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In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents.

CD200 is a widely distributed membrane protein that gives inhibitory signals through its receptor (CD200R) on myeloid cells. CD200 has been acquired by herpesviruses where it has been shown to interact with host CD200R and downmodulate the immune system. It has been hypothesized that poxviruses have acquired CD200; but the potential orthologues show less similarity to their hosts. Myxoma virus M141 protein is a potential CD200 orthologue with a potent immune modulatory function in rabbits. Here, we characterized the rabbit CD200, CD200R and tested the CD200-like sequences for binding CD200R. No binding could be detected using soluble recombinant proteins, full length protein expressed on cells or myxoma virus infected cells. Finally, using knockdown models, we showed that the inhibitory effect of M141 on RAW 264.7 cells upon myxoma virus infection is not due to CD200R. We conclude that the rabbit poxvirus CD200-like proteins cause immunomodulation without utilizing CD200R., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Several herpesviruses have acquired the gene for the CD200 membrane protein from their hosts and can downregulate myeloid activity through interaction of this viral CD200 orthologue with the host receptor for CD200, namely CD200R, which can give inhibitory signals. This receptor is a 'paired receptor', meaning proteins related to the inhibitory CD200R are present but differ in that they can give activating signals and also give a negligible interaction with CD200. We showed that the viral orthologues e127 from rat cytomegalovirus and K14 from human herpesvirus 8 do not bind the activating CD200R-like proteins from their respective species, although they do bind the inhibitory receptors. It is thought that the activating receptors have evolved in response to pathogens targeting the inhibitory receptor. In this case, the CD200 orthologue is not trapped by the activating receptor but has maintained the specificity of the host from which it was acquired, suggesting that the activating members of the CD200R family have evolved to protect against a different pathogen.

Adolescent idiopathic scoliosis (AIS) is a common orthopedic disorder of unknown etiology and pathogenesis. Melatonin and melatonin pathway dysfunction has been widely suspected to play an important role in the pathogenesis. Many different types of animal models have been developed to induce experimental scoliosis mimicking the pathoanatomical features of idiopathic scoliosis in human. The scoliosis deformity was believed to be induced by pinealectomy and mediated through the resulting melatonin-deficiency. However, the lack of upright mechanical spinal loading and inherent rotational instability of the curvature render the similarity of these models to the human counterparts questionable. Different concerns have been raised challenging the scientific validity and limitations of each model. The objectives of this review follow the logical need to re-examine and compare the relevance and appropriateness of each of the animal models that have been used for studying the etiopathogenesis of adolescent idiopathic scoliosis in human in the past 15 to 20 years.

One common way to study human leucocytes and cancer cells in an experimental in vivo situation is to use mice that have been genetically engineered to lack an immune system and prevent human cell rejection. These mice lack CD132 and either RAG2 or the catalytic subunit of the DNA-dependent protein kinase, to make the mice deficient in lymphocytes and natural killer cells. The NOD mouse strain provides a better background for engraftment than other strains due to stronger engagement of the signal-regulatory protein-α (SIRPα) inhibitory receptor with human CD47 (hCD47) resulting in a 'don't-eat-me' signal. To determine the molecular parameters that determine this major functional effect in the NOD mouse we measured the affinity of hCD47 for SIRPα from various mouse strains. Human CD47 bound SIRPα from the NOD mouse with an affinity 65 times greater than SIRPα from other mouse strains. This is due mainly to the NOD SIRPα lacking two amino acids in domain 1 compared with other mouse strains. Remarkably the SIRPα(NOD) binds hCD47 with 10 times the affinity of the syngeneic hCD47/hSIRPα interaction. This affinity is outside the normal range for affinities for leucocyte surface protein interactions and raises questions as to what is the optimal affinity of this interaction for engraftment and what other xenogeneic interactions involved in homeostasis may also not be optimal., (© 2014 The Authors. Immunology published by John Wiley & Sons Ltd.)

Host recognition of conserved pathogen-associated molecular patterns (PAMPs) and their interactions with pattern-recognition receptors, including the Toll-like receptors (TLR) is essential for innate immune response induction. The TLR1 family (TLR1, 2, 6 and 10) is involved in the recognition of gram-positive and gram-negative bacteria and heterodimers of TLR1 or TLR6 with TLR2 are crucial for the identification of several PAMPs. Studies on cell surface expression of TLR in ruminants are hampered by the lack of specific antibodies and no convincingly cross-reactive anti-human antibodies have been described so far. We describe herein four antibodies which recognise bovine TLR2. Differences in TLR2 expression were evident on bovine antigen presenting cells with high level expression on peripheral blood monocytes and monocyte-derived macrophages. Lower levels of expression were evident on dendritic cell populations derived in vitro and ex vivo, and on alveolar macrophages. One of the antibodies recognised TLR2 expression on ovine peripheral blood monocytes. The identification of antibodies specific for bovine and ovine TLR2 will facilitate studies of the role of this important PRR in the initiation of immune responses to important pathogens., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Tumour necrosis factor alpha (TNF-alpha) is an innate pro-inflammatory cytokine involved in protection against intracellular pathogens. Existing methods for measuring TNF-alpha production and function in ruminants are limited to ELISA and many rely on polyclonal antisera. With a view to developing improved detection methods for bovine (bov) TNF-alpha, monoclonal antibodies (mAb) were produced by immunising mice with a plasmid encoding bov TNF-alpha. Two of the resulting mAb, termed CC327 and CC328, were used to develop a sandwich ELISA capable of detecting both native and recombinant bov TNF-alpha. This ELISA did not detect recombinant ovine (ov) TNF-alpha. A luminometric method was applied to the ELISA to improve sensitivity for detection of native bov TNF-alpha in culture supernatants derived from bovine monocyte-derived dendritic cells (DC) infected with Mycobacterium bovis. Both CC327 and CC328 detected intracytoplasmic expression of TNF-alpha in mitogen-activated bovine T lymphocytes. However, only CC328 detected intracytoplasmic ovine TNF-alpha in transfected cells, explaining the failure of the sandwich ELISA to detect recombinant ov TNF-alpha. These mAbs have generated the capability to study the role of TNF-alpha in host immune protection and disease pathogenesis in ruminants., (Copyright 2010 Elsevier B.V. All rights reserved.)

Recombinant bovine IL-4 (rbo IL-4) was transiently expressed in COS-7 cells. Mice were immunised with a plasmid encoding rbo IL-4 and boosted with rbo IL-4. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-4 in an ELISA and these cloned hybridomas were termed CC311, CC312, CC313 and CC314. A pair of mAb (CC313 and CC314) was identified that together could be used to detect both recombinant and native bovine IL-4 by ELISA and a luminometric detection method was applied to the ELISA. Using this method native bovine IL-4 was detected in supernatants of PBMC stimulated with mitogens. In addition, high level secretion of IL-4 by Fasciola hepatica specific Th2 clones, but not by a Babesia bovis specific Th1 clone, was confirmed. The ELISA was also able to detect recombinant ovine IL-4. The pair of mAb used for ELISA could also be used for the detection of IL-4 spot forming cells by ELISPOT. In addition intracytoplasmic expression of IL-4 could be detected. The ability to detect ruminant IL-4 by three methods: ELISA, ELISPOT and by flow cytometric analysis of intracytoplasmic expression will permit studies of the role of this important cytokine in the immunology and pathogenesis of animal diseases.

The objective of the investigation was to provide data on how a prior exposure of cattle to Mycobacterium avium, used here as a model of exposure to an environmental mycobacterium, affected the cellular immune response that follows vaccination with Mycobacterium bovis BCG. The assessment of cellular immune responses included lymphocyte proliferation assays, the delayed hypersensitivity skin test and IFN-gamma synthesis in whole blood cultures. One group of calves was inoculated subcutaneously with M. avium followed 12 weeks later by M. bovis-BCG. The other group was vaccinated subcutaneously with BCG alone. Calves previously exposed to M. avium responded more rapidly, as assessed in the in vitro assays, to purified protein derivative (PPD) from M. avium (PPD-A) or M. bovis (PPD-B) than did calves inoculated with BCG only, indicating that the exposure to M. avium had primed the immune response in these calves. Following inoculation of BCG the intensity of the in vitro responses and the delayed hypersensitivity skin test to PPD-A was higher for the M. avium-primed animals while the responses to PPD-B were similar in the M. avium-primed and BCG-only groups. The results are consistent with a model in which prior exposure to environmental mycobacteria does not necessarily inhibit the immune response to the vaccine strain, BCG. They suggest that M. avium infection primes the immune system of calves and that the detection of an immune response specific for M. bovis BCG is masked by reactivity to antigens also present in M. avium.

Recombinant bovine IL-12 (rbo IL-12) was transiently expressed in COS-7 cells and shown to upregulate the synthesis of IFNgamma by bovine cells stimulated with a suboptimal concentration of mitogen in vitro. Mice were immunised with a plasmid encoding rbo IL-12 and boosted with rbo IL-12 and a number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-12 in an ELISA. Some of these mAb neutralised the ability of rbo IL-12 to induce IFNgamma synthesis by bovine cells. A pair of mAb was identified that together could be used to detect both recombinant and natural bovine IL-12 by ELISA and a luminometric detection method was applied to the ELISA making it more sensitive. Using this method native bovine IL-12 was detected in supernatants of dendritic cells (DC) cultured in vitro with a synthetic lipopeptide known to stimulate secretion of IL-12 by human DC. The ELISA was also able to detect recombinant ovine IL-12 and, less effectively, recombinant human IL-12. In contrast, bovine IL-12 was not detected by a commercial human IL-12 ELISA kit. Intracytoplasmic IL-12 was detected in bovine DC using the antibodies described herein. The ability to detect ruminant IL-12 by three methods: ELISA, bioassay with neutralising mAb and cytoplasmic staining, will permit studies of the role of this important cytokine in the immunology and pathogenesis of animal diseases.

The objective of the study was to develop an assay for bovine IL-10 that could be applied to analyses of immune responses and advance understanding of a variety of diseases of cattle. Recombinant bovine IL-10 (rbo IL-10) was transiently expressed in Cos-7 cells and shown to inhibit the synthesis of IFN gamma by bovine cells stimulated with antigen in vitro. Mice were immunised with a plasmid containing a cDNA insert encoding rbo IL-10 and inoculated with rbo IL-10. A number of monoclonal antibodies (mAb) were generated that reacted with rbo IL-10 in an ELISA. Some of these mAb neutralised the ability of rbo IL-10 to inhibit IFN gamma synthesis by antigen-stimulated bovine cells. A pair of mAb was identified that together could be used to detect both recombinant and natural bovine IL-10 present in supernatant of PBMC stimulated with ConA. A luminescent detection method was applied to the ELISA making it more sensitive. Using this method native IL-10 was detected in supernatants of PBMC, diluted blood and undiluted blood from cattle immunised with Mycobacterium bovis BCG or ovalbumin and incubated in vitro with antigen indicating the applicability of the assay to a number of in vitro culture systems.

Twelve subpanels of monoclonal antibodies (MAb) included within the 6th International Workshop on Human Leukocyte Differentiation Antigens (6th HLDA) were assayed for reactivity with bovine peripheral blood leukocytes. Sixty-nine of the 807 MAb (8.6%) stained bovine cells. These MAb represented 30 different human CD groups. Nine of the MAb to different human CD antigens (CD19, CD23, CD39, CD47, CD86, CD117, CD120b, CDw149, CD165) potentially recognized antigens on cattle cells that had not previously been identified. These were investigated further by two-colour immunofluorescence to compare the cellular expression of the antigen on cattle cells with that reported for the different CD antigens in humans. Four of the MAb that belonged to CD23, CD39, CD47, and CDw149 stained bovine cells in a manner that indicated an almost identical cellular distribution of the antigen to that reported in humans. This implied that these MAb reacted with the homologous cattle molecules. Further work would be necessary to confirm specificity of CD19, CD86, CD117, CD120b and CD165 MAb. Other cross-reacting MAb either recognized antigens already defined in cattle or antigens not yet clustered in humans. The study has identified valuable new reagents for studies of cattle and confirmed that most common cross-reactive MAb are to epitopes on integrins.

Few data are available regarding the induction of memory T-lymphocyte responses in cattle following Bacille Calmette Guérin (BCG) vaccination. Studies of the immune response induced by BCG vaccination provide an insight into the basis of antimycobacterial immunity that could be exploited for the development of more effective vaccination strategies. We used autologous dendritic cells (DC) infected with Mycobacterium bovis Bacille Calmette Guérin (BCG) or pulsed with purified protein derivative from M. bovis (PPD-B) or M. avium (PPD-A) to assess responses of CD4+, CD8+ and WC1+ gammadelta TCR+ lymphocytes from BCG vaccinated and nonvaccinated cattle. Mycobacteria-specific CD4+ and CD8+, but not WC1+ gammadelta TCR+, memory T lymphocytes were demonstrated in BCG-vaccinated cattle. CD4+ and CD8+ lymphocytes proliferated and produced interferon (IFN)-gamma in response to BCG-infected or PPD-B-pulsed DC. Proliferative responses were greater for CD4+ than CD8+ lymphocytes, although secretion of IFN-gamma was higher from the CD8+ T cells. Responses to PPD-A-pulsed DC were lower, with no CD8+ response. Lymphocytes from nonvaccinated calves were also stimulated to proliferate by BCG-infected DC, although the magnitude of proliferation was lower. The findings suggest that immunity to M. bovis induced by BCG vaccination in cattle may involve CD8+ memory T cells which produce IFN-gamma, as well as CD4+ memory T cells.

Immunofluorescent staining and flow cytometric analysis of dendritic cells from cattle afferent lymph has established that within the afferent lymph veiled cells (ALVC) there are two phenotypically distinct, major populations. One is CD11a+, CD5+, CD21- and expresses the bovine WC10 (workshop cluster 10) molecule and the Ag recognized by mAb CC81 but is not recognized by mAbs CC149 and IL-A24. The second ALVC subpopulation is CD11a-, CD5-, CD21+/-, workshop cluster 10- and is not recognized by mAb CC81 but is recognized by mAb CC149. Thus, the two populations, which can be identified by staining for CD11a, are defined by the differential expression of a number of Ag. The ALVC populations had differing capacities to stimulate T cells. CD11a- ALVC were more effective at stimulating proliferative responses in allogeneic CD4+ T cells and CD8+ T cells. This was not related to binding of CTLA4Ig or CD40L fusion proteins, implying similar levels of expression of their ligands, CD80 and CD86 or CD40. Both subsets were able to present OVA to resting memory CD4+ T cells, indicating that both were able to take up and process soluble native protein. In contrast, the CD11a- ALVC were more effective in presenting respiratory syncytial virus Ag to resting CD4+ T cells. Considering the central role of dendritic cells in the initiation of immune responses in naive animals, the two cell types may have different roles in the induction of primary responses induced following infection or immunization.

Six monoclonal antibodies (mAbs) that clustered together in a statistical analysis of data submitted to the Third Workshop on Ruminant Leucocyte Antigens and mAb CC-G33 were tested for ability to stain COS-7 cells transfected with cDNA encoding human CD14. Only mAb CC-G33 recognised the human molecule. The six mAbs were compared with mAb CC-G33 by flow cytometry and three were shown to be directed against bovine CD14.

Dendritic cells were identified in afferent lymph derived by lymphatic cannulation of cattle, stained with monoclonal antibody (mAb) to the bovine workshop cluster 6 (WC6) antigen, which is highly expressed on bovine afferent lymph veiled cells, and sorted with a fluorescence-activated cell sorter. These cells expressed major histocompatibility complex (MHC) class I and II and CD1b but not CD14. They bound human and murine CTLA4-immunoglobulin (CTLA4-Ig) fusion proteins indicating expression of CD80 and or CD86. Dendritic cells induced proliferative responses in allogeneic CD4+ and CD8+ cells sorted from blood but did not induce responses in purified allogeneic WC1+, gamma/delta T cells, which are CD2-, CD4-, CD8- and are the major gamma delta T-cell population in cattle blood, even when interleukin-2 (IL-2) was added to cultures. A WC1-, CD2+ gamma delta T-cell receptor (TCR)+ population predominates in cattle spleens and proliferation of a T-cell line with this phenotype was not induced by allogeneic dendritic cells, with or without added IL-2. The observations imply that the ligand for the gamma delta TCR expressed on the two populations is not present on allogeneic dendritic cells or that the costimulatory molecules expressed on dendritic cells that render them highly effective at stimulating MHC class I- and class II-restricted CD8+ and CD4+ T cells are not recognized by the WC1+ or WC1- gamma/delta T cells. Expression of CD28 by the four cell types was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Purified CD4+ and CD8+ cells both produced CD28 transcripts but neither purified WC1+ cells nor the WC1- gamma delta TCR+ cell line did so. The findings indicate that CD80 and or CD86 are involved in the stimulation of CD4+ and CD8+ alpha beta TCR+ T cells but not in the stimulation of either of the two gamma delta TCR+ populations.