasitological detection in the blood and cerebrospinal fluid (CSF) is often very difficult. Another very important problem is the difficulty of staging the disease, a crucial step in the decision of the treatment to be given. While eflornithine is difficult to administer, melarsoprol is highly toxic with incidences of reactive encephalopathy as high as 20%. Staging, which could be diagnosed as early (stage 1) or late (stage 2), relies on the examination of CSF for the presence of parasite and/or white blood cell (WBC) counting. However, the parasite is rarely found in CSF and WBC count is not standardised (cutoff set between 5 and 20 WBC per µL). In the present study, we hypothesized that an early detection of stage 2 patients with one or several proteins in association with clinical evaluation and WBC count would improve staging accuracy and allow more appropriate therapeutic interventions