Search

Background: Antibodies have been proven effective as diagnostic agents for detecting zoonotic diseases. The variable domain of camel heavy chain antibody (VHH), as an antibody derivative, may be used as an alternative for traditional antibodies in existing immunodiagnostic reagents for detecting rapidly spreading infectious diseases. Objectives: To expedite the isolation of specific antibodies for diagnostic purposes, we constructed a semi‐synthetic camel single domain antibody library based on the phage display technique platform (PDT) and verified the validity of this study. Methods: The semi‐synthetic single domain antibody sequences consist of two parts: one is the FR1‐FR3 region amplified by RT‐PCR from healthy camel peripheral blood lymphocytes (PBLs), and the other part is the CDR3‐FR4 region synthesised as an oligonucleotide containing CDR3 randomised region. The two parts were fused by overlapping PCR, resulting in the rearranged variable domain of heavy‐chain antibodies (VHHs). Y. pestis low‐calcium response V protein (LcrV) is an optional biomarker to detect the Y. pestis infection. The semi‐synthetic library herein was screened using recombinant (LcrV) as a target antigen. Results: After four cycles of panning the library, four VHH binders targeting 1–270 aa residues of LcrV were isolated. The four VHH genes with unique sequences were recloned into an expression vector and expressed as VHH‐hFc chimeric antibodies. The purified antibodies were identified and used to develop a lateral flow immunoassay (LFA) test strip using latex microspheres (LM) for the rapid and visual detection of Y. pestis infection. Conclusions: These data demonstrate the great potential of the semi‐synthetic library for use in isolation of antigen‐specific nanobodies and the isolated specific VHHs can be used in antigen‐capture immunoassays. [ABSTRACT FROM AUTHOR]

Neoantigen delivery using extracellular vesicles (EVs) has gained extensive interest in recent years. EVs derived from tumour cells or immune cells have been used to deliver tumour antigens or antitumor stimulation signals. However, potential DNA contamination from the host cell and the cost of large‐scale EV production hinder their therapeutic applications in clinical settings. Here, we develop an antigen delivery platform for cancer vaccines from red blood cell‐derived EVs (RBCEVs) targeting splenic DEC‐205+ dendritic cells (DCs) to boost the antitumor effect. By loading ovalbumin (OVA) protein onto RBCEVs and delivering the protein to DCs, we were able to stimulate and present antigenic OVA peptide onto major histocompatibility complex (MHC) class I, subsequently priming activated antigen‐reactive T cells. Importantly, targeted delivery of OVA using RBCEVs engineered with anti‐DEC‐205 antibody robustly enhanced antigen presentation of DCs and T cell activation. This platform is potentially useful for producing personalised cancer vaccines in clinical settings. [ABSTRACT FROM AUTHOR]

Nanoparticle‐based electrochemical immunosensors are fascinating and promising screening systems that rely on the affinity binding between antibodies and antigens, allowing the specific detection of diverse types of targets (e. g., proteins, hormones, antibodies). The employment of nanoparticles in the realization of such devices dramatically improves the selectivity, sensitivity, and the transducer surface area for effectively immobilizing the bioreceptor or target molecules. During the last years, the nanoparticles design and fabrication was mainly focused on taking advantage from synergistic effects due to the coupling of different materials. In this review, we summarize the very recent advancements in this regard, with specific emphasis to nanoparticle syntheses procedures, antibody oriented immobilization strategies and transduction techniques. Moreover, the last trends and applications have been extensively discussed along with the remaining challenges and future perspectives. [ABSTRACT FROM AUTHOR]

Aim: An immunoproteomic approach was followed to identify immunoreactive antigens of fish ectoparasite, Argulus siamensis with rohu (Labeo rohita) immune sera for screening of potential vaccine candidates. Materials and results: The whole adult Argulus antigen was run in 2D electrophoresis with IEF in 7 cm IPG strips of pH 4‐7 and SDS‐PAGE with 12% acrylamide concentration. Two parallel gels were run; one was stained with silver stain, and the other was Western blotted to nitrocellulose paper (NCP) and reacted with rohu anti‐A siamensis sera. Fourteen protein spots corresponding to the spots developed in NCP were picked from the silver‐stained gel and subjected to mass spectrometry in MALDI‐TOF/TOF. The MS/MS spectra were analysed in MASCOT software with taxonomy 'other metazoa' and the proteins identified based on similarity with the proteins from heterologous species. The gene ontology analysis revealed a majority of proteins being involved in binding activity in 'molecular function' and belonging to metabolic processes in 'biologic process' categories. The possibility of these proteins as vaccine candidates against Asiamensis is discussed in the paper. Conclusion: Three of the identified proteins namely, bromodomain‐containing protein, anaphase‐promoting complex subunit 5 and elongation factor‐2 could possibly serve as vaccine candidates against argulosis in carps. [ABSTRACT FROM AUTHOR]

Cancer immunotherapies have improved human health, and one among the important technologies for cancer immunotherapy is cancer vaccine. Antigens are the most important components in cancer vaccines. Generally, antigens in cancer vaccines can be divided into two categories: pre‐defined antigens and unidentified antigens. Although, cancer vaccines loaded with predefined antigens are commonly used, cancer vaccine loaded with mixed unidentified antigens, especially whole cancer cells or cancer cell lysates, is a very promising approach, and such vaccine can obviate some limitations in cancer vaccines. Their advantages include, but are not limited to, the inclusion of pan‐spectra (all or most kinds of) antigens, inducing pan‐clones specific T cells, and overcoming the heterogeneity of cancer cells. In this review, the recent advances in cancer vaccines based on whole‐tumor antigens, either based on whole cancer cells or whole cancer cell lysates, are summarized. In terms of whole cancer cell lysates, the focus is on applying whole water‐soluble cell lysates as antigens. Recently, utilizing the whole cancer cell lysates as antigens in cancer vaccines has become feasible. Considering that pre‐determined antigen‐based cancer vaccines (mainly peptide‐based or mRNA‐based) have various limitations, developing cancer vaccines based on whole‐tumor antigens is a promising alternative. [ABSTRACT FROM AUTHOR]

Several technical limitations of Luminex single antigen (LSA) assays have been described so far. This study focused on a reactivity pattern observed in many sera that cannot be explained by eplets described in the Epitope Registry database and sometimes appearing against a self‐HLA allele or antigen. In most cases, this pattern is revealed by a discrepant result when compared with other assays (Luminex PRA, cell‐binding assays such as flow cytometry cross match, LSA from another manufacturer...). We focus here on the Cw1/12/15 pattern appearing on the LABScreen class I LSA provided by One Lambda. We documented its behavior using this LSA after acid denaturation of the beads, using Lifecodes LSA from Immucor, and adsorption of sera either on spleen mononuclear cells from deceased donors or on single HLA transfected cell clones. We studied 33 sera from different patients positive for the three Cw beads, selected from our routine patients' LSA database. Nine patients had transplants from a Cw12 or Cw15 donor without any pejorative evolution of the graft, nor post‐transplant MFI (mean fluorescence intensity) increase of the Cw1/12/15 beads. A significant increase of MFI was observed after acid denaturation of the LABScreen beads. All sera tested by Lifecodes LSA were negative for these Cw beads. Finally, we found no significant difference of MFI after adsorption on cells from either origin. Therefore, the Cw1/12/15 pattern appears to be a false positive reactivity of the LABScreen single antigen assay. [ABSTRACT FROM AUTHOR]

Objective: Simultaneous antibody testing during screening for autoimmune conditions is discouraged. The incidence of positive extractable nuclear antigen (ENA) in the setting of a negative antinuclear antibody (ANA) has been reported as low. Our objective was to characterize the frequency of diagnosis of new ANA‐associated rheumatic disease (AARD) in the setting of a negative ANA with a positive ENA. Methods: This was a 7‐year retrospective study from a multicenter tertiary health network in Australia. Clinical information was sought on patients over 18 years old who had a negative ANA but positive ENA test result. Results were extracted from hospital computer systems. Results: From March 19, 2011, to July 23, 2018, ENA testing was ordered simultaneously with an ANA test on 4,248 occasions in 3,484 patients. ANA was positive in 2,520 patients (59.3%) and ENA was positive in 1,980 patients (46.6%). Among positive ANA patients, ENA was positive in 1,563 patients (62.0%). Among 1,728 negative ANA tests, ENA was positive in 417 (24.1%) (P < 0.001). A total of 328 patients with discordant ANA/ENA results had data available for further analysis, of whom 279 had no pre‐established rheumatologic condition. A new AARD was diagnosed in 17 of 279 patients, yielding a positive predictive value of 6.09% (95% confidence interval 3.59–9.58). Conclusion: Despite the higher‐than‐expected incidence of positive ENA in the setting of a negative ANA, the yield of newly diagnosed rheumatic diseases was low. Our findings support the stepwise addition of ENA requests when an ANA test result is positive and clinical suspicion of an AARD is high. [ABSTRACT FROM AUTHOR]

The aim of this document was to provide a critical review of the current knowledge on hypersensitivity pneumonitis caused by the occupational environment and to propose practical guidance for the diagnosis and management of this condition. Occupational hypersensitivity pneumonitis ( OHP) is an immunologic lung disease resulting from lymphocytic and frequently granulomatous inflammation of the peripheral airways, alveoli, and surrounding interstitial tissue which develops as the result of a non-IgE-mediated allergic reaction to a variety of organic materials or low molecular weight agents that are present in the workplace. The offending agents can be classified into six broad categories that include bacteria, fungi, animal proteins, plant proteins, low molecular weight chemicals, and metals. The diagnosis of OHP requires a multidisciplinary approach and relies on a combination of diagnostic tests to ascertain the work relatedness of the disease. Both the clinical and the occupational history are keys to the diagnosis and often will lead to the initial suspicion. Diagnostic criteria adapted to OHP are proposed. The cornerstone of treatment is early removal from exposure to the eliciting antigen, although the disease may show an adverse outcome even after avoidance of exposure to the causal agent. [ABSTRACT FROM AUTHOR]

Background: Cellular therapies using engineered T cells, haploidentical transplants, and autologous gene therapy are increasing. Specified CD3+ or high CD34+ doses are typically required for subsequent manufacturing, manipulation, or CD34+ selection. Simple, practical, and reliable lymphocyte and hematopoietic progenitor cell (HPC) collection algorithms accounting for subsequent CD34+ selection have not been published.Study Design and Methods: In this analysis of 15 haploidentical donors undergoing tandem lymphocyte and HPC collections, we validated one-step, practical prediction algorithms (Appendix S1, available as supporting information in the online version of this paper) that use conservative facility-specific collection efficiencies, CD34+ selection efficiency, and donor-specific peripheral counts to reliably achieve the target CD3+ and CD34+ product doses. These algorithms expand on our previously published work regarding predictive HPC collection algorithms.Results: Ninety-three percent of lymphocyte and 93% of CD34+ collections achieved the final target CD3+ and CD34+ product dose when our algorithm-calculated process volumes were used. Linear regression analysis of our algorithms for CD3+, preselection CD34+, and postselection CD34+ showed statistically significant models with R2 of 0.80 (root mean square error [RMSE], 31.3), 0.72 (RMSE, 385.7), and 0.56 (RMSE, 326.0), respectively, all with p values less than 0.001.Conclusion: Because achievement of CD3+ or CD34+ dose targets may be critical for safety and efficacy of cell therapies, these simple, practical, and reliable prediction algorithms for lymphocyte and HPC collections should be very useful for collection facilities. [ABSTRACT FROM AUTHOR]

People are expected to have been previously vaccinated with a Vaccinia‐based vaccine, as until 1980 smallpox vaccination was a standard protocol in China. It is unclear whether people with smallpox vaccine still have antibody against vaccinia virus (VACV) and cross‐antibody against monkeypox virus (MPXV). Herein, we assessed the binding antibodies with antigen of VACV‐A33 and MPXV‐A35 in the general population and HIV‐1 infected patients. Firstly, we detected VACV antibody with A33 protein to evaluate the efficiency of smallpox vaccination. The result show that 29% (23 of 79) of hospital staff (age ≥ 42 years) and 63% (60 of 95) of HIV‐positive patients (age ≥ 42 years) from Guangzhou Eighth People's Hospital were able to bind A33. However, among the subjects below 42 years of age, 1.5% (3/198) of the hospital volunteer samples and 1% (1/104) of the samples from HIV patients were positive for antibodies against A33 antigen. Then, we assessed the specific cross‐reactive antibodies against MPXV A35 protein. 24% (19 of 79) hospital staff (age〉 = 42 years) and 44% (42 of 95) of HIV‐positive patients (age〉 = 42 years) were positive. 98% (194/198) of the hospital staff and 99% (103/104) of the HIV patients had no A35‐binding antibodies. Further, we found significant sex differences for the reactivity to A35 antigen were observed in HIV population, but no significant sex differences in hospital staff. Further, we analyzed the positivity rate of anti‐A35 antibody of men who have sex with men (MSM) and non‐MSM in HIV patients (age〉 = 42years). We found that 47% of no‐MSM population and 40% of MSM population were positive for A35 antigen, with no significant difference. Lastly, we found only 59 samples were positive for anti‐A33 IgG and anti‐A35 IgG in all participants. Together, we demonstrated A33 and A35 antigens binding antibodies were detected in HIV patients and general population who were older than 42 years, and cohort studies only provided data of serological detection to support early response to monkeypox outbreak. [ABSTRACT FROM AUTHOR]

Objective: Proteinase 3 (PR3) is the major antigen for antineutrophil cytoplasmic antibodies (ANCAs) in the systemic autoimmune vasculitis, granulomatosis with polyangiitis (GPA). PR3‐targeting ANCAs (PR3‐ANCAs) recognize different epitopes on PR3. This study was undertaken to study the effect of mutations on PR3 antigenicity. Methods: The recombinant PR3 variants, iPR3 (clinically used to detect PR3‐ANCAs) and iHm5 (containing 3 point mutations in epitopes 1 and 5 generated for epitope mapping studies) immunoassays and serum samples from patients enrolled in ANCA‐associated vasculitis (AAV) trials were used to screen for differential PR3‐ANCA binding. A patient‐derived monoclonal ANCA 518 (moANCA518) that selectively binds to iHm5 within the mutation‐free epitope 3 and is distant from the point mutations of iHm5 was used as a gauge for remote epitope activation. Selective binding was determined using inhibition experiments. Results: Rather than reduced binding of PR3‐ANCAs to iHm5, we found substantially increased binding of the majority of PR3‐ANCAs to iHm5 compared to iPR3. This differential binding of PR3‐ANCA to iHm5 is similar to the selective moANCA518 binding to iHm5. Binding of iPR3 to monoclonal antibody MCPR3‐2 also induced recognition by moANCA518. Conclusion: The preferential binding of PR3‐ANCAs from patients, such as the selective binding of moANCA518 to iHm5, is conferred by increased antigenicity of epitope 3 on iHm5. This can also be induced on iPR3 when captured by monoclonal antibody MCPR2. This previously unrecognized characteristic of PR3‐ANCA interactions with its target antigen has implications for studying antibody‐mediated autoimmune diseases, understanding variable performance characteristics of immunoassays, and design of potential novel treatment approaches. [ABSTRACT FROM AUTHOR]

High‐frequency mutation of the TP53 tumor suppressor gene is observed in multiple human cancers, which promotes cancer progression. However, the mutated gene‐encoded protein may serve as a tumor antigen to elicit tumor‐specific immune responses. In this study, we detected widespread expression of shared TP53‐Y220C neoantigen in hepatocellular carcinoma with low affinity and low stability of binding to HLA‐A0201 molecules. We substituted the amino acid sequences VVPCEPPEV with VLPCEPPEV in the TP53‐Y220C neoantigen to yield a TP53‐Y220C (L2) neoantigen. This altered neoantigen was found to increase affinity and stability and induce more cytotoxic T lymphocytes (CTLs), indicating improvements in immunogenicity. In vitro assays showed the cytotoxicity of CTLs stimulated by both TP53‐Y220C and TP53‐Y220C (L2) neoantigens against multiple HLA‐A0201‐positive cancer cells expressing TP53‐Y220C neoantigens; however, the TP53‐Y220C (L2) neoantigen showed higher cytotoxicity than the TP53‐Y220C neoantigen against cancer cells. More importantly, in vivo assays demonstrated greater inhibition of hepatocellular carcinoma cell proliferation by TP53‐Y220C (L2) neoantigen‐specific CTLs relative to TP53‐Y220C neoantigen in zebrafish and nonobese diabetic/severe combined immune deficiency mouse models. The results of this study demonstrate enhanced immunogenicity of the shared TP53‐Y220C (L2) neoantigen, which has the potential as dendritic cells or peptide vaccines for multiple cancers. [ABSTRACT FROM AUTHOR]

Lung transplant (LTX) patients are at high risk of cytomegalovirus (CMV) infection, which is often associated with high mortality and morbidity. Reactivation of CMV causes cell injury due to the cytopathic effect of viral replication and triggering of T cell immunity. The aim of this study was to compare expression of immune checkpoints (ICs) (PD‐1, CTLA‐4, LAG‐3 and TIGIT) in CD4, CD8 and CD56 and activation markers CD137, CD154 and CD69 of end‐stage patients awaiting lung transplant. Eighteen pre‐LTX positive for anti‐CMV IgG titres and 18 healthy subjects were enrolled. IC and activation markers have been evaluated through flow cytometric analysis in HC and pre‐LTX patients. Reactive (QF+) and unreactive (QF−) patients were stratified according to QuantiFERON‐CMV assays. ICs' and activation markers' expression were determined before and after in vitro stimulation with pp‐65 and IE‐1 antigens. Lower expression of PD‐1 was observed in CD4 and CD8 cells of pre‐LTX patients than controls, whereas CTLA4 appeared upregulated in CD56 and CD8 cells. TIGIT is increased on the surface of CD4, CD8 and NK cells after peptide stimulation in QF‐negative patients and PD‐1 is only downregulated after stimulation in the QF‐positive patients. This study provides new evidence of immune dysregulation in patients with end‐stage lung disorders, particularly in relation to immune checkpoint cell biology. The change in QF+ mostly happens on cytotoxic cells NK and CD8, while the changes in QF− were observed in adaptive immune cells, including CD4 and CD8. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Clinical Periodontology is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Materials‐based antigen delivery systems can augment the immune response by improving antigen uptake in antigen‐presenting cells, targeting lymph nodes, prolonging antigen exposure, enhancing cross‐presentation, etc. Recent research revealed that some antigen carriers activate the innate immune pathways without additional adjuvant. Here, a vaccine delivery platform (antigen nanocapsules) constructed by a one‐step in situ polymerization is reported, weaving a biodegradable polymer network around the antigen surface. This simple technology allowed us to study the immunomodulatory effect of various antigen carriers. An antigen nanocapsule (NC7) capable of inducing dendritic cell activation and cross‐presentation is identified. Further mechanistic studies revealed that NC7 activated the cGAS‐STING pathway in a cGAS‐dependent manner. Moreover, the subcutaneously injected NC7 accumulated in the lymph nodes and elicited strong cytotoxic T cell immunity and T cell memory against established cancer. Collectively, the often‐neglected immunomodulatory effect of various cationic antigen carriers, enabling potential application in cancer vaccines, is uncovered. [ABSTRACT FROM AUTHOR]

Background and Objectives: Current manual and automated phenotyping methods are based on visual detection of the antigen–antibody interaction. This approach has several limitations including the use of large volumes of patient and reagent red blood cells (RBCs) and antisera to produce a visually detectable reaction. We sought to determine whether the flow cytometry could be developed and validated to perform RBC phenotyping to enable a high‐throughput method of phenotyping using comparatively miniscule reagent volumes via fluorescence‐based detection of antibody binding. Materials and Methods: RBC phenotyping by flow cytometry was performed using monoclonal direct typing antisera (human IgM): anti‐C, ‐E, ‐c, ‐e, ‐K, ‐Jka, ‐Jkb and indirect typing antisera (human IgG): anti‐k, ‐Fya, ‐Fyb, ‐S, ‐s that are commercially available and currently utilized in our blood transfusion services (BTS) for agglutination‐based phenotyping assays. Results: Seventy samples were tested using both flow‐cytometry‐based‐phenotyping and a manual tube standard agglutination assay. For all the antigens tested, 100% concordance was achieved. The flow‐cytometry‐based method used minimal reagent volume (0.5–1 μl per antigen) compared with the volumes required for manual tube standard agglutination (50 μl per antigen) Conclusion: This study demonstrates the successful validation of flow‐cytometry‐based RBC phenotyping. Flow cytometry offers many benefits compared to common conventional RBC phenotyping methods including high degrees of automation, quantitative assessment with automated interpretation of results and extremely low volumes of reagents. This method could be used for high‐throughput, low‐cost phenotyping for both blood suppliers and hospital BTS. [ABSTRACT FROM AUTHOR]

Background and Objectives: A group AB D‐positive child presented 1 year after haematopoietic stem cell transplant (HSCT) from a group O D‐negative donor as group A D‐negative. Engraftment remained at 100% in white cell lineages. The reason for the unusual result was explored, and the scarcely reported phenomenon of adsorption of secreted antigen was considered. This study also investigated the prevalence of secreted antigen adsorbed onto donor‐derived group O red blood cells (RBCs) in children after HSCT and defined a process for laboratory management. Materials and Methods: Retrospective data analysis of HSCTs carried out over 19 months at Great Ormond Street Hospital was conducted to identify cases of adsorbed A antigen after HSCT. Investigation of RBC reactions with different clones of anti‐A and in vitro experiments was performed to recreate adsorption. Results: Nineteen A to O HSCTs were conducted over 19 months, of which six (31%) displayed weak A antigen on RBCs despite full myeloid engraftment. Negative reactions with anti‐A were obtained when run on an alternative clone. Laboratory protocols for the future management of these cases have been developed. Conclusion: Passive adsorption of secreted antigen is responsible for these results and is more widespread than previously reported, as a third of A to O HSCTs at our centre demonstrated this phenomenon. A process has been implemented into the laboratory to manage this cohort, ensuring component groups compatible with both donor and recipient are given, and the shared care centres are aware of these requirements. [ABSTRACT FROM AUTHOR]

The human cytomegalovirus (HCMV) is an asymptomatic common virus that is typically harmless, but in some cases, it can be life threatening. Thus, there is an urgent need to develop novel diagnostic methods and strengthen the efforts to combat this virus. A microcantilever‐based biosensor functionalised with the UL83‐antibody of HCMV (UL83‐HCMV antibody) has been developed to detect the UL83‐antigen of HCMV (UL83‐HCMV antigen) at different concentrations ranging from 0.3 to 300 ng/ml. The response of the biosensor to the presence of UL83‐HCMV antigen was measured through the shift in resonance frequency before and after antigen–antibody binding. The system shows a low detection limit of 84 pg/ml, which is comparable to traditional sensors, and a detection time of less than 15 min was achieved. The selectivity of the sensor was demonstrated using three different proteins with and without the UL83‐HCMV antigen. The biosensor shows high selectivity for the UL83‐HCMV antigen. Mass loading by the UL83‐HCMV antigen was roughly estimated with a sensitivity of ∼30 fg/Hz. This technique is crucial for the fabrication of portable and low‐cost biosensors that can be used in real‐time monitoring and enables early medical diagnosis. [ABSTRACT FROM AUTHOR]

Recent clinical studies show activating multiple innate immune pathways drives robust responses in infection and cancer. Biomaterials offer useful features to deliver multiple cargos, but add translational complexity and intrinsic immune signatures that complicate rational design. Here a modular adjuvant platform is created using self‐assembly to build nanostructured capsules comprised entirely of antigens and multiple classes of toll‐like receptor agonists (TLRas). These assemblies sequester TLR to endolysosomes, allowing programmable control over the relative signaling levels transduced through these receptors. Strikingly, this combinatorial control of innate signaling can generate divergent antigen‐specific responses against a particular antigen. These assemblies drive reorganization of lymph node stroma to a pro‐immune microenvironment, expanding antigen‐specific T cells. Excitingly, assemblies built from antigen and multiple TLRas enhance T cell function and antitumor efficacy compared to ad‐mixed formulations or capsules with a single TLRa. Finally, capsules built from a clinically relevant human melanoma antigen and up to three TLRa classes enable simultaneous control of signal transduction across each pathway. This creates a facile adjuvant design platform to tailor signaling for vaccines and immunotherapies without using carrier components. The modular nature supports precision juxtaposition of antigen with agonists relevant for several innate receptor families, such as toll, STING, NOD, and RIG. [ABSTRACT FROM AUTHOR]

Human Leucocyte Antigen (HLA) genes and molecules have an important role in transplantation, aetiology of many autoimmune, non-autoimmune and infection diseases. Due to the extremely high polymorphism of HLA genes and their different frequency distributions in various populations, an increasing probability of HLA non-compatible blood products, tissues or organs usage exists. For that reason, the aim of this paper was to give a concise overview of the role of HLA antigens and antibodies in adverse reactions caused by administration of transfusion products. The HLA system can cause detrimental immune reactions in transfusion therapy (platelet immune refractoriness, febrile transfusion reaction, transfusion-related acute lung injury and transfusion-associated graft versus host disease). Anti-HLA antibodies present in the patient are responsible for some of these reactions, while anti-HLA antibodies or HLA reactive cells present in the transfused product are accountable for immune-reactivity in other cases. In order to avoid or reduce the development of these transfusion-related events, anti-HLA antibody-negative or compatible products should be used. This is increasingly facilitated by introduction of more sensitive and specific techniques to determine anti-HLA antibodies and gene polymorphisms. In conclusion, the most common adverse reactions related to administration of incompatible HLA transfusion products are discussed. Basic information about HLA genes and antibodies as well as methods for their detection is also provided in order to give sufficient data for safe and efficient administration of transfusion products. [ABSTRACT FROM AUTHOR]

Novel antibodies have been generated by immunizing with bacterially expressed fragments of the repetitive motif of the Ki-67 gene. One such antibody, MIB1, recognizes a fixation and embedding resistant epitope on the Ki-67 protein if sections are previously microwaved in a citrate buffer. We have investigated the utility of this antibody as a marker of cell proliferation in archival material. The microwave technique is simple but requires careful monitoring since different tissues and fixatives require different irradiation times. Strong nuclear immunoreactivity was detected with all fixatives studied. Cytoplasmic staining was not identified. In a wide range of normal tissues the distribution and number of MIB1 immunoreactive cells matched that of cryostat sections stained with Ki-67. In nude mouse xenografts in which the growth fraction had been defined using a fraction of labelled mitosis method, the labelling index with MIB1 matched that previously determined for Ki-67 and correlated well with the growth fraction. Other markers of proliferation (e.g. proliferating cell nulcear antigen) have been shown to be expressed in DNA repair, thus we investigated expression of MIB1 immunoreactivity in situations of DNA repair in vivo—ultraviolet irradiated human skin. MIB1 staining correlated with semi-conservative DNA synthesis rather than excision repair DNA synthesis. Finally, the morphological and cell cycle distribution of MIB1 expression is identical to that of Ki-67. Thus, MIB1 represents a new anti-Ki-67 antibody which appears to be a robust marker of cell proliferation easily applicable to archival material. [ABSTRACT FROM AUTHOR]

PCNA is a nuclear protein that is synthesized in late G[sub 1] and S phases of the cell cycle and is, therefore, correlated with the cell proliferative state. A new monoclonal antibody (PC10) to genetically engineered PCNA has been shown to label proliferating cells in formalin-fixed paraffin-embedded normal human tissues. Previous studies in lymphomas, using various markers of cell proliferation, have shown a strong correlation between indices of cell proliferation and histological grade. These studies have shown that within each histological subtype there is often a wide range of proliferative indices and that these may be of some prognostic significance. Thirty-one gastrointestinal lymphomas were studied. Our results show that there is a good correlation between PC10 index and histological grade of tumour (0.01 > P > 0.001 ) and also a significant relationship between PC 10 index and S + G[sub 2] + M phase fraction as measured by flow cytometric analysis (r² = 0.62; P< 0.01). Twenty-three cases were available for survival analysis. In these cases a high PC10 score correlated with poor survival (P=0.04). Based on this series, it appears that there is a significant relationship between PC 10 index and histological grade, and between PC 10 index and S + G[sub 2] + M phase as measured by flow cytometric analysis. In addition, our results suggest that a high PC10 index is an adverse prognostic factor in primary gastrointestinal lymphoma. [ABSTRACT FROM AUTHOR]

Anthrax infection is associated with severe illness and high mortality. Protective antigen (PA) is the central component of the anthrax toxin, which is one of two major virulence factors of Bacillus anthracis, the causative agent of anthrax disease. Upon endocytosis, PA opens a pore in the membranes of endosomes, through which the cytotoxic enzymes of the toxin are extruded. The PA pore is formed by a cooperative conformational change in which the membrane‐penetrating loops of PA associate, forming a hydrophobic rim that pierces the membrane. Due to its crucial role in anthrax progression, PA is an important target for monoclonal antibody‐based therapy. cAb29 is a highly effective neutralizing antibody against PA. Here, the cryo‐EM structure of PA in complex with the Fab portion of cAb29 was determined. It was found that cAb29 neutralizes the toxin by clamping the membrane‐penetrating loop of PA to the static surface‐exposed loop of the D3 domain of the same subunit, thereby preventing pore formation. These results provide the structural basis for the antibody‐based neutralization of PA and bring into focus the membrane‐penetrating loop of PA as a target for the development of better anti‐anthrax vaccines. [ABSTRACT FROM AUTHOR]

IgE, the key molecule in atopy has been shown to bind two receptors, FcεRI, the high‐affinity receptor, and FcεRII (CD23), binding IgE with lower affinity. Whereas cross‐linking of IgE on FcεRI expressed by mast cells and basophils triggers the allergic reaction, binding of IgE to CD23 on B cells plays an important role in both IgE regulation and presentation. Furthermore, IgE‐immune complexes (IgE‐ICs) bound by B cells enhance antibody and T cell responses in mice and humans. However, the mechanisms that regulate the targeting of the two receptors and the respective function of the two pathways in inflammation or homeostasis are still a matter of debate. Here, we focus on CD23 and discuss several mechanisms related to IgE binding, as well as the impact of the IgE/antigen‐binding on different immune cells expressing CD23. One recent paper has shown that free IgE preferentially binds to FcεRI whereas IgE‐ICs are preferentially captured by CD23. Binding of IgE‐ICs to CD23 on B cells can, on one hand, regulate serum IgE and prevent effector cell activation and on the other hand facilitate antigen presentation by delivering the antigen to dendritic cells. These data argue for a multifunctional role of CD23 for modulating IgE serum levels and immune responses. [ABSTRACT FROM AUTHOR]

Grass pollen allergens are shown to remain associated with protein material and a yellow pigment during paper chromatography and during dialyses and ultrafiltrations of various types. Dialysable* allergens comprise only a fraction of I per cent of the total activity and the amount of activity extractable by diethylene glycol (DEG) and similar solvents is of the same order. Besides the allergens, the DEG and aqueous extracts contain large amounts of inositol, glucose and fructose, also some yellow pigments and phosphates. Larger amounts of free and combined amino acids are found in the aqueous than in the DEG extracts, but the reverse is true for sucrose. In addition the DEG extracts contain a yellow glucoside different from the dactylen of the aqueous extracts, a glucosan and an arabinose-galactose-pigment complex, only the latter being associated with any activity. The spontaneous release of the crystalline dactylen from originally clear aqueous pollen extracts is found not to be caused by enzymes. The washed crystals are found to be chromatographically and electrophoretically homogeneous and devoid of allergenic activity. [ABSTRACT FROM AUTHOR]

Hyaluronidase (E.C. 3.2.1.35), one of the three major allergens of yellow jacket venom, is a glycoprotein of 45 kDa that is largely responsible for the cross-reactivity of wasp and bee venoms with sera of allergic patients. The asparagine-linked carbohydrate often appears to constitute the common IgE-binding determinant. Using a combination of MALDI MS and HPLC of 2-aminopyridine-labelled glycans, we found core-difucosylated paucimannosidic glycans to be the major species in the 43–45 kDa band of Vespula vulgaris and also in the corresponding bands of venoms from five other wasp species ( V. germanica, V. maculifrons, V. pensylvanica, V. flavopilosa and V. squamosa). Concomitant peptide mapping of the V. vulgaris 43 kDa band identified the known hyaluronidase, Ves v 2 (SwissProt P49370), but only as a minor component. De novo sequencing by tandem MS revealed the predominating peptides to resemble a different, yet homologous, sequence. cDNA cloning retrieved a sequence with 58 and 59% homology to the previously known isoform and to the Dolichovespula maculata and Polistes annularis hyaluronidases. Close homologues of this new, putative hyaluronidase b (Ves v 2b) were also the major isoform in the other wasp venoms. [ABSTRACT FROM AUTHOR]

Thirty patients with haematological malignancies received peripheral blood stem cells from human leucocyte antigen (HLA)-identical sibling donors after non-myeloablative conditioning with fludarabine and total body irradiation. Twenty-seven patients received the transplant as an outpatient procedure. All patients engrafted. The probability of acute graft- versus-host disease (GVHD) grades II–IV and extensive chronic GVHD was 57% and 80%, respectively. Patients alive on day +365 experienced a median of 44 d (range 4–151) of hospitalization during the first year. In the entire cohort, GVHD accounted for 22%, infections for 18%, thrombotic thrombocytopenic purpura (TTP) for 16% and engraftment syndrome for 14% of the time in hospital. The 1-year risk of TTP was 26%. Acute GVHD was a risk factor for the development of TTP ( P = 0·008). With a median follow-up of 602 d, the 2-year estimates for overall survival, progression-free survival, non-relapse mortality and relapse related mortality were 68%, 43%, 22% and 13%, respectively. This transplantation regimen is feasible and induces long-term remissions in heavily pretreated patients. The procedure can be performed in the outpatient setting, but complications could result in a substantial number of admissions during the first year. [ABSTRACT FROM AUTHOR]

Without transplantation from a human leucocyte antigen-identical family donor, refractory severe aplastic anaemia (SAA) has an unfavourable prognosis. Conventional transplantation from a matched unrelated donor carries a high rate of mortality. We transplanted large numbers of highly purified CD34+ cells from matched unrelated ( n = 4), mismatched unrelated ( n = 4) and mismatched related ( n = 1) donors into nine children with refractory SAA. The grafts consisted of granulocyte colony-stimulating factor-mobilized peripheral positively selected CD34+ cells. A median of 15·1 × 106/kg CD34+ stem cells and 11 × 103/kg CD3+ T-lymphocytes were infused. No additional pharmacological graft versus host disease (GVHD) prophylaxis was given. At a median follow-up of 47 (range 37–72) months, eight patients (89%) were in complete remission with >90% donor chimaerism and no evidence of GVHD. One patient died on day +238 as a consequence of GVHD. The use of highly purified mobilized CD34+ stem cells warrants further clinical exploration in children with refractory SAA. [ABSTRACT FROM AUTHOR]

Recent studies have shown that human myeloid leukaemia cells can differentiate into dendritic cell (DC)-like cells (leukaemia-DCs) when cultured with a combination of cytokines. In the present study, we examined whether the transduction of leukaemia-DCs with OX40 ligand (OX40L), a member of the tumour necrosis factor (TNF) family, resulted in augmentation of their antigen presenting activity. Bicistronic retroviral vectors expressing both human OX40L and enhanced green fluorescent protein (EGFP) or EGFP alone were generated and used for transduction. Fresh leukaemic cells from five patients with acute myeloid leukaemia (AML) were isolated and retrovirally transduced with OX40L during the culture with a combination of cytokines from stem cell factor, fms-like tyrosine kinase (Flt)-3 ligand, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and TNF- α. After 7 d, the majority of cells showed DC-like morphology, and expressed higher levels of CD80, CD86 and HLA-DR than fresh leukaemic cells. The transduction efficiency was 8·5–27·2%. Leukaemia-DCs transduced with OX40L elicited higher proliferative response of allogeneic CD4+ T cells than fresh leukaemic cells, non-transduced, or mock-transduced leukaemia-DCs. Co-culture of allogeneic CD4+ T cells with OX40L-transduced leukaemia-DCs was superior in the generation of interferon (IFN)- γ producing CD4+ T cells and in production of IFN- γ. Furthermore, OX40L-transduced leukaemia-DCs could elicit significant proliferative response of human leucocyte antigen-matched T cells from the donor in allogeneic stem cell transplantation. These results indicate that retroviral transduction of leukaemia-DCs with OX40L augments their antigen presenting cell activity and thus renders them more suitable for tumour vaccines or ex vivo stimulation of leukaemia-specific T cells. [ABSTRACT FROM AUTHOR]

The 48 G>C transversion in exon 1 of the RHCE gene leads to Trp16Cys, usually present in the conventional RHCE Ce, while Trp16 is associated with RHCE ce. The presence of Cys16 in RHCE ce is associated with the R0 (Dce) haplotype in Africans, leading to a weak ‘e’ antigen expression on red blood cells (RBCs). VS is a common red cell antigen in individuals of African descent and results from a single point mutation in exon 5 of the RHCE (733C>G), leading to Leu245Val substitution; VS positivity is also associated with weak expression of ‘e’. This study investigated the association of Cys16 and/or VS with the RHCE ce alleles in a cohort of sickle cell disease (SCD) patients phenotyped as R0r or R0R0 and rr. DNA samples from 58 SCD patients were tested for the 48 G>C transversion, encoding Cys16, by allele-specific polymerase chain reaction (PCR). We also amplified exon 5 of the RHCE by PCR and subjected the amplified product to restriction fragment length polymorphism analysis, using BfaI, in order to determine the VS status. Further cDNA analysis was performed on three samples to verify whether the mutations were located on the same or on different alleles. Fifty-six of the 58 SCD patients studied (97%) were heterozygous for 48G/48C (Cys16). Of these, 18 (32%) were also heterozygous for 733C/G (245Val). All of these 18 samples showed weak ‘e’ expression on RBCs when tested with at least one monoclonal antibody to e antigen. cDNA sequencing of three of 18 patient samples showed that the genes encoding Cys16 and Val245 (VS) were on different alleles. We found a high incidence of Cys16 associated with the RHCE ce in our SCD cohort. A high percentage of these patients were also found to be heterozygous for VS. cDNA analysis showed that, in at least three samples, the two mutations were on different alleles, with consequent weakening of expression of the e antigen on RBCs. [ABSTRACT FROM AUTHOR]

We investigated the molecular basis of factor VII (FVII) deficiency in a Japanese woman who suffered occasional epistaxis. The patient had low levels of both FVII coagulant activity (FVII:C) and antigen (FVII:Ag) (5·0% and 7·3% of normal controls respectively). DNA sequence analysis of the FVII gene showed that the patient was homozygous for a mutation that resulted in a Cys for Gly354 substitution, a novel missense mutation in the catalytic domain. Haplotype analysis showed that this missense mutation was inherited from her consanguineous parents. Transient expression experiments showed that secreted FVII Cys354, FVII:C and FVII:Ag levels in conditioned media were reduced to 4% and 5%, respectively, of levels secreted from wild-type FVII. However, the intracellular FVII Cys354 was 67% that of normal recombinant protein. Immunohistochemical analysis showed that intracellular FVII:Ag from FVII 354Cys was present diffusely throughout the cytoplasm. Substitution of FVII 354Gly with amino acids other than Cys (Arg, Asp, Ser and Phe), did not produce a phenotype similar to FVII Cys354Gly. Molecular modelling indicated that FVII Gly354 was located outside the FVII heavy chain, and that Cys135 in the EGF2 domain, Cys262 in the catalytic domain and Cys127 all exist within 10 Å of Gly354. Therefore, we propose that the introduction of an additional free cysteine residue in the FVII molecule results in the formation of illegitimate disulphide bonds and a mis-folded domain, leading to defective secretion. [ABSTRACT FROM AUTHOR]

Despite a successful outcome of the anti-D prophylaxis programme, alloimmunization still occurs. The aim of this study was to estimate the frequency of Rh-specific B lymphocytes in the peripheral blood of nine Rh-alloimmunized individuals at different time intervals after parturition. The donors’ B lymphocytes were transformed with Epstein–Barr virus (EBV) and cultured at different cell densities over a feeder of human fetal fibroblasts. Culture supernatants were screened for human immunoglobulin by enzyme-linked immunosorbent assay (ELISA) and for anti-Rh antibody by using a direct haemagglutination technique. The percentage of CD19+ B lymphocytes in peripheral blood was determined by flow cytometry, and the frequency of Rh-specific B lymphocytes was estimated by limiting-dilution assay (LDA). The frequency of Rh-specific B lymphocytes varied from 1 : 150 to 1 : 27 850 in different donors. There was a decrease in this frequency and level of anti-Rh antibody with increase in time interval between bleeding and last exposure to the antigen. Furthermore, a positive correlation was observed between the titre of Rh-specific antibody and frequency of Rh-specific B cells in each of three subjects bled at multiple time-points postdelivery. The magnitude of the specific antibody response to Rh antigens varies greatly in Rh-alloimmunized women, which partly reflects the difference in frequency of specific B cells in these individuals. [ABSTRACT FROM AUTHOR]

The aim of the study was to perform fetal RHD genotyping in maternal plasma using a fluorescent polymerase chain reaction (PCR) technique. Duplex PCR, amplifying RHD and SRY in the same tube, was undertaken. The effect of varying storage temperatures on the concentration of fetal DNA was investigated in a separate study involving 10 RhD-negative pregnant women. Primers and probes for the RHD gene's exon 7 and the sex-determining region, Y, were designed, and monoplex and duplex PCR were performed. Blood samples from 10 RhD-negative women were split into four and treated in four different ways before measuring the concentration of fetal DNA by quantitative PCR. DNA extracted from the plasma of 114 RhD-negative pregnant women was tested for the presence of fetal RHD. The discrepancy between genotyping and serological RhD typing of the babies postpartum was 8% when counting one positive replicate as a positive result. Duplex PCR, amplifying RHD and SRY in the same tube, showed a reduced sensitivity for amplification of the SRY gene segment. There was a statistically significant reduction of fetal DNA in blood samples stored at room temperature for 48 h compared with the same sample stored at a temperature of < 10 °C for the same length of time. This method is not suitable for routine analysis because of the lack of a positive control for RHD-negative female fetuses and a decrease in PCR sensitivity when performing duplex PCR. Fetal DNA in maternal plasma is better preserved when the blood sample is kept cool. [ABSTRACT FROM AUTHOR]

The antigenic components of adrenal extract were fractionated by chromatography on DEAE-cellulose. Using a stepwise elution procedure, it was found that a component of major interest, termed Pk3b, was eluted by a buffer of pH 6.70 and 0.09 M in phosphate-chloride. This antigenic preparation reacted with several rabbit antisera that had been prepared by isoimmunization with rabbit adrenal homogenate, in contrast to several other fractions, which reacted with only one (or two) of the antisera. Pk3b also showed a high degree of homogeneity, as shown by immunodiffusion, paper electrophoresis, and analytical ultracentrifugation. By the latter technique, an extrapolated sedimentation coefficient of 4.45S was determined. The further comparison of this antigen in immunodiffusion, along with an antiserum and the adrenal extract from the antiserum-providing animal, confirmed again that this is an autoantigen, and that it is one of five autoantigens of the adrenal gland. [ABSTRACT FROM AUTHOR]

The hallmark of DCs is their potent and outstanding capacity to activate naive resting T cells. As such, DCs are the sentinels of the immune system and instrumental for the induction of immune responses. This is one of the reasons, why DCs became the focus of immunotherapeutical strategies to fight infections, cancer, and autoimmunity. Besides the exploration of adoptive DC‐therapy for which DCs are generated from monocytes or purified in large numbers from the blood, alternative approaches were developed such as antigen targeting of DCs. The idea behind this strategy is that DCs resident in patients' lymphoid organs or peripheral tissues can be directly loaded with antigens in situ. The proof of principle came from mouse models; subsequent translational studies confirmed the potential of this therapy. The first clinical trials demonstrated feasibility and the induction of T‐cell immunity in patients. This review will cover: (i) the historical aspects of antigen targeting, (ii) briefly summarize the biology of DCs and the immunological functions upon which this concept rests, (iii) give an overview on attempts to target DC receptors with antibodies or (glycosylated) ligands, and finally, (iv) discuss the translation of antigen targeting into clinical therapy. [ABSTRACT FROM AUTHOR]

Expressing heterologous antigens by plasmids may cause antibiotic resistance. Additionally, antigen expression via plasmids is unstable due to the loss of the plasmid. Here, we developed a balanced‐lethal system. The Listeria monocytogenes (LM) balanced‐lethal system has been previously used as an antigen carrier to induce cellular immune response. However, thus far, there has been no reports on Listeria ivanovii (LI) balanced‐lethal systems. The dal and dat genes from the LI‐attenuated LIΔatcAplcB (LIΔ) were deleted consecutively, resulting in a nutrient‐deficient LIΔdd strain. Subsequently, an antibiotic resistance‐free plasmid carrying the LM dal gene was transformed into the nutrient‐deficient strain to generate the LI balanced‐lethal system LIΔdd:dal. The resultant bacterial strain retains the ability to proliferate in phagocytic cells, as well as the ability to adhere and invade hepatocytes. Its genetic composition was stable, and compared to the parent strain, the balanced‐lethal system was substantially attenuated. In addition, LIΔdd:dal induced specific CD4+/CD8+ T‐cell responses and protected mice against LIΔ challenge. Similarly, we constructed an LM balanced‐lethal system LMΔdd:dal. Sequential immunization with different recombinant Listeria strains will significantly enhance the immunotherapeutic effect. Thus, LIΔdd:dal combined with LMΔdd:dal, or with other balanced‐lethal systems will be more promising alternative for vaccine development. [ABSTRACT FROM AUTHOR]

Summary: Background: Treatment of hepatitis C virus (HCV) infection with direct‐acting antivirals (DAAs) is monitored by assessing plasma HCV‐RNA load. However, detection of HCV core antigen (HCVcAg) may be an alternative. Aim: To evaluate the diagnostic performance of the HCVcAg assay to monitor the efficacy of DAAs in HCV‐infected patients Methods: We performed searches in multiple electronic databases until 6 July 2022, of studies evaluating the HCVcAg detection in plasma or serum compared with the HCV‐RNA test (gold standard). We calculated pooled measurement at 2 and 4 weeks of treatment, and at end‐of‐treatment (EOT), as well as sustained virological response (SVR; 12 weeks after EOT). Results: We selected 16 studies from 2016 to 2022, with 3237 patients and 8958 samples. Overall, the diagnostic performance and clinical utility of the HCVcAg assay were poor at week 2 (sensitivity = 0.40, specificity = 0.96, positive likelihood ratio (PLR) = 9.16, negative likelihood ratio (NLR) = 0.63, and area under the summary receiver operating curve (SROC) = 0.57), fair at week 4 (sensitivity = 0.30, specificity = 0.90, PLR = 3.18, NLR = 0.77, and AUC = 0.79), acceptable at EOT (sensitivity = 0.40, specificity =0.98, PLR = 16.54, NLR = 0.62, and AUC = 0.97) and excellent for SVR (sensitivity = 0.94, specificity = 0.99, PLR = 107.54, NLR = 0.06, and AUC = 0.99). Conclusions: The HCVcAg assay may be helpful for monitoring the efficacy of HCV treatment with DAAs in HCV‐infected patients at EOT and for documenting SVR, but not at weeks 2 and 4 of treatment due to poor diagnostic performance. [ABSTRACT FROM AUTHOR]

Mtb8.4, a secretory T‐cell antigen of Mycobacterium tuberculosis, is important for providing an antigen‐specific immune response. In this study, we showed Mtb8.4 to have both heme‐binding and fibril‐forming properties, using experimental and in silico methods. High absorbance at 410 nm and interaction with hemin‐agarose demonstrated its heme‐binding nature. Titration of Mtb8.4 with heme resulted in 1 : 1 stoichiometry. The heme‐binding pocket in Mtb8.4 was identified by molecular modeling, and binding residues were predicted using molecular docking. The molecular dynamics simulations of apo‐ and heme‐bound Mtb8.4 confirmed that the heme group forms a stable complex. Transmission electron microscopy analyses and dye‐binding assays showed that Mtb8.4 forms fibers. Computational studies predicted that the C‐terminal sequence (93AAQYIGLVESV103) is important for forming fibers. In silico analyses further anticipated the probable epitope (82AMAAQLQAV90) of Mtb8.4. The fiber‐forming properties of Mtb8.4 could be advantageous from a vaccine perspective for aggregate/fibril‐based vaccine delivery or it might influence the epitope presentation of Mtb8.4. [ABSTRACT FROM AUTHOR]

Background: Although the prevalence and mortality of hepatitis is high in the Asia‐Pacific region, few studies are available on the diagnosis, treatment, and cure rates for viral hepatitis among people living with HIV in this area. This study aims to report the cascade of care (CoC) for hepatitis B (HBV) and C (HCV) among people living with HIV receiving combined antiretroviral therapy (ART). Methods: Patients enrolled in the TREAT Asia HIV Observational Database Low Intensity Transfer (TAHOD‐LITE) cohort, on ART, and with follow‐up data from 2010 to 2019 were included. Patients were determined as positive for HCV or HBV co‐infection if they ever tested positive for HCV antibody (anti‐HCV) or HBV surface antigen (HBsAg), respectively. Results: In total, 39% (8612/22 340) of the adult HIV cohort had undergone HBsAg testing, with 8% (672/8612) testing positive. HBV CoC demonstrated that 71% (474/672) of those with HBsAg positive results initiated treatment, 67% (318/474) of those on treatment had HBV DNA testing to evaluate treatment progression, and 18% (58/318) of those tested reached viral suppression. Of the cohort, 37% (8231/22 340) had anti‐HCV testing, of whom 10% (779/8231) tested positive. The HCV CoC showed that 68% (526/779) of those with positive anti‐HCV tests had HCV RNA tests, of whom 51% (267/526) had detectable HCV RNA. Among those with detectable HCV RNA, 65% (174/267) initiated HCV treatment. Of the 40% (69/174) who initiated HCV treatment, 90% (62/69) reached sustained virological response. Conclusion: Our findings identified less frequent testing in the healthcare system and limited access to treatment as gaps in the CoC for viral hepatitis. More routine HCV RNA and HBV DNA testing is required for patients with positive screening tests to identify those in need of treatment. [ABSTRACT FROM AUTHOR]

Noroviruses are major causative agents of nonbacterial acute gastroenteritis in humans. Ten genogroups of noroviruses have been identified to date, among which genogroup I (GI) and genogroup II (GII) noroviruses are major pathogens for humans. GI and GII noroviruses are further classified into nine and 27 genotypes, respectively. Noroviruses are well known to bind to histo‐blood group antigens (HBGAs). Many studies have revealed that virus‐like particles (VLPs) from different genotypes exhibit distinct patterns of HBGA binding, but the assay conditions used in these studies were not identical. To enable comparison of the binding to HBGA of nine GI genotypes, I purified VLPs from insect cells and analysed their HBGA‐binding profiles. Although each genotype exhibited a distinct pattern of HBGA binding, Lewis b antigen was commonly recognized by all of the genogroup I strains, suggesting that this antigen plays a critical role in the pathogenesis of noroviruses. [ABSTRACT FROM AUTHOR]

The ongoing global COVID‐19 pandemic, caused by the SARS‐CoV‐2 virus, has resulted in significant loss of life since December 2019. Timely and precise virus detection has been proven as an effective solution to reduce the spread of the virus and to track the epidemic. Rapid antigen diagnostics has played a significant role in the frontline of COVID‐19 testing because of its convenience, low cost, and high accuracy. Herein, different types of recently innovated in‐lab and commercial antigen diagnostic technologies with emphasis on the strengths and limitations of these technologies including the limit of detection, sensitivity, specificity, affordability, and usability are systematically reviewed. The perspectives of assay development are looked into. [ABSTRACT FROM AUTHOR]

CD4+FOXP3+ Tregs are currently explored to develop cell therapies against immune‐mediated disorders, with an increasing focus on antigen receptor‐engineered Tregs. Deciphering their mode of action is necessary to identify the strengths and limits of this approach. Here, we addressed this issue in an autoimmune disease of the CNS, EAE. Following disease induction, autoreactive Tregs upregulated LAG‐3 and CTLA‐4 in LNs, while IL‐10 and amphiregulin (AREG) were increased in CNS Tregs. Using genetic approaches, we demonstrated that IL‐10, CTLA‐4, and LAG‐3 were nonredundantly required for the protective function of antigen receptor‐engineered Tregs against EAE in cell therapy whereas AREG was dispensable. Treg‐derived IL‐10 and CTLA‐4 were both required to suppress acute autoreactive CD4+ T‐cell activation, which correlated with disease control. These molecules also affected the accumulation in the recipients of engineered Tregs themselves, underlying complex roles for these molecules. Noteworthy, despite the persistence of the transferred Tregs and their protective effect, autoreactive T cells eventually accumulated in the spleen of treated mice. In conclusion, this study highlights the remarkable power of antigen receptor‐engineered Tregs to appropriately provide multiple suppressive factors nonredundantly necessary to prevent autoimmune attacks. [ABSTRACT FROM AUTHOR]

Background and Objectives: D‐negative patients undergoing orthotopic liver transplantation (OLT) might require a large number of red blood cell (RBC) units, which can impact the inventory of D‐negative blood. The blood bank might need to supply these patients with D‐positive RBCs because of inventory constraints. This study evaluates the prevalence of anti‐D formation in D‐negative OLT patients who received D‐positive RBCs perioperatively, as this will assist in successful patient blood management. Materials and Methods: This was a retrospective study performed at a single academic medical centre. Electronic medical records for all 1052 consecutive patients who underwent OLT from January 2007 through December 2017 were reviewed. D‐negative patients who were transfused perioperatively with D‐positive RBCs and had antibody screening at least 30 days after transfusion were included. Results: Of a total of 155 D‐negative patients, 23 (14.8%) received D‐positive RBCs perioperatively. Seventeen patients were included in the study. The median age was 54 years (range 36–67 years); 13 (76.5%) were male. The median number of D‐positive RBC units transfused perioperatively was 7 (range 1–66 units). There was no evidence of D alloimmunization in any patient after a median serologic follow‐up of 49.5 months (range 31 days to 127.7 months). The average number of antibody screening post OLT was 7.29. Conclusion: Our study showed that transfusion of D‐positive RBCs in D‐negative OLT recipients is a safe and acceptable practice in the setting of immunosuppression. This practice allows the conservation of D‐negative RBC inventory. [ABSTRACT FROM AUTHOR]

Standardization of allergen provocation tests of the airways requires standardization of each of the steps involved, including standardization of the extracts used. The value of international standards is emphasized and so is the determination of a clinically relevant potency. The allergen extracts used for bronchial provocation tests must be compared with qualitatively and quantitatively clinically relevant standards using complementary immunochemical and biological methods. Ideally the same extracts should be used for diagnosis (skin test, specific IgE determinations, provocations) and treatment (immuno- therapy). The importance of the biological compared with the non-biological methods is that the biological methods are established in allergenic patients in order to select a clinically relevant potency of the allergen in question, and this potency ought to be confirmed in clinical trials. Once established the biological potency of an in-house preparation can be reproduced by supplementary in vitro methods. Very few commercial allergen preparations are available which fulfil the above-mentioned criteria. [ABSTRACT FROM AUTHOR]

Complement‐mediated interference is a well described phenomenon in single antigen bead (SAB) Luminex assay that leads to falsely low or negative results for anti‐HLA antibody (Ab). In a context of high amount of Ab, the enrichment of the Ab around the bead can lead to complement cascade activation and deposition, thereafter impairing Ab detection. EDTA is now routinely used to circumvent this interference. In this report, we attempted to decipher the role of the phycoerythrin (PE) label conjugated to the secondary Ab in this interference. Indeed, PE is a huge molecule (240 kDa) that could participate to limiting access of the conjugate to its Ab target on the bead. To this purpose, 22 sera displaying complement interference without pre‐treatment with EDTA were compared on SAB assay with three detection strategies: the recommended PE‐conjugated secondary Ab (IgGPE), an Alexa Fluor 532‐conjugated Ab (IgGAF) bearing a tiny 724 Da fluorochrome, and a biotinylated Ab followed by PE‐conjugated streptavidin (IgGBiot). Complement interference occurred with the three detection methods, but its depth, defined by the percentage of MFI loss with neat serum, was the highest for IgGPE. Our study highlighted the partial role of the PE fluorochrome in complement interference in SAB assays. [ABSTRACT FROM AUTHOR]

Familial clustering of the skin disease primary hyperhidrosis suggests a genetic component to the disease. The human leucocyte antigen (HLA) is implicated in a range of diseases, including many comorbidities to hyperhidrosis. No study has investigated whether the HLA genes are involved in the pathogenesis of hyperhidrosis. We, therefore, compared HLA alleles in individuals with and without hyperhidrosis in this study of 65 000 blood donors. In this retrospective cohort study, we retrieved information on individuals with and without hyperhidrosis using self‐reported questionnaires, the Danish National Patient Registry and the Danish National Prescription Registry on participants recruited to the Danish Blood Donor Study between 2010 and 2019. Association tests using logistic regression were conducted for each HLA allele corrected for sex, age, body mass index, smoking and principal components. Overall, 145 of 65 795 (0.2%) participants had hospital diagnosed hyperhidrosis. Similarly, 1379 of 15 530 (8.9%) participants had moderate‐severe self‐reported hyperhidrosis, of whom 447 (2.9%) had severe self‐reported hyperhidrosis. Altogether, 28 participants had both hospital diagnosed and moderate‐severe self‐reported hyperhidrosis. Severe self‐reported hyperhidrosis was associated with HLA‐A*80:01 (adjusted odds ratio 26.97; 95% confidence interval 5.32‐136.70; n = 7; P <.001). Moderate‐severe self‐reported hyperhidrosis and hospital diagnosed hyperhidrosis were not associated with any HLA. The association between hyperhidrosis and HLA‐A*80:01 was based on a very small number of cases and not replicated in other patient subsets, and therefore likely a chance finding. Thus, this study suggests that genes other than the HLA are involved in the pathogenesis of hyperhidrosis. [ABSTRACT FROM AUTHOR]

Systemic lupus erythematosus (SLE) is characterized by the production of pathogenic autoantibodies with nuclear antigen (nAg) specificity. Using (SWRxNZB)F1 (SNF1) mice, we showed higher levels of Immunoglobulin A (IgA) production in the intestine and the nAg reactivity of faecal IgA under lupus susceptibility. Here, we determined whether the faecal IgA abundance and nAg reactivity are higher in, different among, various lupus‐prone preclinical models (MRL/lpr, NZBxNZW‐F1, SNF1, NZM2410 and NZM2328). We also determined whether the faecal IgA nAg reactivity at preseropositive ages correlates with the eventual serum autoantibody levels in males and females of these mouse models. We show that age‐dependent increase in the abundance and nAg reactivity of faecal IgA can vary among different lupus‐prone mouse models. Importantly, faecal IgA in these mice show significant levels of nAg reactivity, starting as early as at juvenile age. Furthermore, the pre‐seropositive stage nAg reactivity of faecal IgA in most lupus‐prone strains correlates well with that of eventual, seropositive stage systemic autoantibody levels. Gender differences in serum autoantibody levels were preceded by similar differences in the faecal IgA abundance and nAg reactivity. These observations suggest that faecal IgA features, nAg reactivity particularly, could serve as a biomarker for early prediction of the eventual systemic autoimmunity in lupus‐prone subjects. [ABSTRACT FROM AUTHOR]

The antigenic trisaccharides, α‐gal epitope and H antigen, containing N‐acetyl‐d‐glucosamine (GlcNAc) were synthesized using a diacetyl strategy, in which NHAc is tentatively converted to NAc2 during oligosaccharide construction. Acetylation of NHAc in GlcNAc significantly improved the reactivity in glycosylation reactions. The diacetyl strategy allowed to achieve the efficient synthesis of α‐gal through a sequential one‐pot and one‐flow procedure. Meanwhile, H antigen was synthesized by stepwise elongation from the reducing end side of GlcNAc. The enhancement of the reactivity by NAc2 protection was observed in both glycosylations at proximal and distal positions to GlcNAc. This diacetyl strategy is expected to be applicable to the synthesis of a wide range of glycans. [ABSTRACT FROM AUTHOR]

Background: To date, several cases of transfusion-transmitted ZIKV infections have been confirmed. Multiple studies detected prolonged occurrence of ZIKV viral RNA in whole blood as compared to plasma samples indicating potential ZIKV interaction with hematopoietic cells. Also, infection of cells from the granulocyte/macrophage lineage has been demonstrated. Patients may develop severe thrombocytopenia, microcytic anemia, and a fatal course of disease occurred in a patient with sickle cell anemia suggesting additional interference of ZIKV with erythroid and megakaryocytic cells. Therefore, we analyzed whether ZIKV propagates in or compartmentalizes with hematopoietic progenitor, erythroid, and megakaryocytic cells.Methods: ZIKV RNA replication, protein translation and infectious particle formation in hematopoietic cell lines as well as primary CD34+ HSPCs and ex vivo differentiated erythroid and megakaryocytic cells was monitored using qRT-PCR, FACS, immunofluorescence analysis and infectivity assays. Distribution of ZIKV RNA and infectious particles in spiked red blood cell (RBC) units or platelet concentrates (PCs) was evaluated.Results: While subsets of K562 and KU812Ep6EPO cells supported ZIKV propagation, primary CD34+ HSPCs, MEP cells, RBCs, and platelets were non-permissive for ZIKV infection. In spiking studies, ZIKV RNA was detectable for 7 days in all fractions of RBC units and PCs, however, ZIKV infectious particles were not associated with erythrocytes or platelets.Conclusion: Viral particles from plasma or contaminating leukocytes, rather than purified CD34+ HSPCs or the cellular component of RBC units or PCs, present the greatest risk for transfusion-transmitted ZIKV infections. [ABSTRACT FROM AUTHOR]

Self-adjuvanting vaccines, consisting of recombinant protein antigens and covalently attached Toll-like receptor (TLR) agonists, have the ability to simultaneously and efficiently deliver antigen and TLR adjuvant to antigen presenting cells (APCs). Here, an enzyme-mediated ligation approach was used to overcome difficulties in producing homogeneous, molecularly defined self-adjuvanting vaccine products under native conditions. This process was optimized to allow the incorporation of the lipopeptide TLR2 agonist fibroblast-stimulating lipopeptide (FSL)-1 onto the N- or C-termini of recombinant protein antigens, employing the enzyme Staphylococcus aureus sortase A (SrtAsa) penta mutant. In addition, because SrtAsamediated ligations are reversible, a tryptophan zipper derived sequence was introduced into both reactants, which was demonstrated to improve ligation efficiency through the formation of a β-hairpin structure that hinders the reverse reaction. Finally, it was demonstrated that N- or C-terminal conjugation, and the incorporation of the β-hairpin structure, did not affect the TLR2-agonist activities of protein antigen TLR agonist conjugates. Overall, this SrtAsa-mediated ligation platform enabled production of antigen TLR2 agonist conjugates with enhanced ligation efficiency, with the conjugates demonstrating potent TLR2 signaling activation (EC50<1nM). [ABSTRACT FROM AUTHOR]