Quantitative Profiling of the Lymph Node Clearance Capacity

Transport of tissue-derived lymphatic fluid and clearance by draining lymph nodes are pivotal for maintenance of fluid homeostasis in the body and for immune-surveillance of the self- and non-self-proteomes. Yet a quantitative analysis of nodal filtration of the tissue-derived proteome present in lymphatic fluid has not been reported. Here we quantified the efficiency of nodal clearance of the composite proteomic load using label-free and isotope-labeling proteomic analysis of pre-nodal and post-nodal samples collected by direct cannulation. These results were extended by quantitation of the filtration efficiency of fluorophore-labeled proteins, bacteria, and beads infused at physiological flow rates into pre-nodal lymphatic collectors and collected by post-nodal cannulation. We developed a linear model of nodal filtration efficiency dependent on pre-nodal protein concentrations and molecular weight, and uncovered criteria for disposing the proteome incoming from defined anatomical districts under physiological conditions. These findings are pivotal to understanding the maximal antigenic load sustainable by a draining node, and promote understanding of pathogen spreading and nodal filtration of tumor metastasis, potentially helping to improve design of vaccination protocols, immunization strategies and drug delivery

Multivariate time-series analysis of biomarkers from a dengue cohort offers new approaches for diagnosis and prognosis

Dengue is a major public health problem worldwide with distinct clinical manifestations: an acute presentation (dengue fever, DF) similar to other febrile illnesses (OFI) and a more severe, life-threatening form (severe dengue, SD). Due to nonspecific clinical presentation during the early phase of dengue infection, differentiating DF from OFI has remained a chal-lenge, and current methods to determine severity of dengue remain poor early predictors. We present a prospective clinical cohort study conducted in Caracas, Venezuela from 2001–2005, designed to determine whether clinical and hematological parameters could distinguish DF from OFI, and identify early prognostic biomarkers of SD. From 204 enrolled suspected dengue patients, there were 111 confirmed dengue cases. Piecewise mixed effects regression and nonparametric statistics were used to analyze longitudinal records. Decreased serum albumin and fibrinogen along with increased D-dimer, thrombin-anti-thrombin complex, activated partial thromboplastin time and thrombin time were prognostic of SD on the day of defervescence. In the febrile phase, the day-to-day rates of change in serum albumin and fibrinogen concentration, along with platelet counts, were significantly decreased in dengue patients compared to OFI, while the day-to-day rates of change of lym-phocytes (%) and thrombin time were increased. In dengue patients, the absolute lympho-cytes to neutrophils ratio showed specific temporal increase, enabling classification of dengue patients entering the critical phase with an area under the ROC curve of 0.79. Secondary dengue patients had elongation of Thrombin time compared to primary cases while the D-dimer formation (fibrinolysis marker) remained always lower for secondary compared to primary cases. Based on partial analysis of 31 viral complete genomes, a high frequency of C-to-T transitions located at the third codon position was observed, suggesting deamina-tion events with five major hot spots of amino acid polymorphic sites outside in non-structural proteins. No association of severe outcome was statistically significant for any of the five major polymorphic sites found. This study offers an improved understanding of dengue hemostasis and a novel way of approaching dengue diagnosis and disease prognosis using piecewise mixed effect regression modeling. It also suggests that a better discrimination of the day of disease can improve the diagnostic and prognostic classification power of clinical variables using ROC curve analysis. The piecewise mixed effect regression model corroborated key early clinical determinants of disease, and offers a time-series approach for future vaccine and pathogenesis clinical studies

Evaluation of a method to measure HHV-6B infection in vitro based on cell size

Abstract Background Human herpesvirus 6 (HHV-6A and HHV-6B) infection of cell cultures can be measured by different methods, including immunofluorescence microscopy, flow cytometry, or quantification of virus DNA by qPCR. These methods are reliable and sensitive but require long processing times and can be costly. Another method used in the field relies on the identification of enlarged cells in the culture; this method requires little sample processing and is relatively fast. However, visual inspection of cell cultures can be subjective and it can be difficult to establish clear criteria to decide if a cell is enlarged. To overcome these issues, we explored a method to monitor HHV-6B infections based on the systematic and objective measurement of the size of cells using an imaging-based automated cell counter. Results The size of cells in non-infected and HHV-6B-infected cultures was measured at different times post-infection. The relatively narrow size distribution observed for non-infected cultures contrasted with the broader distributions observed in infected cultures. The average size of cultures shifted towards higher values after infection, and the differences were significant for cultures infected with relatively high doses of virus and/or screened at longer times post-infection. Correlation analysis showed that the trend observed for average size was similar to the trend observed for two other methods to measure infection: amount of virus DNA in supernatant and the percentage of cells expressing a viral antigen. In order to determine the performance of the size-based method in differentiating non-infected and infected cells, receiver operating characteristic (ROC) curves were used to analyze the data. Analysis using size of individual cells showed a moderate performance in detecting infected cells (area under the curve (AUC) ~ 0.80-0.87), while analysis using the average size of cells showed a very good performance in detecting infected cultures (AUC ~ 0.99). Conclusions The size-based method proved to be useful in monitoring HHV-6B infections for cultures where a substantial fraction of cells were infected and when monitored at longer times post-infection, with the advantage of being relatively fast and easy. It is a convenient method for monitoring virus production in-vitro and bulk infection of cells

Additional file 1: of Evaluation of a method to measure HHV-6B infection in vitro based on cell size

Effect of heat-inactivation and UV-inactivation of HHV-6B in the average size of cells. Control: non-infected SupT1; HHV-6B: live, heat-inactivated, UV-inactivated. Statistical significant differences observed only between control and live virus. (PDF 76 kb

Additional file 3: of Evaluation of a method to measure HHV-6B infection in vitro based on cell size

ROC curves for size data analyzed as percentage of cells above the cutoff at 4 dpi (A) and 7 dpi (B). (PDF 197 kb

A Simple Proteomics-Based Approach to Identification of Immunodominant Antigens from a Complex Pathogen: Application to the CD4 T Cell Response against Human Herpesvirus 6B.

Most of humanity is chronically infected with human herpesvirus 6 (HHV-6), with viral replication controlled at least in part by a poorly characterized CD4 T cell response. Identification of viral epitopes recognized by CD4 T cells is complicated by the large size of the herpesvirus genome and a low frequency of circulating T cells responding to the virus. Here, we present an alternative to classical epitope mapping approaches used to identify major targets of the T cell response to a complex pathogen like HHV-6B. In the approach presented here, extracellular virus preparations or virus-infected cells are fractionated by SDS-PAGE, and eluted fractions are used as source of antigens to study cytokine responses in direct ex vivo T cell activation studies. Fractions inducing significant cytokine responses are analyzed by mass spectrometry to identify viral proteins, and a subset of peptides from these proteins corresponding to predicted HLA-DR binders is tested for IFN-γ production in seropositive donors with diverse HLA haplotypes. Ten HHV-6B viral proteins were identified as immunodominant antigens. The epitope-specific response to HHV-6B virus was complex and variable between individuals. We identified 107 peptides, each recognized by at least one donor, with each donor having a distinctive footprint. Fourteen peptides showed responses in the majority of donors. Responses to these epitopes were validated using in vitro expanded cells and naturally expressed viral proteins. Predicted peptide binding affinities for the eight HLA-DRB1 alleles investigated here correlated only modestly with the observed CD4 T cell responses. Overall, the response to the virus was dominated by peptides from the major capsid protein U57 and major antigenic protein U11, but responses to other proteins including glycoprotein H (U48) and tegument proteins U54 and U14 also were observed. These results provide a means to follow and potentially modulate the CD4 T-cell immune response to HHV-6B

Elevated levels of soluble ST2 protein in dengue virus infected patients

Levels of the soluble form of the interleukin-1 receptor-like 1 protein (IL-1RL-1/ST2) are elevated in the serum of patients with diseases characterized by an inflammatory response. The objective of this study was to determine the concentration of soluble ST2 (sST2) in dengue infected patients during the course of the disease. Twenty-four patients with confirmed dengue infection, classified as dengue fever, and 11 patients with other febrile illness (OFI) were evaluated. Levels of sST2 in serum and laboratory variables usually altered during dengue infections were measured. Dengue infected patients had higher serum sST2 levels than OFI at the end of the febrile stage and at defervescence (p=0.0088 and p=0.0004, respectively). Patients with secondary dengue infections had higher serum sST2 levels compared with patients with primary dengue infections (p=0.047 at last day of fever and p=0.030 at defervescence). Furthermore, in dengue infected patients, we found a significant negative correlation of sST2 with platelet and WBC counts, and positive correlation with thrombin time and transaminases activity. We suggest that sST2 could be a potential marker of dengue infection, could be associated with severity or could play a role in the immune response in secondary dengue virus infection

Mapping of the CD4 T cell responses in PBMCs to HHV-6B proteins.

<p>A. IFN-γ ELISpot responses (SFU/10⁶ cells) by donors #037, 118 and 132 to pools 1–29. Error bars represent the standard deviation of the replicates. B. Heat maps summarizing the response of all donors (n = 5) to all pools tested (53). Positive responses were assessed by DFR2x and ER analyses (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142871#sec002" target="_blank">Methods</a>). Pools for which positive responses were observed by DFR2x in at least 4 donors were selected for further analysis and are indicated by (+). C. IFN-γ ELISpot responses by different donors to individual peptides in pools 5, 11, 23 and 29. D. Heat-maps summarizing the IFN-γ responses to individual peptides present in the pools selected in B. Positive responses to individual peptides were assessed by ER and DFR2x analyses. Fourteen peptides that induced responses in multiple donors were selected for further validation, and are indicated by (+).</p

Validation of the IFN-γ response to selected HHV-6B peptides.

<p>Peptide-expanded T cell cultures to each of the 14 peptides in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142871#pone.0142871.t004" target="_blank">Table 4</a> were generated for 3 donors, and tested for IFN-γ using ELISpot (SFU/10⁶ cells). A. Responses of all expanded T cells to autologous PBMCs pulsed with the peptide (P) or HHV-6B (V). ELISpot negative controls are indicated by (-). Positive responses were assessed by DFR2x and are indicated by (+) on top of the bar. B. Summary of the responses of expanded T cells to autologous PBMCs pulsed with HCMV or HHV-6B for two HCMV seronegative (#118 and 132) and one seropositive (#131) donor (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142871#pone.0142871.t001" target="_blank">Table 1</a>). P-values for HCMV vs HHV-6B (unpaired t-test) are shown. C. Alignment of homologous HHV-6B U57 and HCMV UL86 sequences in the region of known epitopes (boxes indicate the complete stimulating sequence). D. Response of a 6BZ_1084 CD4 T cell clone to autologous BLCLs or PBMCs pulsed with HHV-6B 6BZ_1084 or with HCMV UL86-B peptides, as measured by IFN-γ ELISA. P-values for no-peptide vs peptide (unpaired t-test) are shown.</p

DRB1 haplotype and serologic status of donors used in this study.

<p>1. HHV-6 IgG titer measured by IFA</p><p>2. HCMV IgG status measured by ELISA</p><p>DRB1 haplotype and serologic status of donors used in this study.</p