Research & Publications
Leonard Milstone's laboratory studies have focused mainly on the structure and function of epidermal keratinocytes in tissue culture, in experimental animals and in patients who have ichthyosis. Past work includes: identification and characterization of keratins 4 and 13; discovery of epican, the CD44 proteoglycan characteristically found on stratified squamous epithelia; production of PTHrP by keratinocytes; seminal observations on interferon gamma.
Clinical studies have focused on effects that systemic retinoids have on bones of patients who have ichthyosis or acne, and on characterization of rare genodermatoses, such as dermatosparaxis and ichthyosis with confetti. We regularly participate in Phase I studies of topical treatments for patients with inherited disorders of keratinization.
Extensive Research Description
Discovery of interferon gamma.In 1968, the only known ways to evoke interferon secretion were by live or dead viruses or by a special class of polynucleotides.Because of the presence of lymphocytes in tissue of brains infected with virus, I hypothesized that those cells were making a virus inhibitor.I further postulated, and then demonstrated, that antigen-sensitized immune cells would respond to an antigen, such as the protein component of the tuberculosis bacillus (PPD), with the production of a virus inhibitor.It turned out to be a distinct interferon, and others subsequently named this inhibitor interferon gamma.The in vitro procedure we used to produce interferon gamma now forms the basis of the widely used blood test for tuberculosis, the quantiferon gold assay.
Milstone LM, Waksman BH.Release of virus inhibitor from tuberculin‑sensitized peritoneal cells stimulated by antigen.J Immunol 105:1068‑1071, 1970
Discovery of K4 and K13 keratins.In 1978 other investigators had begun to characterize keratin proteins that formed intermediate filaments and to show that the unit structure of keratin intermediate filaments was a heteropolymer.The abundance of individual keratins in most tissues left open the question of whether the basic assembly unit was a dimer, trimer or multimer.Unfortunately the complexity of keratin proteins isolated from cutaneous tissues made this difficult to resolve.Fortuitously, I found that esophagus epithelium had only two predominant keratin proteins present in equal stoichiometric amounts.We then isolated and characterized an acidic keratin, later known as keratin 13, and a basic keratin, later known as keratin 4, and showed that purified K4 combined with purified K13 in equal amounts to form intermediate filaments.These data favored a unit structure composed of a dimer of acidic and basic pairs.This structure has been abundantly confirmed by many other investigators and a heterodimer is now accepted as the essential unit of all keratin intermediate filaments. Keratins 4 and 13 are mutated in the mucosal disorder called white sponge nevus.
Milstone LM, McGuire J.Different polypeptides form the intermediate filaments in bovine hoof and esophageal epithelium and in aortic endothelium.J Cell Biol 88:312‑316, 1981.
Milstone LM.Isolation and characterization of two polypeptides that form intermediate filaments in bovine esophageal epithelium.J Cell Biol 88:317‑ 322, 1981.
Discovery of epican and characterization of cell surface proteoglycans on keratinocytes. In 1988, before it was well-established that the cadherins were the most dominant adhesive molecules on keratinocytes, I postulated that cell surface proteoglycans might be important in keratinocyte cell-cell adhesion.We characterized proteoglycans on cultured keratinocytes and made monoclonal antibodies to their core proteins.We then isolated the most abundant proteoglycan on keratinocytes and cloned and characterized the heparin/chondrointin sulfate splice variant of CD44.We called this variant, which is exclusively expressed on stratified squamous epithelia, CD44Epican.We then showed that CD44Epican can mediate weak cell-cell adhesion through a hyaluronate bridge.Additional functions for Epican have been identified since then; no single critical function is known.
Haggerty JG, Bretton RH, Milstone LM.Identification and characterization of a cell surface proteoglycan on keratinocytes. J Invest Dermatol 99:374-380, 1992.
Kugelman LC, Ganguly S, Haggerty JG, Weissman SM, Milstone LM.The core protein of epican, a heparan sulfate proteoglycan on keratinocytes, is an alternative form of CD44.J Invest Dermatol 99:887-891, 1992.
Milstone LM, Hough-Monroe L, Kugelman LC, Bender JR, Haggerty JG. Epican, a heparan/chondroitin sulfate proteoglycan form of CD44, mediates cell-cell adhesion.J Cell Sci 107: 3183-3190, 1994.
Epidermis and systemic mineral metabolism.Stimulated by a difficult patient of mine who had pustular psoriasis, I organized a group to study the relationship between epidermal disease and systemic mineral metabolism.In that patient with pustular psoriasis and subsequently in others, we showed that pustular psoriasis of von Zumbush could be triggered by hypocalcemia caused by hypoparathyroidism (iatrogenic or idiopathic).We later collaborated to utilize cultured keratinocytes to purify, characterize and ultimately sequence PTH-RP, a major cause of humoral hypercalcemia of malignancy.We identified bony effects of systemic retinoids given to treat acne and other epidermal diseases.Finally, my lab developed a transgenic animal model to prove that epidermal desquamation could have a significant impact on iron stores in internal organs.
Stewart AF, Battaglini J, Milstone LM.Hypocalcemia‑induced pustular psoriasis of von Zumbusch: new experience with an old syndrome.Ann Int Med 100:677‑680, 1984.
Merendino JJ, Insogna KL, Milstone LM, Broadus AE, Stewart AF.A parathyroid hormone‑like protein from cultured human keratinocytes.Science 231:388‑390, 1986.
Milstone LM, McGuire J, Ablow RC.Premature epiphyseal closure in a child receiving oral 13‑cis retinoic acid.J Amer Acad Dermatol 7:663‑666, 1982.
Milstone LM, Ellison AF, Insogna KL.Serum parathyroid hormone is elevated in some patients with disorders of keratinization. Arch Dermatol 128:926-930, 1992.
Milstone LM, Hu R-H, Dziura JD, Zhou J.Impact of epidermal desquamation on tissue stores of iron.J Dermatol Sci, 67: 9-13, 2012
Functional delivery of oligonucleotides to epidermis in order to edit or modify expression of mutant genes. For the past 15 years I have worked on mutant RNA and DNA as targets for therapeutic intervention in epidermis.Both in my lab and as part of a large consortium, we have worked to improve methods of oligonucleotide delivery and to quantify their biochemical and biological effects. Notable achievements are the first demonstration of localized gene editing in (mouse) epidermis and a pilot demonstration of localized efficacy of a therapeutic siRNA in a human who has a keratin mutation.
Leachman SA, Hickerson RP, Schwartz ME, Bullough EE, Hutcherson SL, Boucher KM, Hansen CD,Eliason MJ, Srivatsa GS, Kornbrust DJ, Smith FJD, McLean WHI, Milstone LM, Kaspar RL.First-in human Mutation-targeted siRNA Phase Ib Trial of an Inherited Skin Disorder.Molec Therapy 18:442-446, 2010
Lara MF, González-González E, Speaker TJ, Hickerson RP, Leake D, Milstone LM, Contag CH, Kaspar RL. Inhibition of CD44 gene expression in human skin models using self-delivery siRNA administered by dissolvable microneedle arrays. Molec Ther, 23:816-823, 2012
Rogers FA, Hu R-H, Milstone LM.Local delivery of gene-modifying triplex-forming molecules to epidermis.J Invest Dermatol, 133:685-691, 2013
Clinical Disorders of Keratinization.As the founder of Yale’s Disorders of Keratinization Clinic and Chair of the Medical and Scientific Advisory Board of F.I.R.S.T. (the Foundation for Ichthyosis and Related Skin Types) I have had many opportunities to contribute to the lay and scientific understanding of those diseases, and to give those patients opportunities to participate in trials of new therapies.Recent projects that I organized include the discovery of a new mechanism for revertant mosaicism in diseased skin and the best available estimate of the incidence of moderate to severe ichthyosis in the United States.The broad collaboration needed to bring new treatments to patients with rare diseases is represented by the 2010 Leachman ref above.
Choate KA, Yu L, Zhou J, Choi M, Elias PM, Farhi A, Nelson-Williams C, Crumrine D, Williams ML, Nopper AJ, Bree A, Milstone LM, Lifton RP.Mitotic recombination in patients with ichthyosis causes reversion of dominant mutations in KRT10.Science, 330: 94-97, 2010
Milstone LM, Miller K, Haberman M, Dickens J.Incidence of moderate to severe ichthyosis in the United States. Arch Dermatol, 148:1080-1081, 2012
Choate KA, Lu, Y, Zhou J, Elias PM, Zaidi S, Paller AS, Farhi A, Nelson-Williams C, Crumrine D, Milstone LM, Lifton RA. Frequent somatic reversion of KRT1 mutations in ichthyosis with confetti. J Clin Invest, 125:1703-1707, 2015