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Galantino-Homer Laminitis Laboratory

LDD Gross Composite

Laminitis is a common and debilitating disease that affects the folded and interdigitating tissues, called the lamellae, which connect the hoof wall to the underlying tissues of the horse’s foot. The lamellae normally allow the transfer of the horse's weight from the skeletal elements of the digit to the hoof wall.

Dr. Galantino-Homer founded the Laminitis Laboratory at New Bolton Center in 2008. The Laminitis Laboratory was formed in part due to the tragic loss of the 2006 Kentucky Derby winner, Barbaro, to laminitis in January, 2007.

Our goal is to employ cell and molecular biology methods to better understand laminitis pathogenesis in order to improve the prevention, diagnosis, and treatment of this disease.

Our studies include the investigation of laminitis pathogenesis using protein biochemistry, gene expression, and histological analysis, identification of diagnostic serum biomarkers for laminitis, characterization of the keratin proteins that determine the mechanical properties of the hoof lamellae and the effect of laminitis on keratins and associated cell adhesion proteins, the impact of cell stress pathways on laminitis, characterization of the epidermal stem cell population in the lamellae, and establishing an in vitro culture system for equine hoof epidermal cells to minimize the use of live horses for laminitis research.

Learn About Our Work

From the Laminitis Discovery Database (LDD) to ongoing, collaborative research studies, the Galantino-Homer Laminitis Laboratory has focused on this all too common disease for more than two decades. Learn more about our work. Explore what we do.

Current projects include collaborations with Lynne Cassimeris (Lehigh University, Cell Biologist, ER stress and keratin studies), Andrew van Eps (Penn Vet Laminitis Research), Julie Engiles (Penn Vet, Veterinary Pathologist, LDD histopathology studies, laminitis and bone pathology), Darko Stefanovski (Penn Vet, Biostatistics), Samantha Brooks (University of Florida, Equine Genetics, gene expression changes with laminitis), and Bettina Wagner (Cornell University, Equine Immunology and Serum Diagnostics, monoclonal antibody production, serum biomarker studies).

Laminitis Research at New Bolton Center projects are funded by grants from private foundations, including the Grayson-Jockey Club Research Foundation, the American Association of Equine Practitioners Foundation, the Animal Health Foundation, and by generous donations to the Laminitis Research Fund.

Laminitis Discovery Database (LDD)

The LDD is an archive of data and sample sets from naturally-occurring laminitis case and unaffected control horses collected by the Galantino-Homer Laminitis Lab since 2008.


Laminitis Discovery Database, Figure 1

 

Figure 1: Laminitis Discovery Database: Distribution of types of naturally-occurring laminitis cases archived, 91 cases total. 51 non-laminitic controls are also archived in the LDD. The archive currently includes sample sets for over 350 feet. PBZ: phenylbutazone; DSLD: Digital Suspensory Ligament Desmitis; PPID: Pituitary Pars Intermedia Dysfunction; EMS: Equine Metabolic Syndrome; UnK: Unknown; SLL: Supporting Limb Laminitis; Inflam: Inflammatory. Totals as of 8/31/18.

 


The complete data and sample set for each foot includes:

  • gross pathology images
  • measurements, and detailed scoring
  • snap-frozen samples from skin
  • lamellar, and coronary regions of each foot
  • frozen serum and plasma
  • formalin-fixed, paraffin and frozen sections for histology
  • stained histology slides
  • histopathology qualitative scoring and morphometry measurements
  • historical information from 51 non-laminitic control horses and 91 laminitic horses

These materials are being used for several published and ongoing multi-institutional collaborative studies and for educational purposes.

The LDD also has archived samples provided by Christopher Pollitt (University of Queensland, Queensland, Australia), from the Hyperinsulinemia and Oligofructose experimental models of laminitis, and by Andrew van Eps (Penn Vet) and James Belknap (the Ohio State University), from the Hyperinsulinemia with and without limb cooling experimental model of laminitis.

Images from the LDD have been used for educational and research seminars and social media posts for veterinary students, scientists (veterinary and biomedical), veterinarians, farriers, and horse owners.

Studies that have utilized the LDD include an investigation of the effect of chronic laminitis on the expression of p63, and epithelial stem cell marker, in epidermal lamellae, a collaborative study with Julie Engiles (Penn Vet) on bone loss during laminitis, and all current studies in the lab. In collaboration with Julie Engiles and Darko Stefanovski, we are conducting comparative histopathology studies using LDD samples from horses with Supporting Limb Laminitis (SLL), Endocrinopathy-associated Laminitis (EL), and sepsis-associated laminitis to provide information to go with the database samples, in order to evaluate data derived from these samples for other studies relative to disease severity, and to insight into the different mechanisms that result in failure of digital support due to laminitis.

  • Representative Publications

    Engiles, J.B., Galantino-Homer, H., Boston, R., McDonald, D., Dishowitz, M., Hankenson, K.D. Osteopathology in the equine distal phalanx associated with the development and progression of laminitis. Vet Pathol. 52(5):928-44, 2015. Epub 2015 June 10.

    Linardi, R., Megee, S., Mainardi, S., Senoo, M., Galantino-Homer, H. Expression and localization of epithelial stem cell and differentiation markers in equine skin, eye and hoof. Veterinary Dermatology 26 (4): 213-e47, 2015; Epub 2015 May 12.

    Carter, R.A., Megee, S.O., Engiles, J., Senoo, M., Galantino-Homer, H.L. Decreased expression of p63, a regulator of epidermal stem cells, in the chronic laminitic equine hoof. Equine Vet J 43 (5): 543-551, 2011; Epub 2011 Mar 11.

Laminitis and Cell Stress

In collaboration with Lynne Cassimeris, a cell biologist from Lehigh University, we have been looking at the cell stress pathways that could be providing the link between the risk factors for laminitis (hyperinsulinemia in EL, excessive weight-bearing in SLL) and the loss of lamellar tissue integrity.

These pathways are being intensively studied for human diseases, including diabetes mellitus and neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases, and we could therefore benefit from the rapid advances in understanding and therapy that are emerging from those studies.

We completed a study of naturally-occurring EL and endoplasmic reticulum (ER) stress and we are currently completing studies investigating the HI experimental model (funded by the Animal Health Foundation with an application submitted with Dr. van Eps to the Grayson-Jockey Club Research Foundation to extend those studies to investigate the preventive effect of limb cooling).


Fig 2 Stress during Laminitis

Figure 2: ER Stress during laminitis. A) BiP, a marker of ER stress (green stain) localizes to abnormal lamellar cells adjacent to the keratinized axis of the primary epidermal lamella (KA) in laminitic lamellae. B) Abnormal histological morphology and PASH staining of lamellar cells in the region that also contains the BiP-positive cells. This region also undergoes abnormal cornification to form the “lamellar wedge” lesion. ER stress appears to either contribute to, or result from, the loss of normal lamellar structure and integrity.


  • Representative Publication

    Cassimeris, L. and Galantino-Homer, H., Detection of endoplasmic reticulum stress and the unfolded protein response in obesity- and endocrinopathy-associated equine laminitis. Under review (6/12/2018), BMC Vet. (Submitted, under review).

Lamellar Keratins and Diagnostic Biomarkers

Keratins are the major structural proteins of the hoof lamellae and are responsible for the mechanical strength (or failure) of the hoof and hoof lamellae. We discovered that the major keratins of the hoof lamellae, K42 and K124, had previously been uncharacterized. We have performed gene expression studies that show that the expression of these keratins is impacted by laminitis.

Because they are so abundant, we also have been interested in using keratins as biomarkers for lamellar damage during laminitis, for both diagnosis and possibly as a prognostic indicator. Toward this aim, we have generated monoclonal antibodies against the lamellar keratins, which we are currently validating as specific markers of lamellar keratin.

These antibodies will only detect lamellar keratin, not keratin from the skin, making them potentially useful serum marker for laminitis diagnosis. Moreover, these will be very useful for cell culture studies since, prior to now, although we had identified non-specific markers for hoof lamellar stem cells, we had no marker to specifically identify that cells grown in culture were biologically similar to cells in the lamellae, a necessary step in the validation of an in vitro disease model system.


Figure 3A Laminitis Lab Figure 3b Laminitis Lab 

Figure 3: Quantification and localization of the basal cell keratin, K14, and lamellar keratin, K124 in epidermal lamellae.

Graph: The four most abundant type I and type II keratin proteins in equine lamellar tissue, quantified by spectral counting. From: Carter et al. J Anim Sci 88 (12): 3843-3855, 2010. A) Localization of the basal cell keratin, K14, to basal cells of the secondary epidermal lamellae. B) Localization of the lamellar-specific keratin, K124, to basal and suprabasal cells of the secondary epidermal lamellae. Scale bars = 50 μm. From: Linardi et al. Veterinary Dermatology 26 (4): 213-e47, 2015.


  • Representative Publications

    Carter, R.A., Shekk, V., de Laat, M.A., Pollitt, C.C., Galantino-Homer, H.L. Novel keratins identified by quantitative proteomic analysis as the major cytoskeletal proteins of equine (Equus caballus) hoof lamellar tissue. J Anim Sci 88 (12): 3843-3855, 2010; Epub 2010 Jul 9.

    Linardi, R., Megee, S., Mainardi, S., Senoo, M., Galantino-Homer, H. Expression and localization of epithelial stem cell and differentiation markers in equine skin, eye and hoof. Veterinary Dermatology 26 (4): 213-e47, 2015; Epub 2015 May 12.

Dermal and Epidermal Pathology in Laminitis

An early study in the lab used mass spectroscopy to quantify protein changes in experimental models of laminitis. These “discovery mode” studies, along with our observations from histopathology studies, have led to hypothesis-driven studies to determine how and why the lamellae fail, including the above mentioned cell stress studies and keratin studies.

Alumna Sarah Colmer, V’17, has returned to New Bolton Center as a hospital intern and has also returned to the Laminitis Lab to complete studies that she started as a student to determine the effect of laminitis on a marker of dermal lamellar tissue damage that had come up in the proteomics study.

On the epidermal side, we are interested in the impact of laminitis on the types of keratins and changing morphology of the epidermal lamellar cells and changes in interactions between keratin and cell-cell adhesion complexes, again based on the earlier study. Even when the levels of one keratin do not change, we find that laminitis causes changes in the types of cells that are expressing that keratin, consistent with changes in the biology and mechanics of these cells.


Laminitis Lab Fig4

Figure 4: Keratin-14 expression increases in suprabasal cells as a marker of epidermal dysplasia in laminitic lamellae. A) K14 expression is restricted to basal cells in non-laminitic lamellae. B) Moderate epidermal dysplasia, K14 expression in suprabasal cells in some secondary epidermal lamellae. C) Severe epidermal dysplasia in a severely laminitic sample, K14 expression in all epidermal cells.



– Submitted/under review –

Cassimeris, L., Galantino-Homer, H., Engiles, J.B. Detection of endoplasmic reticulum stress and the unfolded protein response in obesity- and endocrinopathy-associated equine laminitis. Under review (6/12/2018), BMC Vet.


– Peer-reviewed papers –

Coleman, M., Belknap, J., Eades, S., Fraley, B., Galantino-Homer, H., Geor, R., McCue, M., McIlwraith, W., Moore, R., Peroni, J., Townsend, H., White, N., Cummings, K., Ivanek-Miojevic, R., Cohen, N. Case-control study of pasture-and endocrinopathy-associated laminitis in horses. JAVMA 253(4): 470-478, 2018.

Engiles, J.B., Galantino-Homer, H., Boston, R., McDonald, D., Dishowitz, M., Hankenson, K.D. Osteopathology in the equine distal phalanx associated with the development and progression of laminitis. Vet Pathol. 52(5):928-44, 2015. Epub 2015 June 10.

Linardi, R., Megee, S., Mainardi, S., Senoo, M., Galantino-Homer, H. Expression and localization of epithelial stem cell and differentiation markers in equine skin, eye and hoof. Veterinary Dermatology 26 (4): 213-e47, 2015; Epub 2015 May 12.

Clark, R.K. and Galantino-Homer, H.L. Wheat Germ Agglutinin as a Counterstain for Equine Hoof Lamina Immunofluorescence Studies.  Exp Dermatol. 23: 677-678; Epub 2014 July 16.

Carter, R.A., Megee, S.O., Engiles, J., Senoo, M., Galantino-Homer, H.L. Decreased expression of p63, a regulator of epidermal stem cells, in the chronic laminitic equine hoof. Equine Vet J 43 (5): 543-551, 2011; Epub 2011 Mar 11.

Carter, R.A., Shekk, V., de Laat, M.A., Pollitt, C.C., Galantino-Homer, H.L. Novel keratins identified by quantitative proteomic analysis as the major cytoskeletal proteins of equine (Equus caballus) hoof lamellar tissue. J Anim Sci 88 (12): 3843-3855, 2010; Epub 2010 Jul 9.


– Reviews and Book Chapters –

Galantino-Homer, H. and Engiles, J.B. Insulin Resistance and Laminitis in Broodmares. J Equine Vet Sci. 32 (10): 680-688, 2012.

Galantino-Homer, H. Endocrinopathic laminitis and equine metabolic syndrome. 2012 Annual Conference Proceedings, Society for Theriogenology/American College of Theriogenologists. Clinical Theriogenology 4, 2012.


– Abstracts/Proceedings papers (Recent) –

Cassimeris, L., Galantino-Homer, H. Detection of ER stress markers in laminitic lamellar tissue from horses with endocrinopathy-associated laminitis. Abstract P1507, Annual Meeting of the American Society for Cell Biology in San Francisco, CA, December 3-7, 2016 (poster presentation). http://www.ascb.org/2016meeting/2016program/

Linardi, R.L., Clark, R.K and Galantino-Homer, H.L. A lectin counterstain for immunofluorescence studies of equine hoof lamellar tissue and cultured keratinocytes. Abstract P627, Annual Meeting of the American Society for Cell Biology, Philadelphia, PA, December 6-10, 2014 (poster presentation).

Modelski, M., Kopper, J., Colmer, S.F., Armstrong, C., Clark, R.K., Brooks, S., Lorom, D., Engiles, J. and Galantino-Homer, H. Lamellar tissue-specific expression of novel keratins and the effect of laminitis on keratin isoform expression. Abstract P543, Annual Meeting of the American Society for Cell Biology in Philadelphia, PA, December 6-10, 2014 (poster presentation).

Galantino-Homer, H.L., Clark, R.K., Linardi, R.L. “Characterization of equine hoof lamellar tissue microanatomy with fluorescent markers.” American Association of Equine Practitioners Annual Convention, Salt Lake City, UT, December 6-10, 2014 (oral presentation). American Association of Equine Practitioners Annual Convention Proceedings 60: 73-78, 2014.


– Publications, Galantino-Homer Reproduction Research –

Honaramooz, A., Megee, S., Zeng, W., Destrempes, M.M., Overton, S.A., Luo, J., Galantino-Homer, H., and 10 others. Adeno-associated virus (AAV) –mediated transduction of male germ line stem cells results in transgene transmission after germ cell transplantation. FASEB J. 22(2):374-82. 2008; Epub ahead of print, Sept 14, 2007.

Galantino-Homer, H.L., Zeng, W.,  Megee, S.O., Dallmeyer, M., Voelkl, D., Dobrinski, I. Effects of b-cyclodextrin and cholesterol on porcine sperm viability and capacitation status following cold shock or incubation. Mol. Reprod. Dev. 73(5): 638-650, 2006.

Galantino-Homer, H.L., Florman, H., Storey, B.T., Kopf, G.S. Bovine sperm capacitation: assessment of phosphodiesterase activity and intracellular alkalinization on capacitation-associated protein tyrosine phosphorylation. Mol.Reprod.Dev. 67: 487-500, 2004.

Honaramooz, A., Behboodi, E., Megee, S.O., Overton, S.A., Galantino-Homer, H., Yann, E., Dobrinski, I. Fertility and germline transmission of donor haplotype following germ cell transplantation in immunocompetent goats. Biol.Reprod. 69: 1260-1264, 2003.

Galantino-Homer, H.L. cAMP-dependent protein tyrosine phosphorylation during bovine sperm capacitation. Doctoral dissertation in Cell and Molecular Biology, University of Pennsylvania, 2000.

Visconti, P.E., Galantino-Homer, H., Ning, X.P., Moore, G.D., Valenzuela, J.P., Jorgez, C.J., Alvarez, J.G., Kopf, G.S. Cholesterol efflux-mediated signal transduction in mammalian sperm. J. Biol. Chem. 274(5), 3235-3242, 1999.

Galantino-Homer, H.L., Visconti, P.E., Kopf, G.S. Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by a cyclic adenosine 3,5’-monophosphate-dependent pathway. Biol. Reprod., 56: 707-719, 1997.

Tokuyama, K., H.L. Galantino, R. Green, G.L. Florant. Seasonal glucose uptake in marmots (marmota flaviventris): The role of pancreatic hormones. Comp.Biochem.Physiol. 100A(4): 925-930. 1991.

Galantino-Homer, H. “Glossary” In: Developmental Biology, vol. 9, by S. Gilbert. Sinauer Associates Inc. (Sunderland, MA) 2010.

Bailey, J.L., Lessard, C., Jacques, J., Brèque, C., Dobrinski, I., Zeng, W., Galantino-Homer, H.L. Cryopreservation of boar semen and its future importance to the industry. Theriogenology 70: 1251-1259, 2008.

Vadnais, M.L., Galantino-Homer, H., Althouse, G.C. Current concepts on membrane activities associated with bovine and porcine sperm capacitation. Arch. Androl. 53: 109-123, 2007.

Kopf, G.S., Visconti, P.E., Galantino-Homer,H. Capacitation of the mammalian      spermatozoon. In: Advances in Developmental Biochemistry, vol. 5, Ed. P. Wassarman. JAI Press, Inc. (Stamford, CT) 83-107, 1999.

Kopf, G.S., Ning, X.P., Visconti, P.E., Purdon, M., Galantino-Homer, H., Fornés, M. Signaling mechanisms controlling mammalian sperm fertilization competence and activation. In: The Male Gamete: From Basic Science to Clinical Application. Ed. C. Gagnon. Cache River Press (Vienna, IL) 1999.

Visconti, P.E., Galantino-Homer, H., Moore, G.D., Bailey, J.L., Ning, X.P., Fornes, M., Kopf, G.S. The molecular basis of sperm capacitation. J. Androl., 19: 242-248, 1998.

Kopf, G.S., Visconti, P.E., Moos, J., Galantino-Homer, H.L., Ning, X.P. Integration of tyrosine kinase- and G protein-mediated signal transduction pathways in the regulation of mammalian sperm function. In: Human Sperm Acrosome Reaction.  Eds. P. Fénichel and J. Parinaud. Colloque INSERM/John Libbey Eurotext, Ltd. Vol. 236, 191-216, 1995.


– Ongoing –

American Association of Equine Practitioners Foundation

  • “Transcriptome analysis of supporting limb laminitis”
  • Role: Co-I, PI: Samantha Brooks, U Florida

Our objective is the identification of genes and signaling pathways that are differentially expressed in lamellar tissue from naturally-occurring supporting limb laminitis (SLL) and control cases from the Laminitis Discovery Database (LDD) in association with severity and duration of gross and histopathological evidence of digital destabilization. This study will be the first to apply next generation quantitative total mRNA sequencing from control and SLL (including unaffected, developmental, and acute phase feet) equine hoof lamellar tissue and to apply bioinformatics analysis to identify differential expression of genes associated with specific pathological and physiological functions and thereby better understand SLL pathogenesis.

American Association of Equine Practitioners Foundation

  • “Endoplasmic reticulum stress and epidermal pathology in supporting limb laminitis”
  • Role: PI, Co-Is: Lynne Cassimeris (Lehigh U), Julie Engiles (Penn Vet), Samantha Brooks (UF)

The goal of this study is to utilize natural cases and controls in the Laminitis Discovery Database (LDD) to: 1) determine the effects of the developmental, acute and chronic phases of supporting limb laminitis (SLL) in Thoroughbreds on epidermal histopathology, keratin isoform localization, and localization and phosphorylation of desmoplakin (a desmosomal protein) as indicators of altered epidermal phenotype and loss of epithelial integrity, 2) test our hypothesis that SLL pathogenesis is associated with endoplasmic reticulum (ER) stress, and 3) determine if lamellar-specific keratins can be detected in the serum of SLL cases as tissue- and disease-specific diagnostic/prognostic biomarkers.