C-C motif chemokine ligand (CCL) production in equine peripheral blood mononuclear cells identified by newly generated monoclonal antibodies

Schnabel, Christiane L.; Wemette, Michelle; Babasyan, Susanna; Freer, Heather; Baldwin, Cynthia L.; Wagner, Bettina

doi:10.1016/j.vetimm.2018.09.003

Cited by 23 publications

(26 citation statements)

References 49 publications

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“…The serum-free cell culture supernatant was harvested after 6 days of incubation and rIL-4/K42 fusion protein was purified using a HiTrap NHS-Activated HP affinity column coupled with aIL-4 monoclonal antibodies and an ÄKTA Fast Protein Liquid Chromatography (FPLC) instrument (GE Healthcare, Piscataway, NJ, USA). Immunizations, subsequent cell fusion, and mAb screening and selection were performed as previously described [35–37]. Briefly, one BALB/c mouse was immunized with 2 μg purified rIL-4/K42 fusion protein initially followed by 4 injections every 2–3 weeks of 1 μg protein with an adjuvant (Adjuvant MM, Gerbu, Heidelberg, Germany).…”

Section: Methodsmentioning

confidence: 99%

The expression of equine keratins K42 and K124 is restricted to the hoof epidermal lamellae of Equus caballus

et al. 2019

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The equine hoof inner epithelium is folded into primary and secondary epidermal lamellae which increase the dermo-epidermal junction surface area of the hoof and can be affected by laminitis, a common disease of equids. Two keratin proteins (K), K42 and K124, are the most abundant keratins in the hoof lamellar tissue of Equus caballus. We hypothesize that these keratins are lamellar tissue-specific and could serve as differentiation- and disease-specific markers. Our objective was to characterize the expression of K42 and K124 in equine stratified epithelia and to generate monoclonal antibodies against K42 and K124. By RT-PCR analysis, keratin gene (KRT) KRT42 and KRT124 expression was present in lamellar tissue, but not cornea, haired skin, or hoof coronet. In situ hybridization studies showed that KRT124 localized to the suprabasal and, to a lesser extent, basal cells of the lamellae, was absent from haired skin and hoof coronet, and abruptly transitions from KRT124-negative coronet to KRT124-positive proximal lamellae. A monoclonal antibody generated against full-length recombinant equine K42 detected a lamellar keratin of the appropriate size, but also cross-reacted with other epidermal keratins. Three monoclonal antibodies generated against N- and C-terminal K124 peptides detected a band of the appropriate size in lamellar tissue and did not cross-react with proteins from haired skin, corneal limbus, hoof coronet, tongue, glabrous skin, oral mucosa, or chestnut on immunoblots. K124 localized to lamellar cells by indirect immunofluorescence. This is the first study to demonstrate the localization and expression of a hoof lamellar-specific keratin, K124, and to validate anti-K124 monoclonal antibodies.

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Section: Methodsmentioning

confidence: 99%

The expression of equine keratins K42 and K124 is restricted to the hoof epidermal lamellae of Equus caballus

et al. 2019

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“…The serum-free cell culture supernatant was harvested after 6 days of incubation and rIL-4/K42 fusion protein was purified, using a HiTrap NHS-Activated HP affinity column coupled with aIL-4 monoclonal antibodies and an ÄKTA Fast Protein Liquid Chromatography (FPLC) instrument (GE Healthcare, Piscataway, NJ, USA). Immunizations, subsequent cell fusion, and mAb screening and selection were performed as previously described [31-33]. Briefly, one BALB/c mouse was immunized with 2 μg purified rIL-4/K42 fusion protein initially followed by 4 injections every 2-3 weeks of 1 μg protein with an adjuvant (Adjuvant MM, Gerbu, Heidelberg, Germany).…”

Section: Methodsmentioning

confidence: 99%

The expression of equine keratins K42 and K124 is restricted to the hoof epidermal lamellae ofEquus caballus

Armstrong

Cassimeris

Santos

et al. 2019

Preprint

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HGH)2 29 Abstract 30 The equine hoof inner epithelium is folded into primary and secondary epidermal lamellae which 31 increase the dermo-epidermal junction surface area of the hoof and can be affected by laminitis, 32 a common disease of equids. Two keratin proteins (K), K42 and K124, are the most abundant 33 keratins in the hoof lamellar tissue of Equus caballus. We hypothesize that these keratins are 34 lamellar tissue-specific and could serve as differentiation-and disease-specific markers. Our 35 objective was to characterize the expression of K42 and K124 in equine stratified epithelia and to 36 generate monoclonal antibodies against K42 and K124. By RT-PCR analysis, keratin gene (KRT) 37 KRT42 and KRT124 expression was present in lamellar tissue, but not cornea, haired skin, or 38 hoof coronet. In situ hybridization studies showed that KRT124 localized to the suprabasal and, 39 to a lesser extent, basal cells of the lamellae, was absent from haired skin and hoof coronet, and 40 abruptly transitions from KRT124-negative coronet to KRT124-positive proximal lamellae. A 41 monoclonal antibody generated against full-length recombinant equine K42 detected a lamellar 42 keratin of the appropriate size, but also cross-reacted with other epidermal keratins. Three 43 monoclonal antibodies generated against N-and C-terminal K124 peptides detected a band of the 44 appropriate size in lamellar tissue and did not cross-react with proteins from haired skin, corneal 45 limbus, hoof coronet, tongue, glabrous skin, oral mucosa, or chestnut on immunoblots. K124 46 localized to lamellar cells by indirect immunofluorescence. This is the first study to demonstrate 47 the localization and expression of a hoof lamellar-specific keratin, K124, and to validate anti-48 K124 monoclonal antibodies. 3 49 1. Introduction 50 The skin and its appendages are made of stratified epithelia composed of keratinocytes, 51 defined by expression of keratin intermediate filament proteins (abbreviated K for proteins and 52 KRT for genes) [1]. Keratin filaments resist stretching (strain) and provide tensile strength to 53 epithelia and skin appendages. Tissue-and differentiation-specific variation in specific keratin 54 isoform content determines the physical and mechanical properties of diverse epithelial tissues 55 and of their keratinocyte building blocks [2;3]. Understanding how keratins function to provide 56 mechanical stability is crucial to our understanding of human diseases associated with keratin 57 mutations and abnormal keratin expression [4-8]. Here we describe unique keratins of the equine 58 (Equus caballus) epidermal lamellae, a highly specialized tissue that withstands extreme force 59 and provides a model for understanding how keratins provide mechanical strength to flexible 60 tissues. 61 Each single-toed foot of a 500 kg horse (E. caballus) must withstand peak ground reaction 62 forces of 2-5,000 N while protecting the underlying limb from trauma [9;10]. The equine 63 adaptation to single-toed unguligrade locomotion requires the ...

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“…20 Moreover, equine monocytes isolated from PBMC using a density gradient express CD14, CD16, CCL2 and CCL3. 16,94 Unlike humans and horses, mice do not have duplicated CD16 gene, while CD14 + CD16 + have also been detected in humans and horses but not in mice. 16 The MPS includes antigen-presenting DCs.…”

Section: F I G U R E 1 Summary Of Macrophage Functionsmentioning

confidence: 99%

“…However, they do express the scavenger receptor CD163, 93 as do equine alveolar and peritoneal macrophages (PMs) 20 . Moreover, equine monocytes isolated from PBMC using a density gradient express CD14, CD16, CCL2 and CCL3 16,94 . Unlike humans and horses, mice do not have duplicated CD16 gene, while CD14 + CD16 + have also been detected in humans and horses but not in mice 16 …”

Section: The Mononuclear Phagocyte System Of the Horsementioning

confidence: 99%

The equine mononuclear phagocyte system: The relevance of the horse as a model for understanding human innate immunity

Karagianni

Lisowski

Hume

et al. 2020

Equine Veterinary Journal

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Horses are already recognised as models for several human diseases, including metabolic syndrome, asthma, musculoskeletal diseases, melanoma and autoimmune uveitis. 1-6 More than 100 equine hereditable conditions may serve as models for human disorders, including inflammation, muscular or fertility disorders, osteoarthritis and even depression. 7-10 The horse has the potential to represent an appropriate

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C-C motif chemokine ligand (CCL) production in equine peripheral blood mononuclear cells identified by newly generated monoclonal antibodies

Cited by 23 publications

References 49 publications

The expression of equine keratins K42 and K124 is restricted to the hoof epidermal lamellae of Equus caballus

The expression of equine keratins K42 and K124 is restricted to the hoof epidermal lamellae of Equus caballus

The expression of equine keratins K42 and K124 is restricted to the hoof epidermal lamellae ofEquus caballus

The equine mononuclear phagocyte system: The relevance of the horse as a model for understanding human innate immunity

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