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Background Brucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable. Methodology/Principal findings In this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen. Conclusions B cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations., Author summary Brucellosis is one of the most important zoonosis in the world and has caused tremendous economic losses in agriculture and animal husbandry in many countries. Developing rapid, sensitive and specific diagnostic methods is very important for early detection and treatment of brucellosis patients. In this study, a novel diagnostic technique, nano-ZnO modified paper ELISA, was established. The antigen used in this technique was a fusion protein containing multiple B cell epitopes, which were predicted from Brucella major outer membrane proteins such as Bp26, Omp31, Omp16, Omp2b and Omp25. Comparing to traditional LPS antigen, this multiepitope based antigen displayed considerably higher sensitivity and higher specificity in laboratory. With the strategy described in this paper, more efficient epitopes and protein antigen can be identified in the future. Currently, LPS antigen is only prepared from live Brucella, while protein antigen can be produced in large quantities in prokaryotic expression system. In addition to nano-p-ELISA, this protein antigen can also be used for development other methods such as fluorescent polarization assay (FPA) and immunochromatographic assay (ICA) to meet the varied demand for brucellosis testing.

Extracts of the exuviae (cast skins) of nymphal bed bugs (Cimex lectularius) were analyzed for volatile compounds that might contribute to arrestment of adult bed bugs. Four volatile aldehydes, (E)-2-hexenal, 4-oxo-(E)-2-hexenal, (E)-2-octenal, and 4-oxo-(E)-2-octenal were consistently detected in the headspace of freshly shed exuviae regardless of the developmental stages from which the exuviae were obtained. Quantification of the aldehydes in the solvent extracts of homogenized fresh, 45- or 99-d aged 5th instar exuviae indicated that the aldehydes are present in the exuviae and dissipate over time, through evaporation or degradation. Microscopic observation of the fifth instar exuviae indicated that the dorsal abdominal glands on the exuviae maintained their pocket-like structures with gland reservoirs, within which the aldehydes might be retained. Two-choice olfactometer studies with the volatiles from exuviae or a synthetic blend mimicking the volatiles indicated that adult bed bugs tend to settle close to sources of the aldehydes. Our results imply that the presence and accumulation of bed bug exuviae and the aldehydes volatilizing from the exuviae might mediate bed bugs' interaction with their microhabitats.

Background & aimsIn our previous study, a Seesaw model was proposed for the fluctuation of crucial anti- (IL-10) and pro-inflammatory (Il-6 & IL-17A) cytokines through vitamin D3. In this paper, however, it is intended to extend the mentioned model by assessing the expression mRNA levels of IL-27 and TGF-β1 as well as the changes of plasma levels of IL-27, TGF-β1, IL-17A, IL-10, and IL-6 after treatment by vitamin D3.MethodVenous blood samples were drawn from Healthy Participants (HP, n = 25) and First-Degree Relative Participants (FDRP, n = 25) as control groups and Multiple Sclerosis Participants (MSP, n = 25) before and after eight weeks of supplementation with 50000 IU vitamin D3. The mRNA expression and plasma concentrations were gauged by using Real-Time PCR and ELISA assay, respectively.ResultsThe mRNA surfaces of IL-27, as well as TGF-β1, were up-regulated. However, the plasma levels of TGF-β1, IL-17A, and IL-6 were significantly different among the three groups. In addition, the plasma levels of IL-27, TGF-β1, IL-10, IL-17A, and IL-6 significantly changed following the administration of vitamin D3.ConclusionThe findings of this paper illustrate that anti-inflammatory cytokines could have a key role in immunomodulatory functions due to their anti-inflammatory functions. To conclude, this might contribute to preventing the pathophysiological process of MS. Also, the proposed model could be used as a preventive way on disposed people to multiple sclerosis, particularly in first degree relatives of these patients.

Kaempferol (kae) and its glycosides are widely distributed in nature and show multiple bioactivities, yet few reports have compared them. In this paper, we report the antitumor, antioxidant and anti-inflammatory activity differences of kae, kae-7-O-glucoside (kae-7-O-glu), kae-3-O-rhamnoside (kae-3-O-rha) and kae-3-O-rutinoside (kae-3-O-rut). Kae showed the highest antiproliferation effect on the human hepatoma cell line HepG2, mouse colon cancer cell line CT26 and mouse melanoma cell line B16F1. Kae also significantly inhibited AKT phosphorylation and cleaved caspase-9, caspase-7, caspase-3 and PARP in HepG2 cells. A kae-induced increase in DPPH and ABTS radical scavenging activity, inhibition of concanavalin A (Con A)-induced activation of T cell proliferation and NO or ROS production in LPS-induced RAW 264.7 macrophage cells were also seen. Kae glycosides were used to produce kae via environment-friendly enzymatic hydrolysis. Kae-7-O-glu and kae-3-O-rut were hydrolyzed to kae by β-glucosidase and/or α-L-rhamnosidase. This paper demonstrates the application of enzymatic catalysis to obtain highly biologically active kae. This work provides a novel and efficient preparation of high-value flavone-related products.

Activities of transcription factors (TFs) are temporally modulated to regulate dynamic cellular processes, including development, homeostasis, and disease. Recent developments of bioinformatic tools have enabled the analysis of TF activities using transcriptome data. However, because these methods typically use exon-based target expression levels, the estimated TF activities have limited temporal accuracy. To address this, we proposed a TF activity measure based on intron-level information in time-series RNA-seq data, and implemented it to decode the temporal control of TF activities during dynamic processes. We showed that TF activities inferred from intronic reads can better recapitulate instantaneous TF activities compared to the exon-based measure. By analyzing public and our own time-series transcriptome data, we found that intron-based TF activities improve the characterization of temporal phasing of cycling TFs during circadian rhythm, and facilitate the discovery of two temporally opposing TF modules during T cell activation. Collectively, we anticipate that the proposed approach would be broadly applicable for decoding global transcriptional architecture during dynamic processes., Author summary Many health-related cellular processes, such as immune response and disease progression, involve dynamic changes of gene expression state, which are orchestrated by transcription factors. Dissecting the activities of transcription factors is thus important for understanding cellular processes and for interfering with dysregulated processes. Our ability to analyze transcription factor activities has been facilitated by genome-wide gene expression data from high-throughput assays such as RNA sequencing. Existing methods typically estimate transcription factor activities based on the expression levels of matured mRNAs of target genes. However, because the levels of matured mRNAs are affected by transcriptional and post-transcriptional regulatory activities, the estimated transcription factor activities may not faithfully recapitulate the regulatory activities of transcription factors. In this paper, we proposed and validated an alternative approach for analyzing transcription factor activities using the expression levels of unmatured mRNAs of target genes, allowing us to decode how transcription factor activities are temporally controlled during key biological processes. Our results provide insights into the temporal phasing of key circadian regulator activities in mouse liver, and uncover two temporally opposing modules of transcription factors that dictate the immune responses in T cells. Therefore, this approach can help understand the regulatory principles of dynamic cellular processes.

In this essay, we show that 3 distinct approaches to immunological exhaustion coexist and that they only partially overlap, generating potential misunderstandings. Exploring cases ranging from viral infections to cancer, we propose that it is crucial, for experimental and therapeutic purposes, to clarify these approaches and their interconnections so as to make the concept of exhaustion genuinely operational., Author summary In this essay, we have written a critical review on immunological exhaustion. We believe that this widely used concept often remains in fact imprecise because there exist 3 different approaches to exhaustion, namely, in terms of dysfunction, cause, and marker, and those are not sufficiently well distinguished and articulated in most scientific papers. We also propose to talk about “exhaustion” for and only for the phenomena in which all 3 approaches are aligned. This can be called the “convergence strategy”: T cells would be described as “exhausted” if and only if they are simultaneously dysfunctional, express given markers such as programmed cell death protein 1 (PD-1), and induced by a specific cause such as chronicity. This strategy could be perfectly reasonable, and we see much value in adopting it, because it would force everyone to be more specific and rigorous when talking about exhaustion. Clarifying the characteristics of cells defined as “exhausted,” and using a convergent strategy to define T-cell exhaustion could have major experimental and clinical consequences, including for viral infections such as the Coronavirus Disease 2019 (COVID-19).

The partial success of tumor immunotherapy induced by checkpoint blockade, which is not antigen-specific, suggests that the immune system of some patients contain antigen receptors able to specifically identify tumor cells. Here we focused on T-cell receptor (TCR) repertoires associated with spontaneous breast cancer. We studied the alpha and beta chain CDR3 domains of TCR repertoires of CD4 T cells using deep sequencing of cell populations in mice and applied the results to published TCR sequence data obtained from human patients. We screened peripheral blood T cells obtained monthly from individual mice spontaneously developing breast tumors by 5 months. We then looked at identical TCR sequences in published human studies; we used TCGA data from tumors and healthy tissues of 1,256 breast cancer resections and from 4 focused studies including sequences from tumors, lymph nodes, blood and healthy tissues, and from single cell dataset of 3 breast cancer subjects. We now report that mice spontaneously developing breast cancer manifest shared, Public CDR3 regions in both their alpha and beta and that a significant number of women with early breast cancer manifest identical CDR3 sequences. These findings suggest that the development of breast cancer is associated, across species, with biomarker, exclusive TCR repertoires., Author summary More than 1,100 cancer treatments are currently in clinical testing. Of these, 295 are immune-oncology medicines and vaccines. For Checkpoint Immunotherapy alone, 52,539 patients were enrolled in 2017. Of these treatments, 108 are for breast cancer, the leading cancer diagnosed in women. Although checkpoint immunotherapy has yet to be proven beneficial in most types of breast cancer, current observations of which specific T cell go into the tumor, and how these cells are connected to the response to treatment, have lead to a new understating of the role of the immune system. Yet, and in spite of this intense research into the role of T cells in tumors, very little is known about how clones of T cells behave during tumor formation. The paper we present here studies this clonal behavior over time. We study it during mammary tumor development in mice. We see that a set of T cell clones, which comes from a diversity of different nucleotide sequences, stands out in its behavior compared with other T cell clones. Importantly, we show that these mouse T cell clones have a strong connection to human breast cancer. The specific identification of a subset of T cell clones, together with the behavior of these clones over time, is critical to our understanding of the effect of modifications to the immune system, such as the modifications made by immunotherapy treatments, and to our understanding of how to better control the behavior of the immune system.

Funder: National Institute for Health Research; funder-id: http://dx.doi.org/10.13039/501100000272, Background: Understanding the T cell response to SARS-CoV-2 is critical to vaccine development, epidemiological surveillance and disease control strategies. This systematic review critically evaluates and synthesises the relevant peer-reviewed and pre-print literature published from 01/01/2020-26/06/2020. Methods: For this systematic review, keyword-structured literature searches were carried out in MEDLINE, Embase and COVID-19 Primer. Papers were independently screened by two researchers, with arbitration of disagreements by a third researcher. Data were independently extracted into a pre-designed Excel template and studies critically appraised using a modified version of the MetaQAT tool, with resolution of disagreements by consensus. Findings were narratively synthesised. Results: 61 articles were included. 55 (90%) studies used observational designs, 50 (82%) involved hospitalised patients with higher acuity illness, and the majority had important limitations. Symptomatic adult COVID-19 cases consistently show peripheral T cell lymphopenia, which positively correlates with increased disease severity, duration of RNA positivity, and non-survival; while asymptomatic and paediatric cases display preserved counts. People with severe or critical disease generally develop more robust, virus-specific T cell responses. T cell memory and effector function has been demonstrated against multiple viral epitopes, and, cross-reactive T cell responses have been demonstrated in unexposed and uninfected adults, but the significance for protection and susceptibility, respectively, remains unclear. Conclusion: A complex pattern of T cell response to SARS-CoV-2 infection has been demonstrated, but inferences regarding population level immunity are hampered by significant methodological limitations and heterogeneity between studies, as well as a striking lack of research in asymptomatic or pauci-symptomatic individuals. In contrast to antibody responses, population-level surveillance of the T cell response is unlikely to be feasible in the near term. Focused evaluation in specific sub-groups, including vaccine recipients, should be prioritised.

Leukocyte (white blood cell, WBC) count is an essential factor that physicians use to diagnose infections and provide adequate treatment. Currently, WBC count is determined manually or semi-automatically, which often leads to miscounting. In this paper, we propose an automated method that uses a bioinspired segmentation mimicking the human perception of color. It is based on the claim that a person can locate WBCs in a blood smear image via the high chromatic contrast. First, by applying principal component analysis over RGB, HSV, and L*a*b* spaces, with specific combinations, pixels of leukocytes present high chromatic variance; this results in increased contrast with the average hue of the other blood smear elements. Second, chromaticity is processed as a feature, without separating hue components; this is different to most of the current automation that perform mathematical operations between hue components in an intuitive way. As a result of this systematic method, WBC recognition is computationally efficient, overlapping WBCs are separated, and the final count is more precise. In experiments with the ALL-IDB benchmark, the performance of the proposed segmentation was assessed by comparing the WBC from the processed images with the ground truth. Compared with previous methods, the proposed method achieved similar results in sensitivity and precision and approximately 0.2% higher specificity and 0.3% higher accuracy for pixel classification in the segmentation stage; as well, the counting results are similar to previous works.

All approved coronavirus disease 2019 (COVID-19) vaccines in current use are safe, effective, and reduce the risk of severe illness. Although data on the immunological presentation of patients with COVID-19 is limited, increasing experimental evidence supports the significant contribution of B and T cells towards the resolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Despite the availability of several COVID-19 vaccines with high efficacy, more effective vaccines are still needed to protect against the new variants of SARS-CoV-2. Employing a comprehensive immunoinformatic prediction algorithm and leveraging the genetic closeness with SARS-CoV, we have predicted potential immune epitopes in the structural proteins of SARS-CoV-2. The S and N proteins of SARS-CoV-2 and SARS-CoVs are main targets of antibody detection and have motivated us to design four multi-epitope vaccines which were based on our predicted B- and T-cell epitopes of SARS-CoV-2 structural proteins. The cardinal epitopes selected for the vaccine constructs are predicted to possess antigenic, non-allergenic, and cytokine-inducing properties. Additionally, some of the predicted epitopes have been experimentally validated in published papers. Furthermore, we used the C-ImmSim server to predict effective immune responses induced by the epitope-based vaccines. Taken together, the immune epitopes predicted in this study provide a platform for future experimental validations which may facilitate the development of effective vaccine candidates and epitope-based serological diagnostic assays.

Background Eosinophilic meningitis (EM) is a rare clinical syndrome caused by both infectious and noninfectious diseases. In tropical pacific countries, Angiostrongylus cantonensis is the most common cause. However, the EM definition varies in the literature, and its relation to parasitic meningitis (PM) remains unclear. Methodology/Principal findings Adult and adolescent patients of 13 years old or above with suspected central nervous system (CNS) infections with abnormal CSF findings were prospectively enrolled at a tertiary referral hospital in Hanoi, Vietnam from June 2012 to May 2014. Patients with EM or suspected PM (EM/PM) were defined by the presence of either ≥10% eosinophils or an absolute eosinophil cell counts of ≥10/mm3 in the CSF or blood eosinophilia (>16% of WBCs) without CSF eosinophils. In total 679 patients were enrolled: 7 (1.03%) had ≥10% CSF eosinophilia, 20 (2.95%) had ≥10/mm3 CSF eosinophilia, and 7 (1.03%) had >16% blood eosinophilia. The patients with ≥10% CSF eosinophilia were significantly younger (p = 0.017), had a lower body temperature (p = 0.036) than patients with ≥10/mm3 CSF eosinophilia among whom bacterial pathogens were detected in 72.2% (13/18) of those who were tested by culture and/or PCR. In contrast, the characteristics of the patients with >16% blood eosinophilia resembled those of patients with ≥10% CSF eosinophilia. We further conducted serological tests and real-time PCR to identify A. cantonensis. Serology or real-time PCR was positive in 3 (42.8%) patients with ≥10% CSF eosinophilia and 6 (85.7%) patients with >16% blood eosinophilia without CSF eosinophils but none of patients with ≥10/mm3 CSF eosinophilia. Conclusions The etiology of PM in northern Vietnam is A. cantonensis. The eosinophil percentage is a more reliable predictor of parasitic EM than absolute eosinophil count in the CSF. Patients with PM may present with a high percentage of eosinophils in the peripheral blood but not in the CSF., Author summary Eosinophilic meningitis (EM) is a rare meningitis accompanied by eosinophils in the CSF and caused by multiple etiologies. Angiostrongylus cantonensis, which is a rat lungworm parasite, is the most common cause in tropical Asia. Previous papers have defined EM as CSF eosinophils ≥10% or CSF eosinophils ≥10/mm3. However, the relationship of EM to parasitic meningitis (PM) remains unclear. This prospective study enrolled 679 patients with suspected CNS infection who were admitted to a tertiary referral hospital in Hanoi, Vietnam from June 2012 to May 2014. The characteristics of patients with ≥10% CSF eosinophilia resembled those of patients with >16% blood eosinophilia without CSF eosinophils, whereas those of patients with ≥10/mm3 CSF eosinophilia were comparable with those of patients with typical bacterial meningitis. Serology or real-time PCR for A. cantonensis was positive in 3 out of 7 patients with ≥10% CSF eosinophilia and 6 out of 7 patients with > 16% blood eosinophilia without CSF eosinophils but none of patients with ≥10/mm3 CSF eosinophilia. The percentage, in contrast to the absolute eosinophil count in CSF, is reliable for predicting parasitic EM. Patients with PM may present with eosinophilia in the peripheral blood but not in the CSF.

Gene expression atlases have transformed our understanding of the development, composition and function of human tissues. New technologies promise improved cellular or molecular resolution, and have led to the identification of new cell types, or better defined cell states. But as new technologies emerge, information derived on old platforms becomes obsolete. We demonstrate that it is possible to combine a large number of different profiling experiments summarised from dozens of laboratories and representing hundreds of donors, to create an integrated molecular map of human tissue. As an example, we combine 850 samples from 38 platforms to build an integrated atlas of human blood cells. We achieve robust and unbiased cell type clustering using a variance partitioning method, selecting genes with low platform bias relative to biological variation. Other than an initial rescaling, no other transformation to the primary data is applied through batch correction or renormalisation. Additional data, including single-cell datasets, can be projected for comparison, classification and annotation. The resulting atlas provides a multi-scaled approach to visualise and analyse the relationships between sets of genes and blood cell lineages, including the maturation and activation of leukocytes in vivo and in vitro. In allowing for data integration across hundreds of studies, we address a key reproduciblity challenge which is faced by any new technology. This allows us to draw on the deep phenotypes and functional annotations that accompany traditional profiling methods, and provide important context to the high cellular resolution of single cell profiling. Here, we have implemented the blood atlas in the open access Stemformatics.org platform, drawing on its extensive collection of curated transcriptome data. The method is simple, scalable and amenable for rapid deployment in other biological systems or computational workflows., Author summary Combining data from many different studies is an attractive way of capturing new aspects of the biology being studied. Biological variance attributable to cell type, cellular niche, origin, disease status or environmental stimuli is the basis of most small-n transcriptome studies. In aggregation, these promise to capture emergent dimensions of a biology that is not possible to view from any individual study. However biological signal is easily swamped by technical artifact, especially when data is generated on platforms with profoundly different data structures. This is the case when comparing microarray data to RNAseq, or RNAseq to single cell profiling. Consequently, transcriptome atlases are generally comprised from a small number of donors/conditions surveyed using one technology platform. In this paper we present a simple and scalable data integration method that is platform agnostic. We provide a proof-of-principle by constructing an atlas of blood cells that combines many data sets measured on different platforms, and that in combination, recapitulates the known blood hierarchy. The atlas provides a reference to compare external samples to, allowing users to benchmark new derivation or isolation methods. It also provides a reference point for new data types, such as the classification of single cells. The approach allows for FAIR data reuse and robust identification of molecular signatures across multiple studies and experimental conditions.

The recognition and classification of White Blood Cell (WBC) play a remarkable role in blood-related diseases (i.e., leukemia, infections) diagnosis. For the highly similar morphology of different WBC subtypes, it is too confused to classify the WBC effectively and accurately for visual observation of blood cell smears. This paper proposes a Deep Convolutional Neural Network (DCNN) with feature fusion strategies, named WBC-AMNet, for automatically classifying WBC subtypes based on focalized attention mechanism. To obtain more localized attention of CNN, the fusion features of the first and the last convolutional layer are extracted by focalized attention mechanism combining Squeeze-and-Excitation (SE) and Gather-Excite (GE) modules. The new method performs successfully in classifying monocytes, neutrophils, lymphocytes, and eosinophils on the complex background with an overall accuracy of 95.66%, better than that of general CNNs. The multi-classification accuracy of WBC-AMNet with the background segmentation is over 98% in all cases. In addition, Gradient-weighted Class Activation Mapping (Grad-CAM) is employed to visualize the attention heatmaps of different feature maps.

Metastatic castration resistant prostate cancer (mCRPC) is commonly treated by androgen deprivation therapy (ADT) in combination with chemotherapy. Immune therapy by checkpoint inhibitors, has become a powerful new tool in the treatment of melanoma and lung cancer, and it is currently being used in clinical trials in other cancers, including mCRPC. However, so far, clinical trials with PD-1 and CTLA-4 inhibitors have been disappointing. In the present paper we develop a mathematical model to assess the efficacy of any combination of ADT with cancer vaccine, PD-1 inhibitor, and CTLA-4 inhibitor. The model is represented by a system of partial differential equations (PDEs) for cells, cytokines and drugs whose density/concentration evolves in time within the tumor. Efficacy of treatment is determined by the reduction in tumor volume at the endpoint of treatment. In mice experiments with ADT and various combinations of PD-1 and CTLA-4 inhibitors, tumor volume at day 30 was always larger than the initial tumor. Our model, however, shows that we can decrease tumor volume with large enough dose; for example, with 10 fold increase in the dose of anti-PD-1, initial tumor volume will decrease by 60%. Although the treatment with ADT in combination with PD-1 inhibitor or CTLA-4 inhibitor has been disappointing in clinical trials, our simulations suggest that, disregarding negative effects, combinations of ADT with checkpoint inhibitors can be effective in reducing tumor volume if larger doses are used. This points to the need for determining the optimal combination and amounts of dose for individual patients.

HIV-1 establishes a persistent proviral reservoir by integrating into the genome of infected host cells. Current antiretroviral treatments do not target this persistent population of proviruses which include latently infected cells that upon treatment interruption can be reactivated to contribute to HIV-1 rebound. Deep sequencing of persistent HIV proviruses has revealed that greater than 90% of integrated HIV genomes are defective and unable to produce infectious virions. We hypothesized that intragenic elements in the HIV genome support transcription of aberrant HIV-1 RNAs from defective proviruses that lack long terminal repeats (LTRs). Using an intact provirus detection assay, we observed that resting CD4+ T cells and monocyte-derived macrophages (MDMs) are biased towards generating defective HIV-1 proviruses. Multiplex reverse transcription droplet digital PCR identified env and nef transcripts which lacked 5’ untranslated regions (UTR) in acutely infected CD4+ T cells and MDMs indicating transcripts are generated that do not utilize the promoter within the LTR. 5’UTR-deficient env transcripts were also identified in a cohort of people living with HIV (PLWH) on ART, suggesting that these aberrant RNAs are produced in vivo. Using 5’ rapid amplification of cDNA ends (RACE), we mapped the start site of these transcripts within the Env gene. This region bound several cellular transcription factors and functioned as a transcriptional regulatory element that could support transcription and translation of downstream HIV-1 RNAs. These studies provide mechanistic insights into how defective HIV-1 proviruses are persistently expressed to potentially drive inflammation in PLWH., Author summary People living with HIV establish a persistent reservoir which includes latently infected cells that fuel viral rebound upon treatment interruption. However, the majority of HIV-1 genomes in these persistently infected cells are defective. Whether these defective HIV genomes are expressed and whether they contribute to HIV associated diseases including accelerated aging, neurodegenerative symptoms, and cardiovascular diseases are still outstanding questions. In this paper, we demonstrate that acute infection of macrophages and resting T cells is biased towards generating defective viruses which are expressed by DNA regulatory elements in the HIV genome. These studies describe an alternative mechanism for chronic expression of HIV genomes.

Respiratory syncytial virus (RSV) infection results in millions of hospitalizations and thousands of deaths each year. Variations in the adaptive and innate immune response appear to be associated with RSV severity. To investigate the host response to RSV infection in infants, we performed a systems-level study of RSV pathophysiology, incorporating high-throughput measurements of the peripheral innate and adaptive immune systems and the airway epithelium and microbiota. We implemented a novel multi-omic data integration method based on multilayered principal component analysis, penalized regression, and feature weight back-propagation, which enabled us to identify cellular pathways associated with RSV severity. In both airway and immune cells, we found an association between RSV severity and activation of pathways controlling Th17 and acute phase response signaling, as well as inhibition of B cell receptor signaling. Dysregulation of both the humoral and mucosal response to RSV may play a critical role in determining illness severity., Author summary This paper presents a novel approach to understanding the localized molecular responses to respiratory syncytial virus (RSV) and the system-level correlates of clinical outcomes. To do this, we developed a novel statistical method able to integrate high dimensional molecular data characterizing the host airway microbota and immune and nasal gene expression. We show that this integrative approach facilitates superior performance in estimating clinical outcome as opposed to any single data type. Using this approach, we identified both cell type-specific and shared biomarkers and regulatory pathways associated with RSV severity. Specifically, we identified an association between RSV severity, activation of pathways controlling Th17, and inhibition of B cell receptor signaling, which were present in both the site of infection airway and in peripheral immune cells. These results can guide future efforts to identify biomarkers for identifying or predicting illness severity following infant RSV infection. They may also be useful as biomarkers to inform the efficacy of future interventions (e.g., therapies) or preventative measures to suppress the rate of severe disease (e.g., vaccines).

Antigen recognition by T-cells is guided by the T-cell receptor (TCR) heterodimer formed by α and β chains. A huge diversity of TCR sequences should be maintained by the immune system in order to be able to mount an effective response towards foreign pathogens, so, due to cooperative binding of α and β chains to the pathogen, any constraints on chain pairing can have a profound effect on immune repertoire structure, diversity and antigen specificity. By integrating available structural data and paired chain sequencing results we were able to show that there are almost no constraints on pairing in TCRαβ complexes, allowing naive T-cell repertoire to reach the highest possible diversity. Additional analysis reveals that the specific choice of contacting amino acids can still have a profound effect on complex conformation. Moreover, antigen-driven selection can distort the uniform landscape of chain pairing, while small, yet significant, differences in the pairing can be attributed to various specialized T-cell subsets such as MAIT and iNKT T-cells, as well as other TCR sets specific to certain antigens., Author summary In the present paper we study chain pairing preferences in the T-cell receptor (TCR) heterodimer complex. The TCR molecule is formed by α and β chains and binding of both of these chains to an antigen presented by the major histocompatibility complex (MHC) molecule is required in order to trigger an immune response against foreign pathogens and neoantigens. We show that chain pairing in the TCR complex is nearly random ensuring a highly diverse set of TCRs required for recognition of a vast set of antigens. Our results also show that chain pairing preferences can nevertheless influence TCR complex geometry and biases in TCR chain pairing can be used to identify antigen-driven selection or selection towards specialized subsets of T-cells such as mucosal-associated and natural killer invariant T-cells.

Cell homeostasis requires the correct levels of reactive oxygen species (ROS) to be maintained as these regulate the proliferation and differentiation of cells, and control the immune response and inflammation. High levels of ROS can cause oxidative stress, leading to protein, lipid and DNA damage, or even cell death. Under physiological conditions, the rate of autophagy remains stable; however, it can be accelerated by a number of exogenous stimuli such as oxidative stress, starvation or hypoxia, leading to cell death. The present paper examines the effect of Conidiobolus coronatus infection on the immune response, oxidative stress processes and autophagy in the greater wax moth, Galleria mellonella. Fungal infection was found to result in the disorganization of the cytoskeleton of the larval immune cells and the enhancement of oxidative defense processes. Lipid peroxidation and autophagy were also induced in the hemocytes. Our findings show that G. mellonella is an ideal model for exploring immune mechanisms.

Adult T-cell leukemia/lymphoma (ATLL) originates from human T-cell leukemia virus type 1 (HTLV-1) infection due to the activation of the nuclear factor-κB (NF-κB) signaling pathway to maintain proliferation and survival. An important mechanism of the activated NF-κB signaling pathway in ATLL is the activation of the macroautophagy (herafter referred to as autophagy in the remainder of this manuscript)-lysosomal degradation of p47 (NSFL1C), a negative regulator of the NF-κB pathway. Therefore, we considered the use of chloroquine (CQ) or hydroxychloroquine (HCQ) (CQ/HCQ) as an autophagy inhibitor to treat ATLL; these drugs were originally approved by the FDA as antimalarial drugs and have recently been used to treat autoimmune diseases, such as systemic lupus erythematosus (SLE). In this paper, we determined the therapeutic efficacy of CQ/HCQ, as NF-κB inhibitors, in ATLL mediated by blockade of p47 degradation. Administration of CQ/HCQ to ATLL cell lines and primary ATLL cells induced cell growth inhibition in a dose-dependent manner, and the majority of cells underwent apoptosis after CQ administration. As to the molecular mechanism, autophagy was inhibited in CQ-treated ATLL cells, and activation of the NF-κB pathway was suppressed with the restoration of the p47 level. When the antitumor effect of CQ/HCQ was examined using immunodeficient mice transplanted with ATLL cell lines, CQ/HCQ significantly suppressed tumor growth and improved the survival rate in the ATLL xenograft mouse model. Importantly, HCQ selectively induced ATLL cell death in the ATLL xenograft mouse model at the dose used to treat SLE. Taken together, our results suggest that the inhibition of autophagy by CQ/HCQ may become a novel and effective strategy for the treatment of ATLL.

Immune checkpoint inhibitors have demonstrated, over the recent years, impressive clinical response in cancer patients, but some patients do not respond at all to checkpoint blockade, exhibiting primary resistance. Primary resistance to PD-1 blockade is reported to occur under conditions of immunosuppressive tumor environment, a condition caused by myeloid derived suppressor cells (MDSCs), and by T cells exclusion, due to increased level of T regulatory cells (Tregs). Since TGF-β activates Tregs, TGF-β inhibitor may overcome primary resistance to anti-PD-1. Indeed, recent mice experiments show that combining anti-PD-1 with anti-TGF-β yields significant therapeutic improvements compared to anti-TGF-β alone. The present paper introduces two cancer-specific parameters and, correspondingly, develops a mathematical model which explains how primary resistance to PD-1 blockade occurs, in terms of the two cancer-specific parameters, and how, in combination with anti-TGF-β, anti-PD-1 provides significant benefits. The model is represented by a system of partial differential equations and the simulations are in agreement with the recent mice experiments. In some cancer patients, treatment with anti-PD-1 results in rapid progression of the disease, known as hyperprogression disease (HPD). The mathematical model can also explain how this situation arises, and it predicts that HPD may be reversed by combining anti-TGF-β to anti-PD-1. The model is used to demonstrate how the two cancer-specific parameters may serve as biomarkers in predicting the efficacy of combination therapy with PD-1 and TGF-β inhibitors.

Antimicrobial resistance (AMR) is currently one of the most important challenges to the treatment of bacterial infections. A critical issue to combat AMR is to restrict its spread. In several instances, bacterial plasmids are involved in the global spread of AMR. Plasmids belonging to the incompatibility group (Inc)HI are widespread in Enterobacteriaceae and most of them express multiple antibiotic resistance determinants. They play a relevant role in the recent spread of colistin resistance. We present in this report novel findings regarding IncHI plasmid conjugation. Conjugative transfer in liquid medium of an IncHI plasmid requires expression of a plasmid-encoded, large-molecular-mass protein that contains an Ig-like domain. The protein, termed RSP, is encoded by a gene (ORF R0009) that maps in the Tra2 region of the IncHI1 R27 plasmid. The RSP protein is exported outside the cell by using the plasmid-encoded type IV secretion system that is also used for its transmission to new cells. Expression of the protein reduces cell motility and enables plasmid conjugation. Flagella are one of the cellular targets of the RSP protein. The RSP protein is required for a high rate of plasmid transfer in both flagellated and nonflagellated Salmonella cells. This effect suggests that RSP interacts with other cellular structures as well as with flagella. These unidentified interactions must facilitate mating pair formation and, hence, facilitate IncHI plasmid conjugation. Due to its location on the outer surfaces of the bacterial cell, targeting the RSP protein could be a means of controlling IncHI plasmid conjugation in natural environments or of combatting infections caused by AMR enterobacteria that harbor IncHI plasmids., Author summary Dissemination of antimicrobial resistance (AMR) among different bacterial populations occurs due to mainly the presence of plasmids that encode AMR determinants. IncHI plasmids are one of the groups of bacterial plasmids that confer AMR to several enterobacteria. Recently, resistance to one of the last-resort antibiotics (colistin) for some multidrug-resistant infections has spread very rapidly. IncHI plasmids represent 20% of all plasmids transmitting colistin resistance worldwide and 40% in Europe. When analyzing the interactions of the IncHI1 plasmid R27 with Salmonella, we identified a large-molecular-mass protein that is encoded by this plasmid and is exported to the external medium. The R27 plasmid gene coding for that protein (R0009) is widespread among IncHI plasmids. In this report, we characterize the protein, termed RSP. The presented data show that RSP plays a relevant role in IncHI plasmid conjugation and suggest that the protein is retained on the outer surface of the bacterial cells and facilitates cell-to-cell contact before plasmid DNA transfer. Considering that IncHI plasmids significantly contribute to AMR dissemination within enterobacteria, the findings reported in this paper suggest that the identified protein can be a target to control both IncHI-mediated AMR dissemination and infections caused by AMR enterobacteria that harbor these plasmids.

Immune repertoires provide a unique fingerprint reflecting the immune history of individuals, with potential applications in precision medicine. However, the question of how personal that information is and how it can be used to identify individuals has not been explored. Here, we show that individuals can be uniquely identified from repertoires of just a few thousands lymphocytes. We present “Immprint,” a classifier using an information-theoretic measure of repertoire similarity to distinguish pairs of repertoire samples coming from the same versus different individuals. Using published T-cell receptor repertoires and statistical modeling, we tested its ability to identify individuals with great accuracy, including identical twins, by computing false positive and false negative rates < 10−6 from samples composed of 10,000 T-cells. We verified through longitudinal datasets that the method is robust to acute infections and that the immune fingerprint is stable for at least three years. These results emphasize the private and personal nature of repertoire data., Author summary Immune repertoires are a trove of personal information: unique to each individual, they are also windows into their past and future health. Thanks to their potential for personalized medicine and progress of sequencing technologies, these repertoires are now routinely sequenced. As a consequence they raise the question of identifiability of samples. In this paper, we estimate the quantity of immune cells needed to associate two samples from the same individual: as little as a finger prick worth of blood can serve as an immune fingerprint that can distinguish even identical twins, without giving away genetic information about non-consenting relatives. We show that this fingerprint is stable through time, and is not erased during infections or vaccinations.

Deconvolution of heterogeneous bulk tumor samples into distinct cellular populations is an important yet challenging problem, particularly when only partial references are available. A common approach to dealing with this problem is to deconvolve the mixed signals using available references and leverage the remaining signal as a new cell component. However, as indicated in our simulation, such an approach tends to over-estimate the proportions of known cell types and fails to detect novel cell types. Here, we propose PREDE, a partial reference-based deconvolution method using an iterative non-negative matrix factorization algorithm. Our method is verified to be effective in estimating cell proportions and expression profiles of unknown cell types based on simulated datasets at a variety of parameter settings. Applying our method to TCGA tumor samples, we found that proportions of pure cancer cells better indicate different subtypes of tumor samples. We also detected several cell types for each cancer type whose proportions successfully predicted patient survival. Our method makes a significant contribution to deconvolution of heterogeneous tumor samples and could be widely applied to varieties of high throughput bulk data. PREDE is implemented in R and is freely available from GitHub (https://xiaoqizheng.github.io/PREDE)., Author summary Tumor tissues are mixtures of different cell types. Identification and quantification of constitutional cell types within tumor tissues are important tasks in cancer research. The problem can be readily solved using regression-based methods if reference signals are available. But in most clinical applications, only partial references are available, which significantly reduces the deconvolution accuracy of the existing regression-based methods. In this paper, we propose a partial-reference based deconvolution model, PREDE, integrating the non-negative matrix factorization framework with an iterative optimization strategy. We conducted comprehensive evaluations for PREDE using both simulation and real data analyses, demonstrating better performance of our method than other existing methods.

Plasma cell segmentation is the first stage of a computer assisted automated diagnostic tool for multiple myeloma (MM). Owing to large variability in biological cell types, a method for one cell type cannot be applied directly on the other cell types. In this paper, we present PCSeg Tool for plasma cell segmentation from microscopic medical images. These images were captured from bone marrow aspirate slides of patients with MM. PCSeg has a robust pipeline consisting of a pre-processing step, the proposed modified multiphase level set method followed by post-processing steps including the watershed and circular Hough transform to segment clusters of cells of interest and to remove unwanted cells. Our modified level set method utilizes prior information about the probability densities of regions of interest (ROIs) in the color spaces and provides a solution to the minimal-partition problem to segment ROIs in one of the level sets of a two-phase level set formulation. PCSeg tool is tested on a number of microscopic images and provides good segmentation results on single cells as well as efficient segmentation of plasma cell clusters.

Continuing efforts from large international consortia have made genome-wide epigenomic and transcriptomic annotation data publicly available for a variety of cell and tissue types. However, synthesis of these datasets into effective summary metrics to characterize the functional non-coding genome remains a challenge. Here, we present GenoSkyline-Plus, an extension of our previous work through integration of an expanded set of epigenomic and transcriptomic annotations to produce high-resolution, single tissue annotations. After validating our annotations with a catalog of tissue-specific non-coding elements previously identified in the literature, we apply our method using data from 127 different cell and tissue types to present an atlas of heritability enrichment across 45 different GWAS traits. We show that broader organ system categories (e.g. immune system) increase statistical power in identifying biologically relevant tissue types for complex diseases while annotations of individual cell types (e.g. monocytes or B-cells) provide deeper insights into disease etiology. Additionally, we use our GenoSkyline-Plus annotations in an in-depth case study of late-onset Alzheimer’s disease (LOAD). Our analyses suggest a strong connection between LOAD heritability and genetic variants contained in regions of the genome functional in monocytes. Furthermore, we show that LOAD shares a similar localization of SNPs to monocyte-functional regions with Parkinson’s disease. Overall, we demonstrate that integrated genome annotations at the single tissue level provide a valuable tool for understanding the etiology of complex human diseases. Our GenoSkyline-Plus annotations are freely available at http://genocanyon.med.yale.edu/GenoSkyline., Author summary After years of community efforts, many experimental and computational approaches have been developed and applied for functional annotation of the human genome, yet proper annotation still remains challenging, especially in non-coding regions. As complex disease research rapidly advances, increasing evidence suggests that non-coding regulatory DNA elements may be the primary regions harboring risk variants in human complex diseases. In this paper, we introduce GenoSkyline-Plus, a principled annotation framework to identify tissue and cell type-specific functional regions in the human genome through integration of diverse high-throughput epigenomic and transcriptomic data. Through validation of known non-coding tissue-specific regulatory regions, enrichment analyses on 45 complex traits, and an in-depth case study of neurodegenerative diseases, we demonstrate the ability of GenoSkyline-Plus to accurately identify tissue-specific functionality in the human genome and provide unbiased, genome-wide insights into the genetic basis of human complex diseases.

Efficient application of veterinary blood transfusion approaches for small companion animals requires readily available supply of the donor material. This can be achieved by developing of effective biobanking technologies allowing long-term storage of donor blood components via cryopreservation. Transfusion of an erythrocyte concentrate allows the successful correction of various hematological pathologies, severe bleeding, and etc. While in the past there were several approaches to cryopreserve red blood cells of dogs, to our knowledge there is virtually no data on cryopreservation of feline erythrocytes. In this paper, we performed a comprehensive parameter optimization for low temperature storage of RBCs of both species. Here, the efficiency of single-component and multicomponent cryoprotective media as well as necessary time of pre-incubation with penetrating and non-penetrating cryoprotectants prior to rapid freezing is analyzed. This study showed that glycerol was not sufficient for cryopreservation of red blood cells of the studied species under the investigated conditions. Application of 10% (v/v) ME2SO allowed for a significant reduction of canine and feline erythrocytes hemolysis after thawing. 17.5% hydroxyethyl starch demonstrated the highest cryoprotective activity for both species. It was found that dog RBCs should be incubated in cryoprotective media for 30 min at 22°C prior to freezing, while for cat RBCs 20 min is sufficient. Combination of CPAs was less effective. Presented data may be considered in further studies in veterinary transfusion and blood banking optimization. DFG/EXC62/1 DFG/EXC/62/1 DFG/EXC/REBIRTH

Background Prognostic models in metastatic castrate resistant prostate cancer (mCRPC) may have clinical utility. Using data from PDS, we aimed to 1) validate a contemporary prognostic model (Templeton et al., 2014) 2) evaluate prognostic impact of concomitant medications and PSA decrease 3) evaluate factors associated with docetaxel toxicity. Methods We accessed data on 2,449 mCRPC patients in PDS. The existing model was validated with a continuous risk score, time-dependent receiver operating characteristic (ROC) curves, and corresponding time-dependent Area under the Curve (tAUC). The prognostic effects of concomitant medications and PSA response were assessed by Cox proportional hazards models. One year tAUC was calculated for multivariable prognostic model optimized to our data. Conditional logistic regression models were used to assess associations with grade 3/4 adverse events (G3/4 AE) at baseline and after cycle 1 of treatment. Results Despite limitations of the PDS data set, the existing model was validated; one year AUC, was 0.68 (95% CI 95% CI, .66 to .71) to 0.78 (95%CI, .74 to .81) depending on the subset of datasets used. A new model was constructed with an AUC of .74 (.72 to .77). Concomitant medications low molecular weight heparin and warfarin were associated with poorer survival, Metformin and Cox2 inhibitors were associated with better outcome. PSA response was associated with survival, the effect of which was greatest early in follow-up. Age was associated with baseline risk of G3/4 AE. The odds of experiencing G3/4 AE later on in treatment were significantly greater for subjects who experienced a G3/4 AE in their first cycle (OR 3.53, 95% CI 2.53–4.91, p < .0001). Conclusion Despite heterogeneous data collection protocols, PDS provides access to large datasets for novel outcomes analysis. In this paper, we demonstrate its utility for validating existing models and novel model generation including the utility of concomitant medications in outcome analyses, as well as the effect of PSA response on survival and toxicity prediction.

Routine blood tests provide important basic information for disease diagnoses. The proportions of three subtypes of white blood cells (WBCs), which are neutrophils, monocytes, lymphocytes, is key information for disease diagnosis. However, current instruments for routine blood tests, such as blood cell analyzers, flow cytometers, and optical microscopes, are cumbersome, time consuming and expensive. To make a smaller, automatic low-cost blood cell analyzer, much research has focused on a technique called lens-less shadow imaging, which can obtain microscopic images of cells in a lens-less system. Nevertheless, the efficiency of this imaging system is not satisfactory because of two problems: low resolution and imaging diffraction phenomena. In this paper, a novel method of classifying cells with the shadow imaging technique was proposed. It could be used for the classification of the three subtypes of WBCs, and the correlation of the results of classification between the proposed system and the reference system (BC-5180, Mindray) was 0.93. However, the instrument was only 10 × 10 × 10 cm, and the cost was less than $100. Depending on the lens-free shadow imaging technology, the main hardware could be integrated on a chip scale and could be called an on-chip instrument.

Many mRNA-based vaccines have been investigated for their specific potential to activate dendritic cells (DCs), the highly-specialized antigen-presenting cells of the immune system that play a key role in inducing effective CD4+ and CD8+ T-cell responses. In this paper we report a new vaccine/gene delivery platform that demonstrates the benefits of using a self-amplifying ("replicon") mRNA that is protected in a viral-protein capsid. Purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is used to in vitro assemble monodisperse virus-like particles (VLPs) containing reporter proteins (e.g., Luciferase or eYFP) or the tandem-repeat model antigen SIINFEKL in RNA gene form, coupled to the RNA-dependent RNA polymerase from the Nodamura insect virus. Incubation of immature DCs with these VLPs results in increased activation of maturation markers - CD80, CD86 and MHC-II - and enhanced RNA replication levels, relative to incubation with unpackaged replicon mRNA. Higher RNA uptake/replication and enhanced DC activation were detected in a dose-dependent manner when the CCMV-VLPs were pre-incubated with anti-CCMV antibodies. In all experiments the expression of maturation markers correlates with the RNA levels of the DCs. Overall, these studies demonstrate that: VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro. Finally, preliminary in vivo experiments involving mouse vaccinations with SIINFEKL-replicon VLPs indicate a small but significant increase in antigen-specific T cells that are doubly positive for IFN and TFN induction.

It is well accepted that influenza A virus predisposes individuals to often more severe superinfections with Streptococcus pneumonia. However, the mechanisms that lead to this synergy are not clearly understood. Recent data suggests that competent Th17 immunity is crucial to clearance and protection from invasive pneumococcal disease of the lung. We demonstrate that early influenza infection significantly reduced levels of pneumococcus driven IL-12p70, IL-23 and IL-27 in human monocytes with significant impairment of IL-17A and IFN-γ in HKSP-treated allogeneic mixed lymphocyte cultures. We also provide evidence to suggest that the hemagglutinin component of the virus is at least partially responsible for this downward pressure on IL-17 responses but surprisingly this suppression occurs despite robust IL-23 levels in hemagglutinin-treated monocyte cultures. This study demonstrates that influenza can directly affect the immunological pathways that promote appropriate responses to Streptococcus pneumonia in human immune cells. Importance Influenza virus is highly contagious and poses substantial public health problems due to its strong association with morbidity and mortality. Approximately 250,000–500,000 deaths are caused by seasonal influenza virus annually, and this figure increases during periods of pandemic infections. Most of these deaths are due to secondary bacterial pneumonia. Influenza-bacterial superinfection can result in hospitalisation and/or death of both patients with pre-existing lung disease or previously healthy individuals. The importance of our research is in determining that influenza and its component haemagglutinin has a direct effect on the classic pneumococcus induced pathways to IL-17A in our human ex vivo model. Our understanding of the mechanism which leaves people exposed to influenza infection during superinfection remain unresolved. This paper demonstrates that early infection of monocytes inhibits an arm of immunity crucial to bacterial clearance. Understanding this mechanism may provide alternative interventions in the case of superinfection with antimicrobial resistant strains of bacteria.

Background The treatment for leishmaniasis is currently based on pentavalent antimonials and amphotericin B; however, these drugs result in numerous adverse side effects. The lack of affordable therapy has necessitated the urgent development of new drugs that are efficacious, safe, and more accessible to patients. Natural products are a major source for the discovery of new and selective molecules for neglected diseases. In this paper, we evaluated the effect of apigenin on Leishmania amazonensis in vitro and in vivo and described the mechanism of action against intracellular amastigotes of L. amazonensis. Methodology/Principal Finding Apigenin reduced the infection index in a dose-dependent manner, with IC50 values of 4.3 μM and a selectivity index of 18.2. Apigenin induced ROS production in the L. amazonensis-infected macrophage, and the effects were reversed by NAC and GSH. Additionally, apigenin induced an increase in the number of macrophages autophagosomes after the infection, surrounding the parasitophorous vacuole, suggestive of the involvement of host autophagy probably due to ROS generation induced by apigenin. Furthermore, apigenin treatment was also effective in vivo, demonstrating oral bioavailability and reduced parasitic loads without altering serological toxicity markers. Conclusions/Significance In conclusion, our study suggests that apigenin exhibits leishmanicidal effects against L. amazonensis-infected macrophages. ROS production, as part of the mechanism of action, could occur through the increase in host autophagy and thereby promoting parasite death. Furthermore, our data suggest that apigenin is effective in the treatment of L. amazonensis-infected BALB/c mice by oral administration, without altering serological toxicity markers. The selective in vitro activity of apigenin, together with excellent theoretical predictions of oral availability, clear decreases in parasite load and lesion size, and no observed compromises to the overall health of the infected mice encourage us to supports further studies of apigenin as a candidate for the chemotherapeutic treatment of leishmaniasis., Author Summary Leishmaniasis is an important neglected disease caused by protozoa of the genus Leishmania and affects more than 12 million people worldwide. Pentavalent antimonials and amphotericin B have been used for decades to treat leishmaniasis; however, these drugs result in numerous adverse side effects, have variable efficacy and are subject to parasite resistance. The lack of suitable therapy necessitates the development of novel antileishmanial compounds. In this study, we investigated the antileishmanial activity of apigenin in vitro and in vivo and described the mechanism of action against intracellular amastigotes of Leishmania amazonensis. Apigenin reduced the infection index in a dose-dependent manner and increased reactive oxygen species (ROS) generation. Additionally, apigenin induced an increase in the number of macrophages autophagosomes after the infection, surrounding the parasitophorous vacuole, suggestive of the involvement of host autophagy probably due to ROS generation induced by apigenin. Furthermore, treatment with apigenin was also effective in vivo, showing oral bioavailability and significantly reducing lesion sizes and parasite burden without altering serological toxicity markers.

There are currently over 2.5 million breast cancer survivors in the United States and, according to the American Cancer Society, 10 to 20 percent of these women will develop recurrent breast cancer. Early detection of recurrence can avoid unnecessary radical treatment. However, self-examination or mammography screening may not discover a recurring cancer if the number of surviving cancer cells is small, while biopsy is too invasive and cannot be frequently repeated. It is therefore important to identify non-invasive biomarkers that can detect early recurrence. The present paper develops a mathematical model of cancer recurrence. The model, based on a system of partial differential equations, focuses on tissue biomarkers that include the plasminogen system. Among them, only uPAR is known to have significant correlation to its concentration in serum and could therefore be a good candidate for serum biomarker. The model includes uPAR and other associated cytokines and cells. It is assumed that the residual cancer cells that survived primary cancer therapy are concentrated in the same location within a region with a very small diameter. Model simulations establish a quantitative relation between the diameter of the growing cancer and the total uPAR mass in the cancer. This relation is used to identify uPAR as a potential serum biomarker for breast cancer recurrence.

Visceral leishmaniasis (VL) is a disease caused by Leishmania infantum, which is transmitted by phlebotomine sandflies. Dogs are the main urban reservoir of this parasite and the disease presents similar characteristics in both humans and dogs. In this paper, we investigated the potential pathways involved in plasma cell replacement of normal cell populations in the spleen, with respect to disease severity in dogs from an endemic area for visceral leishmaniasis. To this end, canine spleen samples were grouped into three categories: TYPE1SC- (non-infected dogs or without active infection with organized white pulp), TYPE1SC+ (infected dogs with organized white pulp) or TYPE3SC+ (infected animals with disorganized white pulp). We analyzed the distribution of different plasma cell isotypes (IgA, IgG and IgM) in the spleen. The expression of cytokines and chemokines involved in plasma cell homing and survival were assessed by real time RT-PCR. Polyclonal B cell activation and hypergammaglobulinemia were also evaluated. The proportion of animals with moderate or intense plasmacytosis was higher in the TYPE3SC+ group than in the other groups (Fisher test, P

Genetic polymorphisms of apolipoprotein E (APOE) are associated with various health conditions and diseases, such as Alzheimer's disease, cardiovascular diseases, type 2 diabetes, etc. Hence, genotyping of APOE has broad applications in biomedical research and clinical settings, particularly in the era of precision medicine. The study aimed to develop a convenient and accurate method with flexible throughput to genotype the APOE polymorphisms. A melting curve-based allele-specific PCR method was developed to genotype two single nucleotide polymorphisms (SNPs) of APOE, i.e. rs429358 at codon 112 and rs7412 at codon 158. These two SNPs determine the genotype of APOE2, E3, and E4. PCR-based Sanger sequencing was used as the reference method for APOE genotyping. A 100% concordance rate was obtained in 300 subjects between the melting curve-based allele-specific PCR method and the Sanger sequencing method. This method was applied to a genetic association analysis of APOE and schizophrenia consisting of 711 patients with schizophrenia and 665 control subjects from Taiwan. However, no significant differences in the allele and genotype frequencies were detected between these two groups. Further experiments showed that DNA dissolved from blood collected on Guthrie filter paper and total blood cell lysate without DNA extraction can be used in the melting curve-based allele-specific PCR method. Thus, we suggest that this is a fast, accurate and robust APOE genotyping method with a flexible throughput and suitable for DNA template from different preparations. This convenient method shall meet the different needs of various research and clinical laboratories.

In the current era of big data, the amount of data available is continuously increasing. Both the number and types of samples, or features, are on the rise. The mixing of distinct features often makes interpretation more difficult. However, separate analysis of individual types requires subsequent integration. A tensor is a useful framework to deal with distinct types of features in an integrated manner without mixing them. On the other hand, tensor data is not easy to obtain since it requires the measurements of huge numbers of combinations of distinct features; if there are m kinds of features, each of which has N dimensions, the number of measurements needed are as many as Nm, which is often too large to measure. In this paper, I propose a new method where a tensor is generated from individual features without combinatorial measurements, and the generated tensor was decomposed back to matrices, by which unsupervised feature extraction was performed. In order to demonstrate the usefulness of the proposed strategy, it was applied to synthetic data, as well as three omics datasets. It outperformed other matrix-based methodologies.

Human herpesvirus 6 (HHV-6) is prevalent in healthy persons, causes disease in immunosuppressed carriers, and may be involved in autoimmune disease. Cytotoxic CD8 T cells are probably important for effective control of infection. However, the HHV-6-specific CD8 T cell repertoire is largely uncharacterized. Therefore, we undertook a virus-wide analysis of CD8 T cell responses to HHV-6. We used a simple anchor motif-based algorithm (SAMBA) to identify 299 epitope candidates potentially presented by the HLA class I molecule B*08:01. Candidates were found in 77 of 98 unique HHV-6B proteins. From peptide-expanded T cell lines, we obtained CD8 T cell clones against 20 candidates. We tested whether T cell clones recognized HHV-6-infected cells. This was the case for 16 epitopes derived from 12 proteins from all phases of the viral replication cycle. Epitopes were enriched in certain amino acids flanking the peptide. Ex vivo analysis of eight healthy donors with HLA-peptide multimers showed that the strongest responses were directed against an epitope from IE-2, with a median frequency of 0.09% of CD8 T cells. Reconstitution of T cells specific for this and other HHV-6 epitopes was also observed after allogeneic hematopoietic stem cell transplantation. We conclude that HHV-6 induces CD8 T cell responses against multiple antigens of diverse functional classes. Most antigens against which CD8 T cells can be raised are presented by infected cells. Ex vivo multimer staining can directly identify HHV-6-specific T cells. These results will advance development of immune monitoring, adoptive T cell therapy, and vaccines., Author summary This paper deals with the immune response to a very common virus, called human herpesvirus 6 (HHV-6). Most people catch HHV-6 in early childhood, which often leads to a disease known as three-day fever. Later in life, the virus stays in the body, and an active immune response is needed to prevent the virus from multiplying and causing damage. It is suspected that HHV-6 contributes to autoimmune diseases and chronic fatigue. Moreover, patients with severely weakened immune responses, for example after some forms of transplantation, clearly have difficulties controlling HHV-6, which puts them at risk of severe disease and shortens their survival. This can potentially be prevented by giving them HHV-6-specific "killer" CD8 T cells, which are cells of the immune system that destroy body cells harboring the virus. However, little is known so far about such T cells. Here, we describe 16 new structures that CD8 T cells can use to recognize and kill HHV-6-infected cells. We show that very different viral proteins can furnish such structures. We also observe that such T cells are regularly present in healthy people and in transplant patients who control the virus. Our results will help develop therapies of disease due to HHV-6.

Co-inheritance of α-thalassemia has a significant protective effect on the severity of complications of sickle cell disease (SCD), including stroke. However, little information exists on the association and interactions for the common African ancestral α-thalassemia mutation (−α3.7 deletion) and β-globin traits (HbS trait [SCT] and HbC trait) on important clinical phenotypes such as red blood cell parameters, anemia, and chronic kidney disease (CKD). In a community-based cohort of 2,916 African Americans from the Jackson Heart Study, we confirmed the expected associations between SCT, HbC trait, and the −α3.7 deletion with lower mean corpuscular volume/mean corpuscular hemoglobin and higher red blood cell count and red cell distribution width. In addition to the recently recognized association of SCT with lower estimated glomerular filtration rate and glycated hemoglobin (HbA1c), we observed a novel association of the −α3.7 deletion with higher HbA1c levels. Co-inheritance of each additional copy of the −α3.7 deletion significantly lowered the risk of anemia and chronic kidney disease among individuals with SCT (P-interaction = 0.031 and 0.019, respectively). Furthermore, co-inheritance of a novel α-globin regulatory variant was associated with normalization of red cell parameters in individuals with the −α3.7 deletion and significantly negated the protective effect of α-thalassemia on stroke in 1,139 patients with sickle cell anemia from the Cooperative Study of Sickle Cell Disease (CSSCD) (P-interaction = 0.0049). Functional assays determined that rs11865131, located in the major alpha-globin enhancer MCS-R2, was the most likely causal variant. These findings suggest that common α- and β-globin variants interact to influence hematologic and clinical phenotypes in African Americans, with potential implications for risk-stratification and counseling of individuals with SCD and SCT., Author summary Recent work has shown that inheriting a single copy of the β-globin gene variant which causes sickle cell disease can be associated with medical risks, such as worsening kidney function. In individuals with sickle cell disease, co-inheritance of other globin gene variants, notably α-thalassemia, can modify an individual’s risk of clinical sequelae such as stroke. In this paper, our results suggest that inheritance of the same 3.7kb deletion that causes α-thalassemia in African populations can lower the risk of anemia and chronic kidney disease among African American community-dwelling individuals with sickle cell trait. Another α-globin genetic locus, located upstream within a well-known non-coding regulatory element, was found to modify associations of the α-thalassemia 3.7kb copy number variant with red blood cell traits in the African American general population and, in sickle cell disease patients, with risk of stroke. Using functional fine mapping and reporter assays, we localized rs11865131 and rs11248850 as the two most likely causal variants for these phenotypic associations. Additional molecular studies will be required to understand the complex regulatory mechanism by which these variants influence α-globin production.

Cross-presentation of apoptotic cell associated antigens by immature dendritic cells prevents the activation of self reactive CD8 T cells. Tolerized self reactive CD8 T cells down-regulate IL-7R expression on their surface. Whether over-expression of IL-7R can reverse their fate and function has not been examined. In this paper, we showed forced expression of IL-7R in OT-I T cells by a transgene enhanced CD8 T cell mediated diabetes in the RIP-mOVA model. Although IL-7R Tg (transgenic) did not completely reverse the deletion of OT-I T cells, it provided a significant survival advantage over w.t OT-I T cells. Furthermore, IL7R Tg OT-I T cells isolated from diabetic pancreata displayed increased production of IFN-γ, higher expression of T-bet, and increased externalization of CD107a. We also found that immature DCs containing apoptotic cells expressed high levels of PDL-1 on their surface. Although IL-7R Tg did not change PD1 expression on activated OT-I cells in vivo, the transgene enabled a significantly lower number of OT-I T cells to induce diabetes in the absence of PDL-1. Our results demonstrated that forced expression of IL-7R not only improved the functionality of tolerized CD8 T cells, it also acted in synergy with PDL-1 deficiency to further promote CD8 T cell cytotoxicity to self antigens.

Human babesiosis is caused by the apicomplexan parasite Babesia microti, which is of major public health concern in the United States and elsewhere, resulting in malaise and fatigue, followed by a fever and hemolytic anemia. In this paper we focus on the characterization of a novel B. microti thrombospondin domain (TSP1)-containing protein (BmP53) from the new annotation of the B. microti genome (locus 'BmR1_04g09041'). This novel protein (BmP53) had a single TSP1 and a transmembrane domain, with a short cytoplasmic tail containing a sub-terminal glutamine residue, but no signal peptide and Von Willebrand factor type A domains (VWA), which are found in classical thrombospondin-related adhesive proteins (TRAP). Co-localization assays of BmP53 and Babesia microti secreted antigen 1 (BmSA1) suggested that BmP53 might be a non-secretory membranous protein. Molecular mimicry between the TSP1 domain from BmP53 and host platelets molecules was indicated through different measures of sequence homology, phylogenetic analysis, 3D structure and shared epitopes. Indeed, hamster isolated platelets cross-reacted with mouse anti-BmP53-TSP1. Molecular mimicry are used to help parasites to escape immune defenses, resulting in immune evasion or autoimmunity. Furthermore, specific host reactivity was also detected against the TSP1-free part of BmP53 in infected hamster sera. In conclusion, the TSP1 domain mimicry might help in studying the mechanisms of parasite-induced thrombocytopenia, with the TSP1-free truncate of the protein representing a potential safe candidate for future vaccine studies.

Purified recombinant proteins for use in biomedical research are invaluable to investigate protein function. However, purity varies in protein batches made in mammalian expression systems, such as CHO-cells or HEK293-cells. This study points to caution while investigating effects of proteins related to the transforming growth factor (TGF)-β superfamily. TGF-β itself is a very potent cytokine and has effects on cells in the femtomolar range. Thus, even very small amounts of contaminating TGF-β in purified protein batches may influence the experimental results given that receptors for TGF-β are present. When we attempted to characterize possible receptors for the TGF-β superfamily ligand GDF15, striking similarities between GDF15-induced activities and known TGF-β activities were found. However, differences between batches of GDF15 were a concern and finally led us to the conclusion that the measured effects were caused by TGF-β and not by GDF15. Our results emphasize that purified recombinant proteins must be used with caution and warrant proper controls. Notably, some conclusions made about GDF15 in already published papers may not be supported by the results shown. Awareness about this issue in the scientific community may prevent spreading of false positive results. © 2017 Olsen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The spread of KPC-type carbapenemases is mainly attributed to the global dissemination of Klebsiella pneumoniae (KP) strains belonging to the clonal group (CG) 258, including sequence type (ST) 258 and other related STs. Two distinct clades of CG258-KP have evolved, which differ mainly for the composition of their capsular polysaccharides, and recent studies indicate that clade 1 evolved from an ancestor of clade 2 by recombination of a genomic fragment carrying the capsular polysaccharide (cps) locus. In this paper, we investigated the ability of two ST258-KP strains, KKBO-1 and KK207-1, selected as representatives of ST258-KP clade 2 and clade 1, respectively, to activate an adaptive immune response using ex vivo-stimulation of PBMC from normal donors as an experimental model. Our data showed that KKBO-1 (clade 2) induces a Th17 response more efficiently than KK207-1 (clade 1): the percentage of CD4+IL17+ cells and the production of IL-17A were significantly higher in cultures with KKBO-1 compared to cultures with KK207-1. While no differences in the rate of bacterial internalization or in the bacteria-induced expression of CD86 and HLA-DR by monocytes and myeloid dendritic cells were revealed, we found that the two strains significantly differ in inducing the production of cytokines involved in the adaptive immune response, as IL-1β, IL-23 and TNF-α, by antigen-presenting cells, with KKBO-1 being a more efficient inducer than KK207-1. The immune responses elicited by KK207-1 were comparable to those elicited by CIP 52.145, a highly virulent K. pneumoniae reference strain known to escape immune-inflammatory responses. Altogether, present results suggest that CG258-KP of the two clades are capable of inducing a different response of adaptive immunity in the human host.

Nucleoside diphosphate kinase (NDK), which has the same sequence as oncoprotein (OP) in humans, can induce nucleoside triphosphates in DNA replication by maintenance of the deoxynucleotide triphosphate (dNTP’s) and is known to be regulated by viral infection in the shrimp Litopenaeus vannamei. This paper describes the relationship between NDK and white spot syndrome virus (WSSV) infection. The recombinant NDK was produced by a prokaryotic expression system. WSSV copy numbers and mRNA levels of IE1 and VP28 were significantly increased in shrimp injected with recombinant NDK at 72 h after WSSV infection. After synthesizing dsRNA-NDK and confirming the efficacy of NDK silencing, we recorded the cumulative mortality of WSSV-infected shrimp injected with NDK and dsRNA-NDK. A comparison between the results demonstrated that silencing NDK delayed the death of shrimps. These findings indicate that NDK has an important role influencing the replication of WSSV replication in shrimp. Furthermore, NDK may have potential target as a new therapeutic strategy against WSSV infection in shrimp.

R-/S-2-(2-Hydroxypropanamido) benzoic acid (R-/S-HPABA), a marine-derived anti-inflammatory drug, however, the antiplatelet and antithrombotic effects have not been investigated. In this paper, the in vitro antiplatelet activities and in vivo antithrombotic effects of R-/S-HPABA were investigated, for the first time. The effects of R-/S-HPABA on platelet aggregation induced by adenosine diphosphate (ADP), collagen (COLL) and arachidonic acid (AA) were evaluated. In addition, the in vivo bleeding time, clotting time, collagen-epinephrine induced pulmonary thrombosis and common carotid artery thrombosis were also investigated in rats. R-/S-HPABA significantly inhibited ADP, COLL and AA induced platelet aggregation in rabbit platelet rich plasma in vitro compared with control group, to a degree similar to that of aspirin. Besides, R-/S-HPABA prolonged bleeding time and clotting time as well as increased the recovery rate obviously in pulmonary thrombosis. Moreover, the level of thromboxane B2 (TXB2) was decreased while the production of 6-keto-prostaglandin F1α (6-keto-PGF1α) was increased markedly by R-/S-HPABA. Furthermore, R-/S-HPABA reduced carotid artery thrombosis weight. These results illustrated that R-/S-HPABA could be a potent antiplatelet aggregation and antithrombotic agent.

NK cell effector functions are controlled by a combination of inhibitory receptors, which modulate NK cell activation initiated by stimulatory receptors. Most of the canonical NK cell inhibitory receptors recognize allelic forms of classical and non-classical MHC class I molecules. Furthermore, high expression of MHC-I molecules on effector immune cells is also associated with reverse signaling, giving rise to several immune-regulatory functions. Consequently, the inhibitory function of MHC class I expressed on a human NKL cell line and activated primary NK and T cells on different activating receptors are analyzed in this paper. Our results reveal that MHC-I molecules display specific patterns of “selective” inhibition over cytotoxicity and cytokine production induced by ITAM-dependent receptors and 2B4, but not on NKG2D. This contrasts with the best known “canonical” inhibitory receptors, which constitutively inhibit both functions, regardless of the activating receptor involved. Our results support the existence of a new fine-tuner inhibitory function for MHC-I molecules expressed on cytotoxic effector cells that could be involved in establishing self-tolerance in mature activated NK cells, and could also be important in tumor and infected cell recognition.

Interleukin-12 is a pro-inflammatory cytokine which promotes Th1 and cytotoxic T lymphocyte activities, such as Interferon-[Formula: see text] secretion. For this reason Interleukin-12 could be a powerful therapeutic agent for cancer treatment. However, Interleukin-12 is also excessively toxic. Interleukin-27 is an immunoregulatory cytokine from the Interleukin-12 family, but it is not as toxic as Interleukin-12. In recent years, Interleukin-27 has been considered as a potential anti-tumor agent. Recent experiments in vitro and in vivo have shown that cancer cells transfected with IL-27 activate CD8+ T cells to promote the secretion of anti-tumor cytokines Interleukin-10, although, at the same time, IL-27 inhibits the secretion of Interferon-[Formula: see text] by CD8+ T cells. In the present paper we develop a mathematical model based on these experimental results. The model involves a dynamic network which includes tumor cells, CD8+ T cells and cytokines Interleukin-27, Interleukin-10 and Interferon-[Formula: see text]. Simulations of the model show how Interleukin-27 promotes CD8+ T cells to secrete Interleukin-10 to inhibit tumor growth. On the other hand Interleukin-27 inhibits the secretion of Interferon-[Formula: see text] by CD8+ T cells which somewhat diminishes the inhibition of tumor growth. Our numerical results are in qualitative agreement with experimental data. We use the model to design protocols of IL-27 injections for the treatment of cancer and find that, for some special types of cancer, with a fixed total amount of drug, within a certain range, continuous injection has better efficacy than intermittent injections in reducing the tumor load while the treatment is ongoing, although the decrease in tumor load is only temporary.

This paper aims to study the thrombolytic effect of low-frequency ultrasound combined with targeted urokinase-containing microbubble contrast agents on treatment of thrombosis in rabbit femoral artery; and to determine the optimal combination of parameters for achieving thrombolysis in this model. A biotinylated-avidin method was used to prepare microbubble contrast agents carrying urokinase and Arg-Gly-Asp-Ser (RGDS) peptides. Following femoral artery thrombosis in New Zealand white rabbits, microbubble contrast agents were injected intravenously, and ultrasonic exposure was applied. A 3 × 2 × 2 factorial table was applied to categorize the experimental animals based on different levels of combination of ultrasonic frequencies (Factor A: 1.6 MHz, 2.2 MHz, 2.8 MHz), doses of urokinase (Factor B: 90,000 IU/Kg, 180,000 IU/Kg) and ultrasound exposure time (Factor C: 30 min, 60 min). A total of 72 experimental animals were randomly divided into 12 groups (n = 6/group). Doppler techniques were used to assess blood flow in the distal end of the thrombotic femoral artery during the 120 minutes thrombolysis experiment. The rate of recanalization following thrombolysis was calculated, and thrombolytic efficacy was evaluated and compared. The thrombolytic recanalization rate for all experimental subjects after thrombolytic therapy was 68.1%. The optimal parameters for thrombolysis were determined to be 1) an ultrasound frequency of 2.2 MHz and 2) a 90,000 IU/kg dose of urokinase. Ultrasound exposure time (30 min vs. 60 min) had no significant effect on the thrombolytic effects. The combination of local low-frequency ultrasound radiation, targeted microbubbles, and thrombolytic urokinase induced thrombolysis of femoral artery thrombosis in a rabbit model. The ultrasonic frequency of 2.2 MHz and urokinase dose of 90,000 IU/kg induced optimal thrombolytic effects, while the application of either 30 min or 60 min of ultrasound exposure had similar effects.

Crohn's Disease (CD) results from inappropriate response toward commensal flora. Earlier studies described CD as a Th1 mediated disease. Current models view both phenotypes as a continuum of various permutations between Th1, Th2 and Th17 pathways compounded by a range of Treg disfunctions. In the present paper, we develop a mathematical model, by a system of differential equations, which describe the dynamic relations among these T cells and their cytokines. The model identities four groups of CD patients according to up/down regulation of Th1 and Th2. The model simulations show that immunosuppression by TNF-α blockage benefits the group with Th1High/Th2Low while, by contrast, the group with Th1Low/Th2High will benefit from immune activation.

Malaria detection through microscopic examination of stained blood smears is a diagnostic challenge that heavily relies on the expertise of trained microscopists. This paper presents an automated analysis method for detection and staging of red blood cells infected by the malaria parasite Plasmodium falciparum at trophozoite or schizont stage. Unlike previous efforts in this area, this study uses quantitative phase images of unstained cells. Erythrocytes are automatically segmented using thresholds of optical phase and refocused to enable quantitative comparison of phase images. Refocused images are analyzed to extract 23 morphological descriptors based on the phase information. While all individual descriptors are highly statistically different between infected and uninfected cells, each descriptor does not enable separation of populations at a level satisfactory for clinical utility. To improve the diagnostic capacity, we applied various machine learning techniques, including linear discriminant classification (LDC), logistic regression (LR), and k-nearest neighbor classification (NNC), to formulate algorithms that combine all of the calculated physical parameters to distinguish cells more effectively. Results show that LDC provides the highest accuracy of up to 99.7% in detecting schizont stage infected cells compared to uninfected RBCs. NNC showed slightly better accuracy (99.5%) than either LDC (99.0%) or LR (99.1%) for discriminating late trophozoites from uninfected RBCs. However, for early trophozoites, LDC produced the best accuracy of 98%. Discrimination of infection stage was less accurate, producing high specificity (99.8%) but only 45.0%-66.8% sensitivity with early trophozoites most often mistaken for late trophozoite or schizont stage and late trophozoite and schizont stage most often confused for each other. Overall, this methodology points to a significant clinical potential of using quantitative phase imaging to detect and stage malaria infection without staining or expert analysis.

For the purposes of information retrieval, users must find highly relevant documents from within a system (and often a quite large one comprised of many individual documents) based on input query. Ranking the documents according to their relevance within the system to meet user needs is a challenging endeavor, and a hot research topic–there already exist several rank-learning methods based on machine learning techniques which can generate ranking functions automatically. This paper proposes a parallel B cell algorithm, RankBCA, for rank learning which utilizes a clonal selection mechanism based on biological immunity. The novel algorithm is compared with traditional rank-learning algorithms through experimentation and shown to outperform the others in respect to accuracy, learning time, and convergence rate; taken together, the experimental results show that the proposed algorithm indeed effectively and rapidly identifies optimal ranking functions.

Interleukin-2 (IL2) is a growth factor for several immune cells and its function depends on its binding to IL2Rs in the cell membrane. The most accepted model for the assembling of IL2-IL2R complexes in the cell membrane is the Affinity Conversion Model (ACM). This model postulates that IL2R receptor association is sequential and dependent on ligand binding. Most likely free IL2 binds first to IL2Rα, and then this complex binds to IL2Rβ, and finally to IL2Rγ (γc). However, in previous mathematical models representing this process, the binding of γc has not been taken into account. In this work, the quantitative contribution of the number of IL2Rγ chain to the IL2-IL2R apparent binding affinity and signaling is studied. A mathematical model of the affinity conversion process including the γ chain in the dynamic, has been formulated. The model was calibrated by fitting it to experimental data, specifically, Scatchard plots obtained using human cell lines. This paper demonstrates how the model correctly explains available experimental observations. It was estimated, for the first time, the value of the kinetic coefficients of IL2-IL2R complexes interaction in the cell membrane. Moreover, the number of IL2R components in different cell lines was also estimated. It was obtained a variable distribution in the number of IL2R components depending on the cell type and the activation state. Of most significance, the study predicts that not only the number of IL2Rα and IL2Rβ, but also the number of γc determine the capacity of the cell to capture and retain IL2 in signalling complexes. Moreover, it is also showed that different cells might use different pathways to bind IL2 as consequence of its IL2R components distribution in the membrane.