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SARS‐CoV‐2 mutacije predstavljaju jako veliki problem za učinkovitost terapije monoklonalnim antitijelima.

PD-1/PD-L1 pathway plays a role in inhibiting immune response. Therapeutic antibodies aimed at blocking the PD-1/PD-L1 interaction have entered clinical development and have been approved for a variety of cancers. However, the clinical benefits are reduced to a group of patients. The research in combined therapies, which allow for a greater response, is strongly encouraging. We previously characterized a polyphenol-rich extract from Caesalpinia spinosa (P2Et) with antitumor activity in both melanoma and breast carcinoma, as well as immunomodulatory activity. We hypothesize that the combined treatment with P2Et and anti-PD-L1 can improve the antitumor response through an additive antitumor effect. We investigated the antitumor and immunomodulatory activity of P2Et and anti-PD-L1 combined therapy in B16-F10 melanoma and 4T1 breast carcinoma. We analyzed tumor growth, hematologic parameters, T cell counts, cytokine expression, and T cell cytotoxicity. In the melanoma model, combined P2Et and anti-PD-L1 therapy has the following effects: decrease in tumor size; increase in the number of activated CD4 + and CD8 + T cells; decrease in the number of suppressor myeloid cells; increase in PD-L1 expression; decrease in the frequency of CD8 + T cell expressing PD-1; improvement in the cytotoxic activity of T cells; and increase in the IFN γ secretion. In the breast cancer model, P2Et and PD-L1 alone or in combination show antitumor effect with no clear additive effect. This study shows that combined therapy of P2Et and anti-PD-L1 can improve antitumor response in a melanoma model by activating the immune response and neutralizing immunosuppressive mechanisms.

Summary: The processes underlying the development and maintenance of tertiary lymphoid organs are incompletely understood. Using a Ccr7 knockout/knockin approach, we show that spontaneous bronchus-associated lymphoid tissue (BALT) formation can be caused by CCR7-mediated migration defects of dendritic cells (DCs) in the lung. Plt/plt mice that lack the CCR7 ligands CCL19 and CCL21-serine do not form BALT spontaneously because lung-expressed CCL21-leucine presumably suffices to maintain steady-state DC egress. However, plt/plt mice are highly susceptible to modified vaccinia virus infection, showing enhanced recruitment of immune cells as well as alterations in CCR7-ligand-mediated lymphocyte egress from the lungs, leading to dramatically enhanced BALT. Furthermore, we identify two independent BALT homing routes for blood-derived lymphocytes. One is HEV mediated and depends on CCR7 and L-selectin, while the second route is via the lung parenchyma and is independent of these molecules. Together, these data provide insights into CCR7/CCR7-ligand-orchestrated aspects in BALT formation. : Fleige et al. demonstrate that CCR7 and its ligands CCL19, CCL21-serine, and CCL21-leucine orchestrate multiple steps during induction and maintenance of bronchus-associated lymphoid tissue (BALT) including DC-based initial developmental processes as well as homing of blood-derived lymphocytes via HEVs to established BALT. Keywords: BALT, CCR7, CCL21, DCs, MVA, plt/plt, HEVs

Although anti-tumor necrosis factor (TNF) agents are highly effective in the treatment of psoriasis, 2–5% of treated patients develop psoriasis-like skin lesions called paradoxical psoriasis. The pathogenesis of this side effect and its distinction from classical psoriasis remain unknown. Here we show that skin lesions from patients with paradoxical psoriasis are characterized by a selective overexpression of type I interferons, dermal accumulation of plasmacytoid dendritic cells (pDC), and reduced T-cell numbers, when compared to classical psoriasis. Anti-TNF treatment prolongs type I interferon production by pDCs through inhibition of their maturation. The resulting type I interferon overexpression is responsible for the skin phenotype of paradoxical psoriasis, which, unlike classical psoriasis, is independent of T cells. These findings indicate that paradoxical psoriasis represents an ongoing overactive innate inflammatory process, driven by pDC-derived type I interferon that does not lead to T-cell autoimmunity., The pathogenesis of paradoxical psoriasis in patients receiving anti-TNF treatments for classical psoriasis is unclear. Here, the authors show that anti-TNF drugs enhance the production of type I interferon by plasmacytoid dendritic cells, causing skin lesions that, unlike classical psoriasis, lack T- cell autoimmunity.

Summary The fungus Batrachochytrium dendrobatidis (Bd) causes chytridiomycosis, a lethal epizootic disease of amphibians. Rapid identification of the pathogen and biosecurity is essential to prevent its spread, but current laboratory‐based tests are time‐consuming and require specialist equipment. Here, we describe the generation of an IgM monoclonal antibody (mAb), 5C4, specific to Bd as well as the related salamander and newt pathogen Batrachochytrium salamandrivorans (Bsal). The mAb, which binds to a glycoprotein antigen present on the surface of zoospores, sporangia and zoosporangia, was used to develop a lateral‐flow assay (LFA) for rapid (15 min) detection of the pathogens. The LFA detects known lineages of Bd and also Bsal, as well as the closely related fungus Homolaphlyctis polyrhiza, but does not detect a wide range of related and unrelated fungi and oomycetes likely to be present in amphibian habitats. When combined with a simple swabbing procedure, the LFA was 100% accurate in detecting the water‐soluble 5C4 antigen present in skin, foot and pelvic samples from frogs, newts and salamanders naturally infected with Bd or Bsal. Our results demonstrate the potential of the portable LFA as a rapid qualitative assay for tracking these amphibian pathogens and as an adjunct test to nucleic acid‐based detection methods.

Microfibrillar-associated protein 4 (MFAP4) is a non-structural matrix protein with cell regulatory activities and a potential as seromarker for fibrosis. We aimed to study the occurrence of MFAP4 in the synovial membrane from patients with rheumatoid arthritis (RA) vs osteoarthritis (OA). Formaldehyde-fixed synovial tissue sections, from patients with RA (N = 6) and OA (N = 6) undergoing total hip arthroplasty, were deparaffinized and immunostained with monoclonal antibodies against MFAP4. Elastin was detected using ElastiKit. MFAP4 in serum (sMFAP4) and synovial fluid was measured by an immunoassay. MFAP4 was present in synovial biopsies from both RA and OA patients, particularly prominent in deep arterioles where it colocalized with elastin. Notably however, MFAP4 was absent from the internal elastic lamina in RA arterioles irrespective of disease duration and synovitis activity, while present although with irregular staining patterns in OA specimens. sMFAP4 was increased in RA and OA serum vs healthy controls: median (interquartile range) 29.8 (25.3-39.1) and 25.5 U/L (18.1-43.3) vs 17.7 U/L (13.7-21.2), p = 0.006 and p = 0.02, respectively The concentration of synovial fluid was lower than in serum in both RA and OA. These findings may suggest that MFAP4 is involved in adaptive vessel wall remodeling associated with chronic joint disease.

International audience; Filaggrin (FLG) and corneodesmosin (CDSN) are two key proteins of the human epidermis. FLG loss-of-function mutations are the strongest genetic risk factors for human atopic dermatitis. Studies of the epidermal distribution of canine FLG and CDSN are limited. Our aim was to better characterize the distribution of FLG and CDSN in canine skin. Using immunohistochemistry on beagle skin, we screened a series of monoclonal antibodies (mAbs) specific for human FLG and CDSN. The cross-reactive mAbs were further used using immunoelectron microscopy and Western blotting. The structure of canine CDSN and FLG was determined using publicly available databases. In the epidermis, four anti-FLG mAbs stained keratohyalin granules in the granular keratinocytes and corneocyte matrix of the lower cornified layer. In urea-extracts of dog epidermis, several bands corresponding to proFLG and FLG monomers were detected. One anti-CDSN mAb stained the cytoplasm of granular keratinocytes and cells of both the inner root sheath and medulla of hair follicles. Dog CDSN was located in lamellar bodies, in the extracellular parts of desmosomes and in corneodesmosomes. A protein of 52 kDa was immunodetected. Genomic DNA analysis revealed that the amino acid sequence and structure of canine and human CDSN were highly similar.

Pri neučinkovitosti sintetskih lijekova koji modificiraju tijek bolesti (engl. disease-modifying antirheumatic drugs – DMARD s; u tekstu DMARD -i) u liječenju bolesnika s aktivnim reumatoidnim artritisom (RA ) možemo primijeniti jedan od bioloških ili biosličnih lijekova prema smjernicama Hrvatskoga reumatološkog društva iz 2013. godine. Unatoč postignutoj remisiji i boljoj kontroli bolesti primarna ili sekundarna neučinkovitost lijeka razvije se, prema literaturnim podacima, čak u 60% bolesnika. Radi utvrđivanja primarne odnosno sekundarne neučinkovitosti lijeka retrospektivno smo analizirali podatke bolesnika liječenih biološkim lijekovima u Zavodu za kliničku imunologiju, alergologiju i reumatologiju Klinike za unutarnje bolesti Medicinskog fakulteta Sveučilišta u Zagrebu, Kliničke bolnice Dubrava, od 2008. do 2016. god. Aktivnost bolesti praćena je indeksom DAS 28-CRP. U ispitivanje je bilo uključeno 88 bolesnika, 25 muškaraca i 63 žene. U 39 bolesnika (44%), 10 muškaraca i 29 žena, prvi biološki lijek zamijenjen je drugime. Od 39 bolesnika, njih 30 (77%) postiglo je remisiju na primijenjeni drugi biološki lijek, a u sedam bolesnika (18%) zbog neučinkovitosti lijeka uveden je treći biološki lijek, dok je u dva bolesnika uveden i četvrti, odnosno peti lijek. Najčešći razlog prekida primjene lijeka bila je klinička neučinkovitost (visoka aktivnost bolesti). Nismo pronašli statistički značajnu razliku u titru reumatoidnog faktora, anticitrulinskih protutijela ni pušačkog status., In the case of ineffectiveness of synthetic disease-modifying anti rheumatic drugs (DMARD s) in the treatment of patients with active rheumatoid arthritis (RA ), we can use one of the biological or biosimilar drugs according to the Croatian Society for Rheumatology guidelines from 2013. Despite the achieved remission and better disease control, according to literature data up to 60% of patients develop primary or secondary ineffectiveness of the drug. In order to determine primary or secondary ineffectiveness of the drug in our patients, we retrospectively analyzed data from patients treated with biological drugs at the Division of Clinical Immunology, Allergology, and Rheumatology of the Department of Internal Medicine of the University of Zagreb School of Medicine, Clinical Hospital Dubrava, in the period 2008–2016. The study included 88 patients, 25 men and 63 women. The activity of the disease was monitored using the DAS 28(CRP) index. In 39 patients (44%), 10 men and 29 women, the first biological drug was replaced with another. Out of these 39 patients, 30 (77%) achieved remission on the second line of treatment. Seven (18%) patients had to be given a third biological drug because of the ineffectiveness of the second drug, while two patients had to be given a fourth or fifth biological drug. The most common cause of discontinuation of the drug was clinical ineffectiveness, which means that the high activity of the disease was maintained. We did not find a statistically significant difference in the titer of rheumatoid factor (RF) and/or anti-citrulatory peptide (anti-CC P) or smoking status in patients treated with a single biological agent and those in which two or more biological drugs had to be used.

Treatment of inflammatory bowel diseases and rheumatic disorders with anti-tumor necrosis factor alpha (TNFα) drugs is expensive, while a significant proportion of patients does not show adequate clinical response. Therapeutic drug monitoring (TDM) enables patient-specific anti-TNFα therapy. The role of laboratory tests in clinical care has recently been described in a value proposition framework. It describes care processes, stakeholders, costs, risks, benefits and patient outcomes based on the use of a laboratory test in a clinical care pathway. We have applied this concept to the use of TDM for anti-TNFα drugs, describing evidence that supports the intervention and its cost effectiveness, steps that need to be adjusted in the care pathway, possible treatment algorithms and measures to assess adoption of this framework into clinical practice. For effective TDM, an assay for measurement of drug levels together with appropriate target ranges and an anti-drug-antibody assay have to be implemented. Also, instead of only reporting the drug concentration, laboratorians, pharmacists and clinicians should deliver added value by introducing a TDM-based treatment algorithm into clinical practice. Thus, to maximize effectiveness of TDM of anti-TNFα therapy in routine care, adjustment of current care pathways and cooperation of many stakeholders are needed.

Incomplete O-glycosylation is a feature associated with malignancy resulting in the expression of truncated glycans such as the sialyl-Tn (STn) antigen. Despite all the progress in the development of potential anti-cancer antibodies, their application is frequently hindered by low specificities and cross-reactivity. In this study, a novel anti-STn monoclonal antibody named L2A5 was developed by hybridoma technology. Flow cytometry analysis showed that L2A5 specifically binds to sialylated structures on the cell surface of STn-expressing breast and bladder cancer cell lines. Moreover, immunoblotting assays demonstrated reactivity to tumour-associated O-glycosylated proteins, such as MUC1. Tumour recognition was further observed using immunohistochemistry assays, which demonstrated a high sensitivity and specificity of L2A5 mAb towards cancer tissue, using bladder and colorectal cancer tissues. L2A5 staining was exclusively tumoural, with a remarkable reactivity in invasive and metastasis sites, not detectable by other anti-STn mAbs. Additionally, it stained 20% of cases of triple-negative breast cancers, suggesting application in diseases with unmet clinical needs. Finally, the fine specificity was assessed using glycan microarrays, demonstrating a highly specific binding of L2A5 to core STn antigens and additional ability to bind 2–6-linked sialyl core-1 probes. In conclusion, this study describes a novel anti-STn antibody with a unique binding specificity that can be applied for cancer diagnostic and future development of new antibody-based therapeutic applications. We thank Dinora Ferreira, Vitor Domingos and Maria Rosário Tito for assistance with animals in the IHMT/ NOVA animal room. We acknowledge the contribution of Prof Ten Feizi and Dr Yan Liu in the glycan microarray analysis and their input in the MIRAGE document; Dr Yibing Zhang for preparation and analysis of the NGL probes; Pengtao Zhang for the preparation of the lipid reagent and the NGL probes and Prof Makoto Kiso and Prof Hideharu Ishida for the synthetic glycosylceramides (GSC) included in the microarray. We also thank Mylène Carrascal for valuable input and expertise.This work was supported by the Bluepharma innovation award “Trifunctional antibodies and dendritic cell-based technologies: a combined approach to cancer immunotherapy” 2013 and the Scientific merit prize Santander - Totta – Lisbon New University - “Antibody engineering for breast cancer therapy” 2013. LRL is supported by the grant PD/BD/52476/2013 from the Portuguese Foundation for Science and Technology (FCT). The work is partially supported by FCT-MCTES through the grants PD/ BD/105727/2014 (VC), IF/00033/2012 (ASP), SFRH/BPD/101827/2014 (LL) and SFRH/BPD/111048/2015 (JAF), the Wellcome Trust Biomedical Resource grant (WC) and the NSFC-Shandong Joint Fund (U1606403) (CL). The authors also acknowledge FCT funding for CI-IPOP research unit (PEst-OE/SAU/UI0776/201). The Applied Molecular Biosciences Unit-UCIBIO is financed by national funds from FCT-MCTES (UID/Multi/04378/2013) and co-financed by the European Regional Development Fund under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). FCT is co-financed by European Social Fund (ESF) under Human Potential Operation Programme (POPH) from National Strategic Reference Framework (NSRF). The funders did not play a role in the study design, data collection or decision to publish.

Summary Since the discovery of the lectin pathway of complement activation, numerous clinical cohorts have been examined for one or more proteins, with the intention of uncovering the functions of the proteins or with the aim of discovering new biomarkers or diagnostic tools. To unveil the abnormal, it is pivotal to know the normal. Our aim was to describe the concentrations of the 11 known proteins of the lectin pathway in serum and plasma and to uncover possible gender differences, age and diurnal variations, which must be taken into account for investigation in different cohorts. We examined the concentrations of all lectin pathway proteins mannan-binding lectin (MBL), H-ficolin, L-ficolin, M-ficolin, collectin-K1, collectin-L1, MBL-associated serine protease 2 (MASP-2), MASP-3, MBL-associated protein of 44 kDa (MAp44) and MAp19 in 300 Danish blood donors in serum and ethylenediamine tetraacetic acid (EDTA) plasma in established assays, and we further developed a new assay to measure MASP-1 in the same samples. We found significant differences in concentrations between serum and plasma for all proteins except for MBL and MASP-3. H-ficolin, M-ficolin and MAp19 displayed convincing diurnal variation. H-ficolin, in particular, halved from morning to the middle of the night. There were gender differences for most proteins, whereas age did not seem to influence concentration. The present study underlines the necessity of considering which material to use, correct matching and a trial design that takes the nature of the protein into account in order for the outcome of cohort studies to have significant relevance.

International audience; GA101, also known as obinutuzumab or Gazyva (Gazyvaro), is a glycoengineered type II humanized antibody that targets the CD20 antigen expressed at the surface of B-cells. This novel anti-CD20 antibody is currently assessed in clinical trials with promising results as a single agent or as part of therapeutic combinations for the treatment of B-cell malignancies. Detailed understanding of the mechanisms of GA101-induced cell death is needed to get insight into possible resistance mechanisms occurring in patients. Although multiple in vitro and in vivo mechanisms have been suggested to describe the effects of GA101 on B-cells, currently available data are ambiguous. The aim of our study was to clarify the cellular mechanisms involved in GA101-induced cell death in vitro, and more particularly the respective roles played by lysosomal and mitochondrial membrane permeabilization. Our results confirm previous reports suggesting that GA101 triggers homotypic adhesion and caspase-independent cell death, two processes that are dependent on actin remodeling and involve the production of reactive oxygen species. With respect to lysosomal membrane permeabilization (LMP), our data suggest that lack of specificity of available antibodies directed against cathepsin B may have confounded previously published results, possibly challenging current LMP-driven model of GA101 action mode.

Nedavni uspješni rezultati više raznih novih imunoterapijskih pristupa u onkoloških bolesnika, kao, primjerice, blokada imunosnih inhibitornih molekularnih interakcija s monoklonskim protutijelima protiv molekula kontrolnih točaka, mogu se smatrati medicinskim iskorakom u kliničkoj onkologiji. Cilj je ovoga rada dati pojednostavnjeni prikaz imunosnog uređivanja raka i kliničke primjene monoklonskih protutijela protiv molekula kontrolnih točaka u onkoloških bolesnika. Interakcije između imunosnog sustava i autolognih tumora složene su, ali rezultati dobiveni primjenom monoklonskih protutijela protiv molekula kontrolnih točaka upućuju na prihvatljivu kliničku primjenjivost, učinkovitost i sigurnost u liječenju bolesnika s određenim tipovima raka. Klinička primjena monoklonskih protutijela protiv molekula kontrolnih točaka CTLA-4, PD-1 i PD-L1, ovisno o tome protiv kojih se tumora ta protutijela testiraju i primjenjuju, jest u rasponu od odobrenja regulatornih agencija i primjene u metastatskoj bolesti u bolesnika s određenim vrstama tumora pa do faza testiranja u kliničkim studijama, a radi istraživanja kliničke učinkovitosti i dobivanja odobrenja., The recent successful results of several relatively new immunotherapeutic anti-cancer strategies such as the blockade of immune inhibitory pathways by monoclonal antibodies against checkpoint molecules can be considered as a medical breakthrough in clinical cancer immunotherapy. This paper presents a basic overview of cancer immunoediting and the clinical application of monoclonal antibodies against checkpoint molecules in cancer patients. Interactions between the immune system and the malignancy are complex, but the results obtained by using the above mentioned therapeutic approaches indicate acceptable clinical utility, efficacy and safety against several types of cancer. Clinical application of monoclonal antibodies against checkpoint molecules CTLA-4, PD-1, and PD-L1, depending on which tumors these antibodies are tested and applied against, ranges from their already usage having been approved by regulatory agencies for patients with particular metastatic tumors to their testing in clinical studies with the aim of demonstrating their efficiency and consequently obtaining approval.

Middle East respiratory syndrome (MERS) represents an important respiratory disease accompanied by lethal outcome in one third of human patients. In recent years, several investigators developed protective antibodies which could be used as prophylaxis in prospective human epidemics. In the current study, eight human monoclonal antibodies (mAbs) with neutralizing and non-neutralizing capabilities, directed against different epitopes of the MERS-coronavirus (MERS-CoV) spike (MERS-S) protein, were investigated with regard to their ability to immunohistochemically detect respective epitopes on formalin-fixed paraffin-embedded (FFPE) nasal tissue sections of MERS-CoV experimentally infected alpacas. The most intense immunoreaction was detected using a neutralizing antibody directed against the receptor binding domain S1B of the MERS-S protein, which produced an immunosignal in the cytoplasm of ciliated respiratory epithelium and along the apical membranous region. A similar staining was obtained by two other mAbs which recognize the sialic acid-binding domain and the ectodomain of the membrane fusion subunit S2, respectively. Five mAbs lacked immunoreactivity for MERS-CoV antigen on FFPE tissue, even though they belong, at least in part, to the same epitope group. In summary, three tested human mAbs demonstrated capacity for detection of MERS-CoV antigen on FFPE samples and may be implemented in double or triple immunohistochemical methods. info:eu-repo/semantics/acceptedVersion

Human papillomavirus is detected in over 50% of oropharyngeal squamous cell carcinomas. Human papillomavirus–positive oropharyngeal squamous cell carcinomas differ from human papillomavirus–negative tumors, and both expression patterns are classified as distinct entities. The Bmi-1 oncogene is a well-known member of the mammalian polycomb-group family. HESC5:3 and HES77 are newly developed monoclonal antibodies produced against undifferentiated embryonic stem cells. Our aim was to explore their roles in both human papillomavirus–positive and –negative oropharyngeal squamous cell carcinomas. Our cohort comprised 202 consecutive oropharyngeal squamous cell carcinoma patients diagnosed and treated with curative intent. We used tissue microarray tumor blocks to study the immunohistochemical expression of Bmi-1, HESC5:3, and HES77. We compared the expressions of these stem cell markers with p16 immunoexpression and human papillomavirus status, as well as with other characteristics of the tumor, and with patients’ clinical data and follow-up data. Human papillomavirus– and p16-positive tumors expressed less Bmi-1 and more HESC5:3 than the negative tumors. HES77 expression was high in human papillomavirus–positive oropharyngeal squamous cell carcinoma, but it did not correlate with p16 positivity. In our multivariable model, Bmi-1 and HESC5:3 were still associated with human papillomavirus, but the association between human papillomavirus and HES77 remained absent. In conclusion, Bmi-1, HESC5:3, and HES77 may have a different role in human papillomavirus–positive and human papillomavirus–negative tumors. There was no correlation between Bmi-1, HESC5:3, and HES77 expression and survival.

Objectives Type VIII collagen is involved in angiogenesis and remodeling of arteries. We hypothesized that type VIII collagen was upregulated in diseases associated with vascular remodeling, e.g. pulmonary fibrosis and cancer. In this paper we present the development and validation of a competitive enzyme-linked immunosorbent assay (ELISA) targeting type VIII collagen in serum and plasma. Design and methods A monoclonal antibody was raised against the C-terminal of type VIII collagen (C8-C) and a competitive ELISA was developed. The assay was evaluated in relation to epitope specificity, technical performance, and in relevant disease cohorts. The developed ELISA was applied for the assessment of type VIII collagen in serum from patients diagnosed with chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and various cancers. Results The C8-C ELISA was technically stable and applicable for measurements in serum and plasma samples. Concentrations of C8-C was increased in serum from patients diagnosed with COPD (n = 68) (p

Histone acetylation is generally associated with gene activation and chromatin decondensation. Recent mass spectrometry analysis has revealed that histone H4 lysine 20, a major methylation site, can also be acetylated. To understand the function of H4 lysine 20 acetylation (H4K20ac), we have developed a specific monoclonal antibody and performed ChIP-seq analysis using HeLa-S3 cells. H4K20ac was enriched around the transcription start sites (TSSs) of minimally expressed genes and in the gene body of expressed genes, in contrast to most histone acetylation being enriched around the TSSs of expressed genes. The distribution of H4K20ac showed little correlation with known histone modifications, including histone H3 methylations. A motif search in H4K20ac-enriched sequences, together with transcription factor binding profiles based on ENCODE ChIP-seq data, revealed that most transcription activators are excluded from H4K20ac-enriched genes and a transcription repressor NRSF/REST co-localized with H4K20ac. These results suggest that H4K20ac is a unique acetylation mark associated with gene repression.

A naturally-occurring H275Y oseltamivir resistant variant of influenza A (H1N1) virus emerged in 2007, subsequently becoming prevalent worldwide, via an undetermined mechanism. To understand the antigenic properties of the H275Y variant, oseltamivir resistant and susceptible strains of H1N1 viruses were analyzed by hemagglutination inhibition (HI) and microneutralization assays. HI analysis with H1-positive sera obtained from seasonal flu vaccine immunized and non-immunized individuals, and H1-specific monoclonal antibodies, revealed that resistant strains exhibited a reduced reactivity to these antisera and antibodies in the HI assay, as compared to susceptible strains. Neutralization assay testing demonstrated that oseltamivir resistant H1N1 strains are also less susceptible to antibody inhibition during infection. Mice inoculated with a resistant clinical isolate exhibit 4-fold lower virus-specific antibody titers than mice infected with a susceptible strain under the same conditions. Resistant and sensitive variants of 2009 pandemic H1N1 virus did not exhibit such differences. While HA1 and NA phylogenetic trees show that both oseltamivir resistant and susceptible strains belong to clade 2B, NA D354G and HA A189T substitutions were found exclusively, and universally, in oseltamivir resistant variants. Our results suggest that the reduced susceptibility to antibody inhibition and lesser in vivo immunogenicity of the oseltamivir resistant 2008-2009 H1N1 influenza A virus is conferred by coupled NA and HA mutations, and may contribute to the prevalence of this H1N1 variant. © 2011 Elsevier B.V., link_to_OA_fulltext

The enzymatic activity of the pathogenic botulinum neurotoxins type A and E (BoNT/A and E) leads to potentially lethal paralytic symptoms in humans and their prompt detection is of crucial importance. A chip assay based on Surface Plasmon Resonance monitoring of the cleavage products is a simple method that we have previously established to detect BoNT/A activity. We have now developed a similar format assay to measure BoNT/E activity. A monoclonal antibody specifically recognizing SNAP25 cleaved by BoNT/E was generated and used to measure the appearance of the neo-epitope following injection of BoNT/E over SNAP-25 immobilized on a chip. This assay detects BoNT/E activity at 1 LD50/ml within minutes and linear dose-responses curves were obtained using a multiplexed biosensor. A threshold of 0.01 LD50/ml was achieved after 5 h of cleavage. This assay is 10-fold more sensitive than the in vivo assay for direct detection of BoNT/E in serum samples. The SNAP25 chip assay is able to discriminate in an automated manner the presence of BoNT/E, BoNT/A or a combination of both toxins.

Histone modifications play an important role in epigenetic gene regulation and genome integrity. It remains largely unknown, however, how these modifications dynamically change in individual cells. By using fluorescently labeled specific antigen binding fragments (Fabs), we have developed a general method to monitor the distribution and global level of endogenous histone H3 lysine modifications in living cells without disturbing cell growth and embryo development. Fabs produce distinct nuclear patterns that are characteristic of their target modifications. H3K27 trimethylation-specific Fabs, for example, are concentrated on inactive X chromosomes. As Fabs bind their targets transiently, the ratio of bound and free molecules depends on the target concentration, allowing us to measure changes in global modification levels. High-affinity Fabs are suitable for mouse embryo imaging, so we have used them to monitor H3K9 and H3K27 acetylation levels in mouse preimplantation embryos produced by in vitro fertilization and somatic cell nuclear transfer. The data suggest that a high level of H3K27 acetylation is important for normal embryo development. As Fab-based live endogenous modification labeling (FabLEM) is broadly useful for visualizing any modification, it should be a powerful tool for studying cell signaling and diagnosis in the future.

International audience; The ectodomain of influenza A virus M2 protein (M2e) is composed of 24 amino acids and induces antibodies with inhibitory effect against a broad spectrum of influenza A subtypes in vitro and in vivo. Although relatively conserved, 21 M2e variants emerged in recent influenza A strains, most of the mutations appeared in the middle part of M2e domain. In this study, we characterized the in vitro inhibition efficacy of a monoclonal antibody (mAb) M2e8-7 recognizing the N terminus highly conserved epitope SLLTEVET (aa 2-9) which is common for both M1 and M2 proteins. Peptide binding assay showed that mAb M2e8-7 reacted strongly with M2e and 19 M2e variant peptides. The mAb M2e8-7 potently inhibited the replication of influenza A virus H1 and H3 subtypes in MDCK cells. Two important amino acids in M2e epitope, Threonine at position five and the Glutamic acid at position six, were identified to lead antibody-escaping variants. These results brought new insight in developing vaccine and therapeutic agents against influenza A virus infections.

BACKGROUND: Natural killer (NK) cells participate in the immune response against solid organ allo- and xenografts and are tightly regulated through signals mediated by inhibiting and activating receptors expressed on their cell surface. Human NK cytotoxicity against porcine endothelial cells (pEC) is mediated by the interaction of the activating human NK receptor hNKG2D and its corresponding ligand on pEC, porcine UL-16 binding protein 1 (pULBP1). The aim of the present study was to characterize the regulation of pULBP1 cell-surface expression on primary porcine aortic endothelial cells (PAEC). METHODS: A monoclonal antibody (mAb; aE5-63) directed against pULBP1 was generated by immunizing C57BL/6 mice with the pEC line PEDSV.15, and used in cellular ELISA to determine pULBP1 cell surface expression. PAEC were either left untreated or stimulated with human or porcine cytokines [interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha)], human serum, cultured under hypoxic conditions, or infected with human or porcine cytomegalovirus (CMV). RESULTS: Neither human nor porcine IFN-gamma stimulation changed pULBP1 expression, whereas both human and porcine TNF-alpha stimulation as well as human and porcine CMV infection significantly decreased pULBP1 expression on PAEC. Coculture of PAEC with human serum strongly increased pULBP1 expression depending on the binding of human anti-pig antibodies. Exposure of PAEC to hypoxia only slightly increased pULBP1 expression. CONCLUSIONS: In conclusion, (i) the novel anti-pULBP1 IgM mAb aE5-63 represents a useful tool to study pULBP1/hNKG2D-mediated responses in xenotransplantation, and (ii) the expression of pULBP1, a human-pig cross-species functional hNKG2D ligand, on the surface of PAEC is modulated by various stimuli associated with transplantation.

Antigenic profiles of post-2002 H5N1 viruses representing major genetic clades and various geographic sources were investigated using a panel of 17 monoclonal antibodies raised from five H5N1 strains. Four antigenic groups from seven clades of H5N1 virus were distinguished and characterized based on their cross-reactivity to the monoclonal antibodies in hemagglutination inhibition and cell-based neutralization assays. Genetic polymorphisms associated with the variation of antigenicity of H5N1 strains were identified and further verified in antigenic analysis with recombinant H5N1 viruses carrying specific mutations in the hemagglutinin protein. Modification of some of these genetic variations produced marked improvement to the immunogenicity and cross-reactivity of H5N1 strains in assays utilizing monoclonal antibodies and ferret antisera raised against clade 1 and 2 H5N1 viruses, suggesting that these sites represent antigenically significant amino acids. These results provide a comprehensive antigenic profile for H5N1 virus strains circulating in recent years and will facilitate the recognition of emerging antigenic variants of H5N1 virus and aid in the selection of vaccine strains. Copyright © 2008, American Society for Microbiology. All Rights Reserved., link_to_OA_fulltext

The humoral response to hepatitis C virus (HCV) may contribute to controlling infection. We previously isolated human monoclonal antibodies to conformational epitopes on the HCV E2 glycoprotein. Here, we report on their ability to inhibit infection by retroviral pseudoparticles incorporating a panel of full-length E1E2 clones representing the full spectrum of genotypes 1–6. We identified one antibody, CBH-5, that was capable of neutralizing every genotype tested. It also potently inhibited chimeric cell culture-infectious HCV, which had genotype 2b envelope proteins in a genotype 2a (JFH-1) background. Analysis using a panel of alanine-substitution mutants of HCV E2 revealed that the epitope of CBH-5 includes amino acid residues that are required for binding of E2 to CD81, a cellular receptor essential for virus entry. This suggests that CBH-5 inhibits HCV infection by competing directly with CD81 for a binding site on E2.

In a randomised phase 3 trial, panitumumab significantly improved progression-free survival (PFS) in patients with refractory metastatic colorectal cancer (mCRC). This analysis characterises the association of PFS with CRC symptoms, health-related quality of life (HRQoL), and overall survival (OS). CRC symptoms (NCCN/FACT CRC symptom index, FCSI) and HRQoL (EQ-5D) were assessed for 207 panitumumab patients and 184 best supportive care (BSC) patients who had at least one post-baseline patient-reported outcome (PRO) assessment. Patients alive at week 8 were included in the PRO and OS analyses and categorised by their week 8 progression status as follows: no progressive disease (no PD; best response of at least stable disease) vs progressive disease (PD). Standard imputation methods were used to assign missing values. Significantly more patients were progression free at weeks 8–24 with panitumumab vs BSC. After excluding responders, a significant difference in PFS remained favouring panitumumab (HR=0.63, 95% CI=0.52–0.77; P

The feline immunodeficiency virus (FIV) targets activated CD4-positive helper T cells preferentially, inducing an AIDS-like immunodeficiency in its natural host species, the domestic cat. The primary receptor for FIV is CD134, a member of the tumor necrosis factor receptor superfamily, and all primary viral strains tested to date use CD134 for infection. We examined the expression of CD134 in the cat using a novel anti-feline CD134 monoclonal antibody (MAb), 7D6, and showed that as in rats and humans, CD134 expression is restricted tightly to CD4+, and not CD8+, T cells, consistent with the selective targeting of these cells by FIV. However, FIV is also macrophage tropic, and in chronic infection the viral tropism broadens to include B cells and CD8+T cells. Using 7D6, we revealed CD134 expression on a B220-positive (B-cell) population and on cultured macrophages but not peripheral blood monocytes. Moreover, macrophage CD134 expression and FIV infection were enhanced by activation in response to bacterial lipopolysaccharide. Consistent with CD134 expression on human and murine T cells, feline CD134 was abundant on mitogen-stimulated CD4+T cells, with weaker expression on CD8+T cells, concordant with the expansion of FIV into CD8+T cells with progression of the infection. The interaction between FIV and CD134 was probed using MAb 7D6 and soluble CD134 ligand (CD134L), revealing strain-specific differences in sensitivity to both 7D6 and CD134L. Infection with isolates such as PPR and B2542 was inhibited well by both 7D6 and CD134L, suggesting a lower affinity of interaction. In contrast, GL8, CPG, and NCSU were relatively refractory to inhibition by both 7D6 and CD134L and, accordingly, may have a higher-affinity interaction with CD134, permitting infection of cells where CD134 levels are limiting.

In vitro studies have shown that soluble MHC (sMHC) released by cell lines is bound to nano-vesicles termed exosomes. It is thought that exosomes may represent the major reservoir of sMHC class I and II molecules in biological fluids. However, most studies have been confined to in vitro assays performed with cell lines. We show here that sMHC in the serum or plasma differs from exosome-bound sMHC in five ways: In contrast to exosome-associated sMHC, circulating sMHC is of low density, has a low apparent molecular mass (40-300 kDa) and is not detergent-labile. Moreover, the majority of MHC class II isoforms and MHC class I in blood are not physically linked and circulating HLA-DR is accessible to an antibody specific for the HLA-DR alpha-chain intracellular epitope, which is masked by its association with cellular or exosomal membranes. Finally, utilizing transcriptional activator of murine MHC class II (C2ta) promoter-mutant mice, we showed that the release of sMHC class II into the circulation is dependent on the C2ta pI promoter, but not pIII or pIV. This suggests that myeloid dendritic cells and/or macrophages, which preferentially use promoter pI of the C2ta gene, produce most of the sMHC class II found in the circulation.

BACKGROUND: Psoriasis is considered as a chronic immune-mediated disease characterized by inflammation and proliferation of the epidermis. OBJECTIVES: Targeting intercellular adhesion molecule 1 (ICAM-1) is an attractive therapeutic option as this molecule is critically involved in leucocyte adhesion and extravasation as well as in lymphocyte activation. METHODS: We have selected the fully human monoclonal antibody MOR102 (#5) against ICAM-1 from the Human Combinatorial Antibody Library (HuCAL). This antibody, as human IgG4 [corrected] was tested for its ability to interfere with lymphocyte activation and adhesion in vitro as well as for its antipsoriatic efficacy in vivo using the psoriasis-severe combined immunodeficient (SCID) mouse model. RESULTS: The antibody demonstrated efficient inhibition of lymphocyte adhesion to ICAM-1 in vitro, with an IC(50) of approximately 0.4 microg mL(-1) (3 nmol L(-1)). In addition, MOR102 (#5) reduced lymphocyte proliferation in mixed lymphocyte cultures by approximately 50%. The in vivo efficacy of MOR102 (#5) was tested on grafts derived from lesional skin of patients with chronic plaque-stage psoriasis transplanted on to SCID mice. Intraperitoneal injection of 10 mg kg(-1) of MOR102 (#5) antibody every alternate day over a period of 4 weeks resulted in reconstitution of orthokeratotic differentiation and a significant (P < 0.05) reduction in epidermal thickness as well as marked reduction in the inflammatory infiltrate. Therapeutic activity may be related to the targeting of ICAM-1 on keratinocytes and thus preventing efficient activation of local T cells. CONCLUSIONS: Based on the efficacy of the fully human monoclonal antibody MOR102 (#5) shown in vitro as well as in vivo in the psoriasis-SCID mouse model, initiation of clinical studies is indicated.

The junctional adhesion molecule-C (JAM-C) was recently described as an adhesion molecule localized at interendothelial contacts and involved in leukocyte transendothelial migration. The protein JAM-C interacts with polarity complex molecules and regulates the activity of the small GTPase Cdc42. The angiogenesis process involves rearrangement of endothelial junctions and implicates modulation of cell polarity. We tested whether JAM-C plays a role in angiogenesis using tumor grafts and hypoxia-induced retinal neovascularization. Treatment with a monoclonal antibody directed against JAM-C reduces tumor growth and infiltration of macrophages into tumors. The antibody decreases angiogenesis in the model of hypoxia-induced retinal neovascularization in vivo and vessel outgrowth from aortic rings in vitro. Importantly, the antibody does not induce pathologic side effects in vivo. These findings show for the first time a role for JAM-C in angiogenesis and define JAM-C as a valuable target for antitumor therapies.

Summary The ready access to commercially available multiplex assays and the importance of inflammation in disease pathogenesis has resulted in an abundance of studies aimed at identifying surrogate biomarkers for different clinically important questions. Establishing a link between a biomarker and disease pathogenesis, however, is quite complex, and in some instances this complexity is compounded by post-translational modifications and the use of immunoassays that do not always discriminate between the different forms of the same protein. Herein, we provide a detailed description of an assay system that has been established to discriminate the agonist form of CXCL10 from the NH2-terminal truncated form of the molecule generated by dipeptidylpeptidase IV (DPP4) cleavage. We demonstrate the utility of this assay system for monitoring agonist and antagonist forms of CXCL10 in culture supernatant, patient plasma and urine samples. Given the important role of CXCL10 in chronic inflammatory diseases and its suggested role as a predictive marker in managing patients with chronic hepatitis C, asthma, atopic dermatitis, transplantation, tuberculosis, kidney injury, cancer and other diseases, we believe that our method will be of general interest to the research and medical community.

A patient with agammaglobulinemia developed acute hepatitis that progressed to chronic liver disease with high levels of hepatitis B virus (HBV) DNA in the absence of detectable HBsAg. Sequencing of the a determinant region of HBsAg revealed multiple amino acid substitutions that, unusually, also included a substitution at position 122 that defines subtype specificity. All of these mutations had a profound effect on the antigenicity of this region, which led to the complete failure of variant detection by commercially available routine diagnostic assays or laboratory-based monoclonal antibody assays.

A rapid antigen test for the diagnosis of severe acute respiratory syndrome (SARS) is essential for control of this disease at the point of management. The nucleocapsid (N) protein of SARS-associated coronavirus (SARS-CoV) is abundantly expressed in infected-cell culture filtrate as demonstrable by Western blotting using convalescent-phase sera from patients with SARS. We used monoclonal antibodies specifically directed against N protein to establish a sensitive antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV. The assay employed a mixture of three monoclonal antibodies for capture and rabbit polyclonal antibodies for detection of serum antigen in 32 cases of clinically probable SARS as defined by the World Health Organization during the epidemic in Guangzhou, China. Recombinant N protein was used as a standard to establish a detection sensitivity of approximated 50 pg/ml. The linear range of detection in clinical specimens was from 100 pg/ml to 3.2 ng/ml. Using a panel of sera collected at different points in time, the amount of circulating N antigen was found to peak 6 to 10 days after the onset of symptoms. The sensitivity of the assay was 84.6% in 13 serologically confirmed SARS patients with blood taken during the first 10 days after the onset of symptoms (11 of 13). The specificity of the assay was 98.5% in 1,272 healthy individuals (1,253 of 1,272). There was no cross-reaction with other human and animal coronaviruses in this assay. In conclusion, a sensitive and quantitative antigen capture ELISA was established for the early diagnosis and disease monitoring of SARS-CoV infection., published_or_final_version

By analogy with its human nectin1 counterpart, murine nectin1 serves as a cellular receptor for the entry of herpes simplex virus (HSV) into murine cells. HSV entry mediated by either receptor is dependent on the viral glycoprotein D (gD). Whereas human nectin1 binds gD at high affinity and in a saturable manner, murine nectin1 binds gD in a barely detectable fashion, depending on the sensitivity of the assay. The immunoglobulin type V domain of murine nectin differs from its human counterpart in 11 amino acids. To identify the key residues responsible for the high-affinity binding of gD to human nectin1, we replaced each of the 11 divergent amino acids with the human counterparts singly or in groups in an incremental manner. Replacement in murine nectin1 of six amino acids that lie within the gD binding region of human nectin1 (previously mapped to residues 64 to 94, likely the CC′C″ surface) increased the gD binding activity to a limited extent. In contrast, the single P138L substitution, which lies distal from the gD binding site, markedly increased gD binding. This substitution, when coupled with downstream substitutions, exerted the greatest effect. Three-dimensional modeling of the nectin1 V domain suggested that P138 in murine nectin1 might decrease the stability of the V domain by reducing the size of β-strand G. The results support the notion that the overall structure of V nectin1 plays a pivotal role in its ability to bind HSV gD.

Using a tumor model of spontaneously arising insulinomas expressing a defined tumor-associated antigen, we investigated whether tumor growth promotes cross-presentation and tolerance of tumor-specific T cells. We found that an advanced tumor burden enhanced cross-presentation of tumor-associated antigens to high avidity tumor-specific T cells, inducing T cell proliferation and limited effector function in vivo. However, contrary to other models, tumor-specific T cells were not tolerized despite a high tumor burden. In fact, in tumor-bearing mice, persistence and responsiveness of adoptively transferred tumor-specific T cells were enhanced. Accordingly, a potent T cell–mediated antitumor response could be elicited by intravenous administration of tumor-derived peptide and agonistic anti-CD40 antibody or viral immunization and reimmunization. Thus, in this model, tumor growth promotes activation of high avidity tumor-specific T cells instead of tolerance. Therefore, the host remains responsive to T cell immunotherapy.

In this study, polyclonal and monoclonal antibodies to native and denatured chicken ovalbumin (OVA) were produced to compare their dependency on continuous and three-dimensional epitopes. These antibodies were characterized with respect to reactivity to native and denatured OVA by enzyme-linked immunosorbent assay (ELISA) employing surface-bound OVA and streptavidin-capture ELISA to determine whether effects of different coating influence antibody specificity and with respect to epitope specificity by peptide ELISA, using overlapping peptides, covering the complete OVA sequence. Polyclonal antibodies to native OVA reacted strongly with native and denatured OVA in both assays, but did not react with the overlapping peptides. Polyclonal antibodies to denatured OVA reacted strongly with both OVA forms and with several of the overlapping peptides. Monoclonal antibodies to native OVA reacted preferentially with three-dimensional epitopes on native OVA and not with denatured OVA. Monoclonal antibodies to denatured OVA showed reactivity to both OVA forms. Two of these monoclonal antibodies, HYB 94-06 and 94-07, showed reactivity to overlapping peptides and their epitopes were identified as flexible structures constituting amino acids 130-135 and 136-141, respectively. Moreover, comparison of antibody reactivity to N OVA revealed that in the streptavidin-capture ELISA, antibody reactivity was notably reduced compared to ELISA employing surface-bound OVA. Collectively, immunization with native OVA preferentially generates highly specific antibodies reacting with three-dimensional epitopes, whereas immunization with denatured OVA generates antibodies occasionally reacting with continuous epitopes. Moreover, as differences in monoclonal antibody reactivity was found between the two assays, monoclonal antibodies always should be selected by an assay mimicking the desired use of the final antibodies as closely as possible.

The majority of pancreatic neoplasms are characterized by a generally lethal progress within a short period of time after primary diagnosis and the mortality of patients is expected to increase further. Due to lack of efficient screening programs and moderate response to treatments, novel compounds for treatment are needed. We investigated the CLDN18.2 expression in affected patients as in vitro feasibility study for a potential treatment with the novel antibody IMAB362. Therefore, we analyzed the expression of CLDN18.2 in normal pancreatic tissues (N = 24), primary lesions (N = 202), metastases (N = 84) and intra-individually matched samples (N = 48) of patients with pancreatic ductal adenocarcinoma (PDAC), neuroendocrine neoplasia (NEN) and acinar cell carcinoma. A standardized method for evaluation by immunohistochemistry was developed. The specific staining was evaluated by two independent raters and analysis of staining intensities (range 0-3+) and relative proportions of tumor cells were performed. One hundred three (59.2%) samples of primary PDAC were found positive. The vast majority of positive samples were characterized to highly express CLDN18.2: 54.6% (N = 95) with staining intensities of ≥ 2+. NEN were positive in 20% of cases (all ≥ 2+). Metastases of pancreatic neoplasms were also frequently found positive with comparable high rates (69.4% of lymph node and 65.7% of liver metastases). The rate of CLDN18.2 positivity is high in pancreatic neoplasms whereby the expression is not limited to the primaries but is also maintained upon metastasis. Thus, a considerable number of patients with pancreatic neoplasms would be in principle eligible for a CLDN18.2-targeting approach.

B cells play a major role in the pathogenesis of many autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, and type I diabetes mellitus, as indicated by the efficacy of B cell–targeted therapies in these diseases. Therapeutic effects of the most commonly used B cell–targeted therapy, anti-CD20 mAb, are contingent upon long-term depletion of peripheral B cells. In this article, we describe an alternative approach involving the targeting of CD79, the transducer subunit of the B cell AgR. Unlike anti-CD20 mAbs, the protective effects of CD79-targeted mAbs do not require cell depletion; rather, they act by inducing an anergic-like state. Thus, we describe a novel B cell–targeted approach predicated on the induction of B cell anergy.

Inflammation is mediated mainly by leukocytes that express both Toll-like receptor 4 (TLR4) and Fc γ receptors (FcγR). Dysregulated activation of leukocytes via exogenous and endogenous ligands of TLR4 results in a large number of inflammatory disorders that underlie a variety of human diseases. Thus, differentially blocking inflammatory cells while sparing structural cells, which are FcγR-negative, represents an elegant strategy when targeting the underlying causes of human diseases. Here, we report a novel tethering mechanism of the Fv and Fc portions of anti-TLR4 blocking antibodies that achieves increased potency on inflammatory cells. In the presence of ligand (e.g. lipopolysaccharide (LPS)), TLR4 traffics into glycolipoprotein microdomains, forming concentrated protein platforms that include FcγRs. This clustering produces a microenvironment allowing anti-TLR4 antibodies to co-engage TLR4 and FcγRs, increasing their avidity and thus substantially increasing their inhibitory potency. Tethering of antibodies to both TLR4 and FcγRs proves valuable in ameliorating inflammation in vivo. This novel mechanism of action therefore has the potential to enable selective intervention of relevant cell types in TLR4-driven diseases.

The immunoglobulin-like receptors that mediate entry of herpes simplex virus type 1 (HSV-1) into human cells were found to mediate the direct cell-to-cell spread of wild-type virus. The receptors here designated Nectin1α and -δ and Nectin2α were originally designated HIgR, PRR1/HveC, and PRR2α/HveB, respectively. We report the following. (i) Wild-type HSV-1 spreads from cell to cell in J cells expressing nectin1α or nectin1δ but not in parental J cells that are devoid of entry receptors. A monoclonal antibody to nectin1, which blocks entry, also blocked cell-to-cell spread in nectin1-expressing J cells. Moreover, wild-type virus did not spread from a receptor-positive to a receptor-negative cell. (ii) The antibody to nectin1 blocked transmission of wild-type virus in a number of human cell lines, with varying efficiencies, suggesting that nectin1 is the principal mediator of wild-type virus spread in a variety of human cell lines. (iii) Nectin1 did not mediate cell fusion induced by the syncytial strains HSV-1(MP) and HFEM-syn. (iv) Nectin2α could serve as a receptor for spread of a mutant virus carrying the L25P substitution in glycoprotein D, but not of wild-type virus, in agreement with its ability to mediate entry of the mutant but not of wild-type virus.

By immunizing prion knockout mice ( Prnp −/−) with recombinant murine prion protein (PrP c ), we obtained a panel of mAbs specific for murine PrP c . These mAbs can be applied to immunoblotting, cell surface immunofluorescent staining, and immunohistochemistry at light and electron microscopy. These mAbs recognize both the normal (PrP c ) and protease-resistant (PrP res ) isoforms of PrP. Some mAbs are species restricted, while others react with PrP from a broad range of mammals including mice, humans, monkeys, cows, sheep, squirrels, and hamsters. Moreover, some of the mAbs selectively recognize different PrP glycoforms as well as the metabolic fragments of PrP c . These newly generated PrP c antibodies will help to explore the biology of PrP c and to establish the diagnosis of prion diseases in both humans and animals.

Varicella-zoster virus (VZV) is the etiological agent of chickenpox and shingles. Due to the virus's restricted host and cell type tropism and the lack of tools for VZV proteomics, it is one of the least-characterized human herpesviruses. We generated 251 monoclonal antibodies (MAbs) against 59 of the 71 (83%) currently known unique VZV proteins to characterize VZV protein expression in vitro and in situ . Using this new set of MAbs, 44 viral proteins were detected by Western blotting (WB) and indirect immunofluorescence (IF); 13 were detected by WB only, and 2 were detected by IF only. A large proportion of viral proteins was analyzed for the first time in the context of virus infection. Our study revealed the subcellular localization of 46 proteins, 14 of which were analyzed in detail by confocal microscopy. Seven viral proteins were analyzed in time course experiments and showed a cascade-like temporal gene expression pattern similar to those of other herpesviruses. Furthermore, selected MAbs tested positive on human skin lesions by using immunohistochemistry, demonstrating the wide applicability of the MAb collection. Finally, a significant portion of the VZV-specific antibodies reacted with orthologs of simian varicella virus (SVV), thus enabling the systematic analysis of varicella in a nonhuman primate model system. In summary, this study provides insight into the potential function of numerous VZV proteins and novel tools to systematically study VZV and SVV pathogenesis.

The specificities of a panel of erythrocyte-reactive MoAbs derived from NZB mice with autoimmune haemolytic anaemia (AIHA) were determined by immunoprecipitation and immunoblotting. Of the eight antibodies, two (IgG1 MoAb 105-2H and IgG2a MoAb 34-3C) immunoprecipitated a 105-kD component identified as the erythrocyte anion channel band 3. A similar band was also immunoprecipitated by the IgG2b MoAb 34-2B when used at relatively high concentrations, but none of the remaining hybridoma antibodies precipitated any labelled erythrocyte components. In immunoblotting experiments only 34-2B reacted with band 3, indicating that the epitope recognized by this MoAb is robust and differs from the determinant(s) recognized by 105-2H and 34-3C. The remaining MoAbs to react by immunoblotting were the IgM antibodies IE10 and 4C8, both of which bound to a doublet corresponding to band 4.1 from the internal erythrocyte membrane skeleton. Of the three MoAbs which gave negative results in immunoprecipitation and immunoblotting, the IgM antibodies 103-7E and 106-10E reacted poorly with intact erythrocytes by flow cytometry, but the IgG1 antibody 31-9D bound well. ELISAs demonstrated that all four IgM MoAbs are polyreactive, since they bound to histones from a panel of nuclear antigens, and additionally 103-7E reacted with phosphatidyl choline. It is concluded that band 3 is an important autoantigen in NZB AIHA. However, since 3/5 haemolytic MoAbs failed to precipitate this antigen, either these antibodies represent minor components of the total autoantibody response, or responses to diverse possibly non-protein surface antigens also contribute to the pathogenesis of the disease.

Mice with homologous disruption of the gene coding for the ligand-binding chain of the interferon (IFN) gamma receptor and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in the differentiation of functional CD4+ T cell subsets in vivo and resistance to infection. Wild-type 129/Sv/Ev mice are resistant to infection with this parasite, developing only small lesions, which resolve spontaneously within 6 wk. In contrast, mice lacking the IFN-gamma receptor develop large, progressing lesions. After infection, lymph nodes (LN) and spleens from both wild-type and knockout mice showed an expansion of CD4+ cells producing IFN-gamma as revealed by measuring IFN-gamma in supernatants of specifically stimulated CD4+ T cells, by enumerating IFN-gamma-producing T cells, and by Northern blot analysis of IFN-gamma transcripts. No biologically active interleukin (IL) 4 was detected in supernatants of in vitro-stimulated LN or spleen cells from infected wild-type or deficient mice. Reverse transcription polymerase chain reaction analysis with primers specific for IL-4 showed similar IL-4 message levels in LN from both types of mice. The IL-4 message levels observed were comparable to those found in similarly infected C57BL/6 mice and significantly lower than the levels found in BALB/c mice. Anti-IFN-gamma treatment of both types of mice failed to alter the pattern of cytokines produced after infection. These data show that even in the absence of IFN-gamma receptors, T helper cell (Th) 1-type responses still develop in genetically resistant mice with no evidence for the expansion of Th2 cells.

When endogenous mouse mammary tumor virus (MMTV) superantigens (SAg) are expressed in the first weeks of life an efficient thymic deletion of T cells expressing MMTV SAg-reactive T cell receptor (TcR) V beta segments is observed. As most inbred mouse strains and wild mice contain integrated MMTV DNA, knowing the precise extent of MMTV influence on T cell development is required in order to study T cell immunobiology in the mouse. In this report, backcross breeding between BALB.D2 (Mtv-6, -7, -8 and -9) and 38CH (Mtv-) mice was carried out to obtain animals either lacking endogenous MMTV or containing a single MMTV locus, i.e. Mtv-6, -7, -8 or -9. The TcR V beta chain (TcR V beta) usage in these mice was analyzed using monoclonal antibodies specific for TcR V beta 2, V beta 3, V beta 4, V beta 5, V beta 6, V beta 7, V beta 8, V beta 11, V beta 12 and V beta 14 segments. Both Mtv-8+ mice and Mtv-9+ mice deleted TcR V beta 5+ and V beta 11+ T cells. Moreover, we also observed the deletion of TcR V beta 12+ cells by Mtv-8 and Mtv-9 products. Mtv-6+ and Mtv-7+ animals deleted TcR V beta 3+ and V beta 5+ cells, and TcR V beta 6+, V beta 7+ and V beta 8.1+ cells, respectively. Unexpectedly, TcR V beta 8.2+ cells were also deleted in some backcross mice expressing Mtv-7. TcR V beta 8.2 reactivity to Mtv-7 was shown to be brought by the 38CH strain and to result from an amino acid substitution (Asn-->Asp) in position 19 on the TcR V beta 8.2 fragment. Reactivities of BALB.D2 TcR V beta 8.2 and 38CH TcR V beta 8.2 to the exogenous infectious viruses, MMTV(SW) and MMTV(SHN), were compared. Finally, the observation of increased frequencies of TcR V beta 2+, V beta 4+ and V beta 8+ CD4+ T cell subsets in Mtv-8+ and Mtv-9+ mice, and TcR V beta 4+ CD4+ T cells in Mtv-6+ and Mtv-7+ mice, when compared with the T cell repertoire of Mtv- mice, is consistent with the possibility that MMTV products contribute to positive selection of T cells., Journal Article, info:eu-repo/semantics/published