Determination Of Surface Research Articles

Aberrant CD3-Positive, CD8-Low, CD7-Negative Lymphocytes May Appear During Viral Infections and Mimic Peripheral T-Cell Lymphoma.

Flow cytometry (FC) facilitates diagnosis of peripheral T-cell non-Hodgkin lymphoma (T-NHL), but overlapping features between reactive and neoplastic T-cell proliferations often hamper a rapid assessment. One hundred forty peripheral blood samples submitted to diagnostic FC for T-cell immunophenotyping were retrospectively analyzed. A T-cell population with a conspicuous aberrant surface epitope expression pattern was observed in 18 cases and diagnostic follow up was performed. The aberrant T-cell population exhibited a low scatter profile, a CD7-negative/low, CD8-low and CD3-positive immunophenotype, and monoclonal T-cell receptor expansion. T-NHL was ruled out by follow up in all cases. Epstein-Barr virus infection was diagnosed in 12 cases, cytomegalovirus infection in three cases; one patient had been vaccinated. The irregular subpopulation disappeared spontaneously within days or weeks. We describe a novel peripheral blood T-cell subpopulation with a low light scatter and CD8-low, CD7-negative/low and CD3-positive marker expression profile, which indicates reactive T-cell expansion in patients who present with peripheral lymphadenopathy and/or B symptoms.

Effect of miR-100 on Migration of Rat Bone Marrow Mesenchymal Stem Cells

To explore the effect of miR-100 on the migration of rat bone marrow mesenchymal stem cells(rBMMSC). The rBMMSC were isolated by density gradient centrifugation, cell surface epitopes of CD105, CD45, CD34, CD29 and CD44 were analyzed by flow cytometry. The rBMMSC were transfected with miR-100 mimic or inhibitor, then the expression of miR-100 in transfected cells was detected by real-time PCR. Migration test was used to observe the effect of miR-100 on cell migration ability. The secretion level of chemokine SDF-1 in culture supernatant of cells was quantitatively detected by ELISA. The isolated cells were identified as BMMSC. After rBMMSC were transfected with miR-100 mimic or inhibitor, as compared with control group,the expression of miR-100 in rBMMSC significantly increased or decreased respectively. In the migration experiment, the rBMMSC migration was significantly inhibited in the miR-100 mimic group (P<0.01), while the rBMMSC migration was significantly enhanced in the miR-100 inhibitor group (P<0.01). The concentration of SDF-1 in the supernatant of the miR-100 mimic group and the miR-100 inhibitor group did not change significantly compared with the control group (P>0.05). miR-100 can significantly inhibit the migration of rBMMSC, but not significantly correlated with the SDF-1.

Clinical features of chronic hepatitis B patients with low hepatitis B surface antigen levels and determinants of hepatitis B surface antigen seroclearance.

Background and AimA low hepatitis B surface antigen (HBsAg) level is reported to be predictive of future HBsAg seroclearance. A hospital‐based cohort study was conducted to clarify the clinical features of patients with low HBsAg levels and to demonstrate the usefulness of low HBsAg levels for predicting HBsAg seroclearance.MethodsA total of 1459 patients with chronic hepatitis B were included in the study. Of these, 587 had repeated measurements for HBsAg levels and two or more records of HBsAg‐positive results. HBsAg levels were measured with a commercially available HBsAg assay. Based on a cut‐off index (COI) of 2000, a high HBsAg level was defined as HBsAg ≥2000 COI, and a low HBsAg level was defined as HBsAg <2000 COI.ResultsThe proportion of patients with low HBsAg levels at baseline tended to increase with age. Patients with low HBsAg levels at baseline had significantly older age, lower transaminase levels, and lower hepatitis B virus (HBV) DNA levels than those with high HBsAg levels. The annual HBsAg seroclearance rate was 1.30%/year. The cumulative incidences of HBsAg seroclearance differed significantly by HBsAg level at baseline (<2000 vs ≥2000 COI), age (≥50 vs <50 years), and HBV DNA level (<4.0 vs ≥4.0 log copies/mL). Cox proportional hazards regression analyses showed that low HBsAg level (<2000 COI) and low HBV DNA level (<4.0 log copies/mL) were significantly associated with HBsAg seroclearance.ConclusionAging was one of the factors affecting HBsAg level. HBsAg seroclearance was significantly associated with low HBsAg level and low HBV DNA level at baseline.

Extracellular Vesicle Isolation and Characterization from Periprosthetic Joint Synovial Fluid in Revision Total Joint Arthroplasty.

Extracellular vesicles (EVs) comprise an as yet insufficiently investigated intercellular communication pathway in the field of revision total joint arthroplasty (RTJA). This study examined whether periprosthetic joint synovial fluid contains EVs, developed a protocol for their isolation and characterized them with respect to quantity, size, surface markers as well as documented their differences between aseptic implant failure (AIF) and periprosthetic joint infection (PJI). EV isolation was accomplished using ultracentrifugation, electron microscopy (EM) and nanoparticle tracking analysis evaluated EV presence as well as particle size and quantity. EV surface markers were studied by a bead-based multiplex analysis. Using our protocol, EM confirmed the presence of EVs in periprosthetic joint synovial fluid. Higher EV particle concentrations and decreased particle sizes were apparent for PJI. Multiplex analysis confirmed EV-typical surface epitopes and revealed upregulated CD44 and HLA-DR/DP/DQ for AIF, as well as increased CD40 and CD105. Our protocol achieved isolation of EVs from periprosthetic joint synovial fluid, confirmed by EM and multiplex analysis. Characterization was documented with respect to size, concentration and epitope surface signature. Our results indicate various differences between PJI and AIF EVs. This pilot study enables new research approaches and rising diagnostic opportunities in the field of RTJA.

Hybrid Antigens Expressing Surface Loops of ZnuD From Acinetobacter baumannii Is Capable of Inducing Protection Against Infection.

Acinetobacter baumannii is an important human pathogen causing substantial mortality in hospitalized patients for which treatment with antibiotics has become problematic due to growing antibiotic resistance. In an attempt to develop alternative strategies for dealing with these serious infections surface antigens are being considered as targets for vaccines or immunotherapy. The surface receptor proteins required for zinc acquisition in Gram-negative bacterial pathogens have been proposed as vaccine targets due to their crucial role for growth in the human host. In this study we selected the putative ZnuD outer membrane receptor from A. baumannii as a target for vaccine development. Due to challenges in production of an integral outer membrane protein for vaccine production, we adopted a recently described hybrid antigen approach in which surface epitopes from the Neisseria meningitidis TbpA receptor protein were displayed on a derivative of the C-lobe of the surface lipoprotein TbpB, named the loopless C-lobe (LCL). A structural model for ZnuD was generated and four surface loops were selected for hybrid antigen production by computational approaches. Hybrid antigens were designed displaying the four selected loops (2, 5, 7, and 11) individually or together in a single hybrid antigen. The hybrid antigens along with ZnuD and the LCL scaffold were produced in the E. coli cytoplasm either as soluble antigens or as inclusion bodies, that were used to generate soluble antigens upon refolding. Mice were immunized with the hybrid antigens, ZnuD or LCL and then used in an A. baumannii sepsis model to evaluate their ability to protect against infection. As expected, the LCL scaffold did not induce a protective immune response, enabling us to attribute observed protection to the displayed loops. Immunization with the refolded ZnuD protein protected 63% of the mice while immunization with hybrid antigens displaying individual loops achieved between 25 and 50% protection. Notably, the mice immunized with the hybrid antigen displaying the four loops were completely protected from infection.

Recent advances in molecular imprinting technology for the separation and analysis of proteins

Proteins, which have complex structures and fall under diverse categories, are closely associated with different kinds of life activities. Majority of the proteins exist in low concentrations in complex biological samples, because of which their direct analysis without sample pretreatment is difficult. Thus, selective separation of the target proteins from complex biological samples is essential for efficient analysis. Molecularly imprinted polymers, containing numerous imprinted cavities that are complementary with the templates in terms of shape, size, and functional groups, have great potential for use in the selective recognition and separation of the target proteins from complex biological samples. However, the large size, structural flexibility, and complex structures of proteins hinder their efficient separation and analysis by conventional molecular imprinting technology. In this review, several novel molecular imprinting techniques, including surface imprinting, epitope imprinting, and metal chelate imprinting, are introduced. The emergence of these novel technologies has contributed to advances in protein analysis. The applications of these molecular imprinting techniques to the separation and analysis of proteins in the last three years are summarized herein. Finally, the promising future of molecular imprinting techniques in the area of proteins is prospected.

Weak Fragment Crystallizable (Fc) Domain Interactions Drive the Dynamic Assembly of IgG Oligomers upon Antigen Recognition.

Activation of membrane receptors through clustering is a common mechanism found in various biological systems. Spatial proximity of receptors may be transduced across the membrane to initiate signaling pathways or alternatively be recognized by peripheral proteins or immune cells to trigger external effector functions. Here we show how specific immunoglobulin G (IgG) binding induces clustering of monomeric target molecules in lipid membranes through Fc-Fc interactions. We visualize and characterize the dynamic IgG oligomerization process and the molecular interactions involved using high-speed atomic force microscopy, single-molecule force spectroscopy, and quartz crystal microbalance experiments. We found that the Fc-Fc interaction strength is precisely tuned to be weak enough to prevent IgG oligomerization in solution at physiological titers, but enabling IgG oligomerization when Fabs additionally bind to their cognate surface epitopes, a mechanism that ultimately targets IgG-mediated effector functions such as classical complement activation to antigenic membranes.

Designing a New Multi-Epitope Pertussis Vaccine with Highly Population Coverage Based on a Novel Sequence and Structural Filtration Algorithm.

Pertussis vaccine is produced from physicochemically inactivated toxin for many years. Recent advancements in immunoinformatics [N. Tomar and R. K. De, "Immunoinformatics: an integrated scenario," Immunology, vol. 131, no. 2, pp. 153-168, 2010] and structural bioinformatics can provide a new multidisciplinary approach to overcome the concerns including unwanted antibodies and incomplete population coverage. In this study we focused on solving the challenging issues by designing a multi-epitope vaccine (MEV) using rational bioinformatics analyses. The frequencies of All HLA DP, DQ, and DR alleles were evaluated in almost all countries. Strong binder surface epitopes on the pertussis toxin were selected based on our novel filtration strategy. Finally, the population coverage of MEV was determined in the candidate country. Filtration steps yielded 312 strong binder epitopes. Finally, 8 surface strong binder epitopes were selected as candidate epitopes. The population coverage of the MEV in France and the world was 98 and 100 percent, respectively. Our algorithm successfully filtered many unwanted strong binder epitopes. Considering the HLA type of all individuals in a country, we theoretically provided the maximum chance to all humans to be vaccinated efficiently. Application of a MEV would be led to production of highly efficient target specific antibodies, significant reduction of unwanted antibodies, and avoid possible raising of auto-antibodies as well.

Abstract NT-113: SC-003, AN ANTIBODY-DRUG CONJUGATE TARGETING DIPEPTIDASE 3, EXHIBITS POTENT ANTI-TUMOR ACTIVITY IN PATIENT-DERIVED XENOGRAFT MODELS OF HIGH GRADE SEROUS OVARIAN CANCER

Abstract Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. Ovarian cancer describes a group of malignancies that remain a serious threat to women's health and claim more than 14,000 deaths in the US each year. Among these, high-grade serous ovarian cancers (HGSC) represent the most common and aggressive form, and tumor recurrence is near-universal following initial response to carboplatin and paclitaxel. While multiple treatment options are available for platinum-sensitive recurrent HGSC, such as continued use of carboplatin/paclitaxel with or without bevacizumab, as well as several FDA-approved PARP inhibitors, there are few effective treatment options for platinum-resistant HGSC. Its inherent heterogeneity, characterized by genomic instability and numerous DNA copy number aberrations, poses challenges to the discovery of novel therapeutic targets. Patient-derived xenograft (PDX) models are an important platform for target identification and efficacy testing of therapeutic agents, as they sustain the intratumoral heterogeneity observed in advanced human tumors more faithfully than established cell lines. The isolation of distinct tumor cell populations by flow cytometry and subsequent testing of their tumorigenic potential can identify tumor-initiating cells (TICs), which have the potential to propagate the tumor over multiple generations in immune-deficient NOD/SCID mice. Thorough characterization of these TIC populations by transcriptome profiling and flow cytometry has identified TIC-enriched cell surface proteins that are targetable by antibody drug conjugates (ADCs), including EFNA4 in mixed Müllerian and ovarian cancers and PTK7 in triple-negative breast and ovarian cancers. ADCs consist of a monoclonal antibody directed against a cell surface epitope linked to a cytotoxic agent, such as auristatin, maytansinoid, or pyrrolobenzodiazepine (PBD). Here, we describe the generation and characterization of a PDX bank for ovarian cancers that led to the identification of dipeptidase 3 (DPEP3) as a TIC-associated target in HGSC. We show that DPEP3 is enriched in the TIC fraction of platinum-sensitive and platinum-resistant HGSC PDX models, where it localizes to the plasma membrane and is detected by flow cytometry and immunohistochemistry (IHC). In contrast, DPEP3 expression is low or absent in most normal adult tissues, thus providing a therapeutic window for an antibody-based therapeutic agent. In order to target DPEP3-expressing ovarian cancer cells, we developed SC-003, an ADC consisting of a humanized monoclonal antibody linked to a PBD dimer via a cleavable linker. We show that SC-003 specifically binds to DPEP3-expressing cells and, upon internalization, elicits cytotoxicity via release of its PBD warhead following lysosomal degradation of the antibody component. A single dose of SC-003 induced tumor regression in DPEP3-positive HGSC PDX models, including platinum-resistant PDX models. Mechanistically, we show that the anti-tumor effect of SC-003 is mediated by a significant reduction in TIC frequency. Moreover, combination with an anti-PD1 antibody potentiated SC-003 efficacy in a syngeneic mouse model engineered to overexpress human DPEP3. In summary, these findings support the clinical development of SC-003 as a novel therapeutic agent for HGSC. Citation Format: Wolf R. Wiedemeyer, Sheila Bheddah, Christine Saechao, Dorothy French, Erik Huntzicker, Aaron Kempema, Julia Gavrilyuk, David Liu, Vikram Sisodiya, Alina He, Zhaomei Zhang, Monette A. Aujay, Kristyn Hayashi, Sandro Vivona, Xi Zhao, Kimberly Walter, Laura R. Saunders, Johannes Hampl, Shravanthi Madhavan, Marybeth Pysz, Alexander J. Bankovich, Holger Karsunky, Scott J. Dylla. SC-003, AN ANTIBODY-DRUG CONJUGATE TARGETING DIPEPTIDASE 3, EXHIBITS POTENT ANTI-TUMOR ACTIVITY IN PATIENT-DERIVED XENOGRAFT MODELS OF HIGH GRADE SEROUS OVARIAN CANCER [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-113.

Immunoinformatics Approach for Multiepitope Vaccine Prediction from H, M, F, and N Proteins of Peste des Petits Ruminants Virus.

Background Small ruminant morbillivirus or peste des petits ruminants virus (PPRV) is an acute and highly contagious viral disease of goats, sheep, and other livestock. This study aimed at predicting an effective multiepitope vaccine against PPRV from the immunogenic proteins haemagglutinin (H), matrix (M), fusion (F), and nucleoprotein (N) using immunoinformatics tools. Materials and Methods The sequences of the immunogenic proteins were retrieved from GenBank of the National Center for Biotechnology Information (NCBI). BioEdit software was used to align each protein from the retrieved sequences for conservancy. Immune Epitope Database (IEDB) analysis resources were used to predict B and T cell epitopes. For B cells, the criteria for electing epitopes depend on the epitope linearity, surface accessibility, and antigenicity. Results Nine epitopes from the H protein, eight epitopes from the M protein, and ten epitopes from each of the F and N proteins were predicted as linear epitopes. The surface accessibility method proposed seven surface epitopes from each of the H and F proteins in addition to six and four epitopes from the M and N proteins, respectively. For antigenicity, only two epitopes 142PPERV146 and 63DPLSP67 were predicted as antigenic from H and M, respectively. For T cells, MHC-I binding prediction tools showed multiple epitopes that interacted strongly with BoLA alleles. For instance, the epitope 45MFLSLIGLL53 from the H protein interacted with four BoLA alleles, while 276FKKILCYPL284 predicted from the M protein interacted with two alleles. Although F and N proteins demonstrated no favorable interaction with B cells, they strongly interacted with T cells. For instance, 358STKSCARTL366 from the F protein interacted with five alleles, followed by 340SQNALYPMS348 and 442IDLGPAISL450 that interacted with three alleles each. The epitopes from the N protein displayed strong interaction with BoLA alleles such as 490RSAEALFRL498 that interacted with five alleles, followed by two epitopes 2ATLLKSLAL10 and 304QQLGEVAPY312 that interacted with four alleles each. In addition to that, four epitopes 3TLLKSLALF11, 356YFDPAYFRL364, 360AYFRLGQEM368, and 412PRQAQVSFL420 interacted with three alleles each. Conclusion Fourteen epitopes were predicted as promising vaccine candidates against PPRV from four immunogenic proteins. These epitopes should be validated experimentally through in vitro and in vivo studies.

Islet autoantibodies in disease prediction and pathogenesis.

Type 1 diabetes (T1D) is now predictable by measuring specific islet autoantibodies (IAbs). Almost all children who developed multiple IAbs will progress to T1D with time, while individuals with single IAb have a very low risk although it is an important earlier biomarker. The poor prediction of single IAb has been found to be associated with IAb affinity. Majority of single IAb generated in current standard IAb radio-binding assay (RBA) are of low affinity, which have been demonstrated low risk in T1D development. New generation of nonradioactive IAb assay with electrochemiluminescence (ECL) technology has been shown to discriminate high-affinity from low-affinity IAbs and greatly improve sensitivity and disease specificity. With a high-affinity IAb assay, like ECL assay, single IAb will be expected to be a reliable biomarker for T1D early prediction. Although appearance of IAbs is most reliable biomarkers for T1D, there are no direct evidences that IAbs contribute to β-cell damage. With recent studies on ZnT8, a merging protein on β-cell surface membrane associated with insulin secretion, a subclass of ZnT8 autoantibodies directed to extra-cellular epitopes of ZnT8 on β-cell surface has recently been identified in T1D patients and these cell surface autoantibodies have been found to appear very early, before other IAbs. These findings lead us to a hypothesis that the immunogenic epitopes on β-cell surface might be early targets for autoimmune disease and IAbs to cell surface epitopes might be involved in β-cell destruction, which will change the paradigm of IAbs in T1D pathogenesis.

Improved methods for fluorescent labeling and detection of single extracellular vesicles using nanoparticle tracking analysis

Growing interest in extracellular vesicles (EV) has necessitated development of protocols to improve EV characterization as a precursor for myriad downstream investigations. Identifying expression of EV surface epitopes can aid in determining EV enrichment and allow for comparisons of sample phenotypes. This study was designed to test a rigorous method of indirect fluorescent immunolabeling of single EV with subsequent evaluation using nanoparticle tracking analysis (NTA) to simultaneously determine EV concentration, particle size distribution, and surface immunophenotype. In this study, EV were isolated from canine and human cell cultures for immunolabeling and characterized using NTA, transmission electron microscopy, and Western blotting. Indirect fluorescent immunolabeling utilizing quantum dots (Qd) resulted in reproducible detection of individual fluorescently labeled EV using NTA. Methods were proposed to evaluate the success of immunolabeling based on paired particle detection in NTA light scatter and fluorescent modes. Bead-assisted depletion and size-exclusion chromatography improved specificity of Qd labeling. The described method for indirect immunolabeling of EV and single vesicle detection using NTA offers an improved method for estimating the fraction of EV that express a specific epitope, while approximating population size distribution and concentration.

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation.

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Decreased Circulating Myeloid Derived Suppressor Cells at First Follow-up Predict Favorable Response to Immunotherapy in a Randomized Trial of Nivolumab and Bevacizumab in Recurrent GBM

Abstract INTRODUCTION A potential barrier to successful immunotherapy response in glioblastoma (GBM) is myeloid-derived suppressor cells (MDSCs): an immature immunosuppressive cell elevated in the circulation and tumor of GBM patients. Given a limited set of biomarkers predictive of response to immunotherapy in GBM, we explored the change in MDSC levels with immunotherapy to predict treatment response. METHODS A retrospective analysis was performed on patients in a randomized, phase 2 study of nivolumab and bevacizumab at GBM first recurrence. Clinical and radiographic data were analyzed for disease progression or treatment response via Response Assessment in Neuro-Oncology (RANO) criteria. Blood was collected prior to treatment and at first imaging follow-up. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples and analyzed. Characterization of circulating immune cells was performed using flow cytometry. RESULTS A total of 9 patients were identified as responders or nonresponders at a median time of 8.7 wk (range 6.9-10.0) after therapy initiation. MDSCs, as a percentage of total PBMCs, were elevated at baseline in responders compared to nonresponders (4.9% ± 0.7 vs 2.6% ± 0.2, P = .019), which reversed at follow up (1.8% ± 0.4 vs 5.8% ± 1.1, P = .032). There was a 6.4 fold decrease in MDSCs as a percentage of total PBMCs between baseline and first imaging follow-up in responders as compared to nonresponders (P = .001). When looking at subtypes of MDSCs, a 4.9 fold decrease in granulocytic MDSCs (G-MDSCs) was noted in responders over nonresponders (P = .010). Further investigation of this cohort by simultaneous single-cell analysis of transcriptome and surface epitopes is ongoing. CONCLUSION Differences in circulating MDSC levels that were specific to responders and nonresponders were seen both before and after therapy initialization, with decreases in MDSCs (specifically G-MDSCs) being correlated with treatment response. Characterization of MDSCs in the peripheral blood may be helpful in identifying GBM patients likely to benefit from immunotherapy.

Obsessive-Compulsive Disorder: Autoimmunity and Neuroinflammation.

Here, we propose to review the immuno-inflammatory hypothesis in OCD given the concurrent incidence of autoimmune comorbidities, infectious stigma, and raised levels of inflammatory markers in a significant subset of patients. A better understanding of the immune dysfunction in OCD may allow stratifying the patients in order to design personalized pharmaco/psychotherapeutic strategies. A persistent low-grade inflammation involving both innate and adaptive immune system with coexisting autoimmune morbidities and stigma of infectious events has been prominently observed in OCD. Hence, specific treatments targeting inflammation/infection are a feasible alternative in OCD. This review highlights that OCD is associated with low-grade inflammation, neural antibodies, and neuro-inflammatory and auto-immune disorders. In some subset of OCD patients, autoimmunity is likely triggered by specific bacterial, viral, or parasitic agents with overlapping surface epitopes in CNS. Hence, subset-profiling in OCD is warranted to benefit from distinct immune-targeted treatment modalities.

Delineating Surface Epitopes of Lyme Disease Pathogen Targeted by Highly Protective Antibodies of New Zealand White Rabbits.

Lyme disease (LD), the most prevalent vector-borne illness in the United States and Europe, is caused by Borreliella burgdorferi No vaccine is available for humans. Dogmatically, B. burgdorferi can establish a persistent infection in the mammalian host (e.g., mice) due to a surface antigen, VlsE. This antigenically variable protein allows the spirochete to continually evade borreliacidal antibodies. However, our recent study has shown that the B. burgdorferi spirochete is effectively cleared by anti-B. burgdorferi antibodies of New Zealand White rabbits, despite the surface expression of VlsE. Besides homologous protection, the rabbit antibodies also cross-protect against heterologous B. burgdorferi spirochetes and significantly reduce the pathology of LD arthritis in persistently infected mice. Thus, this finding that NZW rabbits develop a unique repertoire of very potent antibodies targeting the protective surface epitopes, despite abundant VlsE, prompted us to identify the specificities of the protective rabbit antibodies and their respective targets. By applying subtractive reverse vaccinology, which involved the use of random peptide phage display libraries coupled with next-generation sequencing and our computational algorithms, repertoires of nonprotective (early) and protective (late) rabbit antibodies were identified and directly compared. Consequently, putative surface epitopes that are unique to the protective rabbit sera were mapped. Importantly, the relevance of newly identified protection-associated epitopes for their surface exposure has been strongly supported by prior empirical studies. This study is significant because it now allows us to systematically test the putative epitopes for their protective efficacy with an ultimate goal of selecting the most efficacious targets for development of a long-awaited LD vaccine.

In silico rational design of a novel tetra-epitope tetanus vaccine with complete population coverage using developed immunoinformatics and surface epitope mapping approaches

Presentation of many unwanted epitopes within tetanus toxoid vaccine to lymphocyte clones may lead to production of many unwanted antibodies. Moreover an ideal vaccine must cover all individuals in a population that is dependent to the kinds of human leukocyte antigen alleles. Concerning these issues, our study was aimed to in silico design of a multi-epitope tetanus vaccine (METV) in order to improve population coverage and protectivity of tetanus vaccine as well as reduction of complications. Concerning these issues, a novel rational filtration was implemented to design a novel METV using immunoinformatics and surface epitope mapping approaches.Prediction of epitopes for tetanus toxin was performed in the candidate country in which the frequency had been gathered from almost all geographical distributions. The most strong binder epitopes for major histocompatibility complex class II were selected and among them the surface epitopes of native toxin were selected. The population coverage of the selected epitopes was estimated. The final candidate epitopes had highly population coverage. Molecular docking was performed to prediction of binding affinity of our candidate epitopes to the HLA-DRB1 alleles.At first, 680 strong binder epitopes were predicted. Among them 11 epitopes were selected. Finally, 4 epitopes had the most population coverage and suggested as a tetra-epitope tetanus vaccine. 99.41% of inessential strong binders were deleted using our tree steps filtration. HLA-DP had the most roles in epitope presentation. Molecular docking analysis proved the strong binding affinity of candidate epitopes to the HLA-DRB1 alleles.In conclusion, we theoretically reduced 99.41% of unwanted antibodies using our novel filtration strategies. Our tetra-epitope tetanus vaccine showed 100% population coverage in the candidate country. Furthermore, it was demonstrated that HLA-DP and HLA-DQ had more potential in epitope presentation in comparison to HLA-DRB1.

Mesenchymal stem cells for cartilage regeneration in dogs.

Articular cartilage damage and osteoarthritis (OA) are common orthopedic diseases in both humans and dogs. Once damaged, the articular cartilage seldom undergoes spontaneous repair because of its avascular, aneural, and alymphatic state, and the damage progresses to a chronic and painful situation. Dogs have distinctive characteristics compared to other laboratory animal species in that they share an OA pathology with humans. Dogs can also require treatment for naturally developed OA; therefore, effective treatment methods for OA are desired in veterinary medicine as well as in human medicine. Recently, interest has grown in regenerative medicine that includes the use of mesenchymal stem cells (MSCs). In cartilage repair, MSCs are a promising therapeutic tool due to their self-renewal capacity, ability to differentiate into cartilage, potential for trophic factor production, and capacity for immunomodulation. The MSCs from dogs (canine MSCs; cMSCs) share various characteristics with MSCs from other animal species, but they show some deviations, particularly in their differentiation ability and surface epitope expression. In vivo studies of cMSCs have demonstrated that intraarticular cMSC injection into cartilage lesions results in excellent hyaline cartilage regeneration. In clinical situations, cMSCs have shown great therapeutic effects, including amelioration of pain and lameness in dogs suffering from OA. However, some issues remain, such as a lack of regulations or guidelines and a need for unified methods for the use of cMSCs. This review summarizes what is known about cMSCs, including their in vitro characteristics, their therapeutic effects in cartilage lesion treatment in preclinical in vivo studies, their clinical efficacy for treatment of naturally developed OA in dogs, and the current limitations of cMSC studies.

Role of Multivalency and Antigenic Threshold in Generating Protective Antibody Responses.

Vaccines play a vital role in protecting our communities against infectious disease. Unfortunately, some vaccines provide only partial protection or in some cases vaccine-mediated immunity may wane rapidly, resulting in either increased susceptibility to that disease or a requirement for more booster vaccinations in order to maintain immunity above a protective level. The durability of antibody responses after infection or vaccination appears to be intrinsically determined by the structural biology of the antigen, with multivalent protein antigens often providing more long-lived immunity than monovalent antigens. This forms the basis for the Imprinted Lifespan model describing the differential survival of long-lived plasma cell populations. There are, however, exceptions to this rule with examples of highly attenuated live virus vaccines that are rapidly cleared and elicit only short-lived immunity despite the expression of multivalent surface epitopes. These exceptions have led to the concept that multivalency alone may not reliably determine the duration of protective humoral immune responses unless a minimum number of long-lived plasma cells are generated by reaching an appropriate antigenic threshold of B cell stimulation. Examples of long-term and in some cases, potentially lifelong antibody responses following immunization against human papilloma virus (HPV), Japanese encephalitis virus (JEV), Hepatitis B virus (HBV), and Hepatitis A virus (HAV) provide several lessons in understanding durable serological memory in human subjects. Moreover, studies involving influenza vaccination provide the unique opportunity to compare the durability of hemagglutinin (HA)-specific antibody titers mounted in response to antigenically repetitive whole virus (i.e., multivalent HA), or detergent-disrupted “split” virus, in comparison to the long-term immune responses induced by natural influenza infection. Here, we discuss the underlying mechanisms that may be associated with the induction of protective immunity by long-lived plasma cells and their importance in future vaccine design.

Microfluidic chip combined with magnetic-activated cell sorting technology for tumor antigen-independent sorting of circulating hepatocellular carcinoma cells.

PurposeWe aimed to generate a capture platform that integrates a deterministic lateral displacement (DLD) microfluidic structure with magnetic-activated cell sorting (MACS) technology for miniaturized, efficient, tumor antigen-independent circulating tumor cell (CTC) separation.MethodsThe microfluidic structure was based on the theory of DLD and was designed to remove most red blood cells and platelets. Whole Blood CD45 MicroBeads and a MACS separator were then used to remove bead-labeled white blood cells. We established HepG2 human liver cancer cells overexpressing green fluorescent protein by lentiviral transfection to simulate CTCs in blood, and these cells were then used to determine the CTC isolation efficiency of the device. The performance and clinical value of our platform were evaluated by comparison with the Abnova CytoQuest™ CR system in the separating of blood samples from 12 hepatocellular carcinoma patients undergoing liver transplantation in a clinical follow-up experiment. The isolated cells were stained and analyzed by confocal laser scanning microscopy.ResultsUsing our integrated platform at the optimal flow rates for the specimen (60 µl/min) and buffer (100 µl/min per chip), we achieved an CTC yield of 85.1% ± 3.2%. In our follow-up of metastatic patients, CTCs that underwent epithelial–mesenchymal transition were found. These CTCs were missed by the CytoQuest™ CR bulk sorting approach, whereas our platform displayed increased sensitivity to EpCAMlow CTCs.ConclusionsOur platform, which integrates microfluidic and MACS technology, is an attractive method for high-efficiency CTC isolation regardless of surface epitopes.