Isolation of M. leprae and M. lepromatosis protein antigens and the impact on global leprosy

By guest bloggers William Levis and Frank Martiniuk of NYU School of Medicine, Departments of Medicine and Dermatology


In the 1980’s, we made significant advances in the serodiagnosis of leprosy using carbohydrate antigens (Levis et al, 1986).  Since then, with the rapid advances on isolation and advances in protein technology, an international consortium of Dutch (Geluk et al, 2011), Brazilian and US scientists led by Duthie (Rada et al, 2011) and Spencer (Spencer et al, 2012) have added to this early work, making leprosy the most exciting immunologic disease for the study of antibodies and T-cell subsets including the old TH1-TH2 paradigm (Modlin, 1994) and the newer subsets of TH17, TH9 and TH22 now under intense study in human inflammatory and neoplastic skin diseases (Martiniuk et al, 2012; Lowes et al, 2013).

The recent Duthie and Spencer serologic test for leprosyis a major advance in leprosy diagnosis and management, as it can monitor therapy and detect subclinical cases that allow for early detection and treatment,  resulting in reduction of residual deformity.  The current difficult and technically challenging Fite histochemical method cannot detect subclinical cases.  Unpublished data also indicate the Duthie/Spencer test can detect but not distinguish what appears to be an emerging microbe, M. lepromatosis (Lowes et al, 2013; personal communication (John Spencer)).  The newly discovered mycobacterial species, M. lepromatosis, originally identified in Mexico, has also been reported in two Chinese immigrants from Asia (where two-thirds of all global leprosy exists ) with Lucio’s (Levis et al, 2012).

Lucio’s has been described as far back as 1948, and until recently, most, but not all, cases have traditionally been from Mexico.  With a phenomenon as uncommon as Lucio’s it is difficult to perform accurate global surveillance.  However,  it may be an emerging virulent mutant of the original M. leprae — or is this only a recently discovered species (or strain) that escaped detection until Han and associates identified the first cases at M. D. Anderson Cancer Center in Houston, Texas? (Han et al, 2009).

Further investigation of these peculiar cases is required before it can be concluded that M. lepromatosis is a more virulent emerging species of M. leprae; this should include sequencing the whole genome.   Duthie, Spencer and the Dutch/Brazilian investigators including Orangelife, Inc. are continuing to work on combination of carbohydrate and protein antigens for diagnosis, contact survelliance, vaccine development, and distinguishing M. leprae and M. lepromatosis.




Geluk A, Duthie MS, Spencer JS. (2011) Postgenomic Mycobacterium leprae antigens for cellular and serological diagnosis of M. leprae exposure, infection and leprosy disease. Lepr Rev 82:402-21


Han XY Sizer KC, Tan H-H. (2012) Identification of the leprosy agent Mycobacterium lepromatosis in Singapore. J Drugs Dermatol 11:168-172.


Han XY, Sizer KC, Thompson EJ, Kabanja J, Li J, Hu P, Gómez-Valero L, Silva FJ. (2009) Comparative sequence analysis of Mycobacterium leprae and the new leprosy-causing Mycobacterium lepromatosis. J Bacteriol 191:6067-74


Levis WR, Meeker HC, Schuller-Levis G, Sersen E, Schwerer B. (1986) IgM and IgG antibodies to phenolic glycolipid I from Mycobacterium leprae in leprosy: insight into patient monitoring, erythema nodosum leprosum, and bacillary persistence. J Invest Dermatol 86:529-34


Levis WR, Zhang S, Martiniuk F. (2012) Mycobacterium lepromatosis: emerging strain or species? J Drugs Dermatol 11:158


Lowes MA, Russell CB, Martin DA, Towne JE, Krueger JG. (2013)  The IL-23/T17 pathogenic axis in psoriasis is amplified by keratinocyte responses. Trends Immunol  Jan 3 S1471-4906 [Epub ahead of print]

Martiniuk F, Giovinazzo J, Tan AU, Shahidullah R, Haslett P, Kaplan G and Levis WR. (2012) Lessons of leprosy: The emergence of TH17 cytokines during type II reactions (ENL) is teaching us about T-cell plasticity. J Drugs Derm 11:507-511


Modlin RL. (1994) Th1-Th2 paradigm: insights from leprosy. J Invest Dermatol 102:828-32


Rada E, Duthie MS, Reed SG, Aranzazu N, Convit J. (2011) Serologic follow-up of IgG responses against recombinant mycobacterial proteins ML0405, ML2331 and LID-1 in a leprosy hyperendemic area in Venezuela. Mem Inst Oswaldo Cruz 107 Suppl 1:90-4


Spencer JS, Duthie MS, Geluk A, Balagon MF, Kim HJ, Wheat WH, Chatterjee D, Jackson M, Li W, Kurihara JN, Maghanoy A, Mallari I, Saunderson P, Brennan PJ, Dockrell HM. (2012) Identification of serological biomarkers of infection, disease progression and treatment efficacy for leprosy. Mem Inst Oswaldo Cruz 107 Suppl 1:79-89.

Editors’ Picks from Experimental Dermatology (February and March issues)

Chrysanthemum extract apigenin improves barrier repair after tape stripping

Apigenin, a chrysanthemum extract, has been used for skin care in Asia for ages but knowledge on how this agent acts is scarce. It was shown before that apigenin exhibits preventive activity against UVB-induced skin tumors (Tong et al., 2007),and that an apigenin-enriched diet attenuates the development of atopic dermatitis-like lesions (Yano et al 2009).

The Man group (2013) now reports the influence of apigenin on early skin barrier repair after tape stripping in murine skin. Treatment with apigenin resulted in elevated filaggrin expression, increased density of lamellar bodies, and more intense immunostaining for antimicrobial peptides. In cultured human keratinocytes, addition of apigenin resulted in elevated mRNA levels of the lipid-synthesizing enzymes HMGCoA, SPT1, and FAS. In summary, this report demonstrates a positive effect of apigenin on skin barrier repair and highlights that various mechanisms may contribute to this effect. Treatment of diseases with barrier repair dysfunction may therefore be a future field of application for apigenin.

Selected by J. Brandner, Hamburg, Germany

Maihua Hou, Richard Sun, Melanie Hupe, Peggy L. Kim, Kyungho Park, Debra Crumrine, Tzu-kai Lin, Juan Luis Santiago, Theodora M. Mauro, Peter M. Elias and Mao-Qiang Man (2013) Topical Apigenin Improves Epidermal Permeability Barrier Homeostasis in Normal Murine Skin by Divergent Mechanisms. Exp Dermatol Accepted manuscript online: 30 JAN 2013 doi: 10.1111/exd.12102, will be published in Vol 22, Nr. 3, March 2013

Tong X, Van Dross RT, Abu-Yousif A, Morrison AR, Pelling JC (2007) Apigenin prevents UVB-induced cyclooxygenase 2 expression: coupled mRNA stabilization and translational inhibition.  Mol Cell Biol. Jan;27(1):283-96.

Yano S, Umeda D, Yamashita S, Yamada K, Tachibana H (2009) Dietary apigenin attenuates the development of atopic dermatitis-like skin lesions in NC/Nga mice.  J Nutr Biochem. Nov;20(11):876-81



Functional melanocortin 1 receptor Mc1r is not necessary for an inflammatory response to UV radiation in adult mouse skin

Melanocortin receptor type 1 (MC1R) is recognized for its role in the regulation of melanin pigmentation. In addition, a majority of investigators believes that it also plays a crucial role in anti-inflammatory responses and in induction of protective responses counteracting the damaging effects of ultraviolet radiation (UVR).

Most recently, researchers from George Washington University and Jagiellonian University (2013), using C57BL/6-c, C57BL/6, and C57BL/6-Mc1re/e  mouse strains, have reported the surprising finding that loss of function in the MC1R neither impacts  inflammatory responses to UV nor affects  UVR-induced immunosuppression. These findings are in striking contrast with the generally accepted opinion that constitutive or ligand-induced MC1R activity plays an important role in modulation of cutaneous immune activity in response to UVR. Interestingly, the authors also showed that UVR induced similar DNA damage in the epidermis and dermis of C57BL/6-c, C57BL/6, and C57BL/6-Mc1re/e strains of mice.

By challenging the existing dogmas on the precise role of MC1R in non-pigmentary responses to the UVR, these results will undoubtly stimulate further research to validate the presented data or to determine to which degree these phenomena extend beyond the C57BL/6 mouse model.

Selected by A. Slominski, Memphis, USA

Wolnicka-Glubisz A, De Fabo E,  Noonan F. (2013) Functional melanocortin 1 receptor Mc1r is not necessary for an inflammatory response to UV radiation in adult mouse skin. Exp Dermatol Accepted manuscript online: 25 JAN 2013 doi: 10.1111/exd.12100.





Human follicular dermal cells support keratinocyte growth: trichogenicity is a virtue?

Autologous transplantation of artificially assembled epidermal sheet has been used for the treatment of severe burns, intractable ulcers, and genodermatoses. Rapid in vitro expansion of patient-derived keratinocytes is a key step for successful treatment. Among currently available keratinocyte culture techniques, the one established by Rheinwald and Green (R&G) in 1975 is still considered to be very efficient. Yet, this protocol requires inactivated 3T3 murine feeder cells and “specially mixed” culture medium (I hope that everyone agrees that it is a bit of a hassle to prepare).

Hill et al. (2013) demonstrated that human dermal papilla (DP) and dermal sheath (DS) cells support human keratinocyte growth in orthodox MEM medium at a level comparable to that achieved with R&G’s condition.  The authors also showed that secreted protein acidic and rich in cysteine (SPARC) expression levels correlated with improved keratinocyte support. Yet, forced expression of SPARC in human dermal fibroblasts was not sufficient to endow these cells with such supportive properties.

DP and DS cells possess hair- inductive capacities, which distinguish those cells from other dermal cells and may enable supporting keratinocyte growth. In this sense, trichogenicity is probably a virtue.

Selected by M. Ohyama, Tokyo, Japan

R.P. Hill, A. Gardner, H.C. Crawford, R. Richer, A. Dodds, W.A. Owens, C. Lawrence, S. Rao, B. Kara, S.E. James and C.A. Jahoda (2013) Human hair follicle dermal sheath and papilla cells support keratinocyte growth in monolayer co-culture. Exp Dermatol Accepted manuscript online: 1 FEB 2013 doi: 10.1111/exd.12107, will be published in Vol 22, Nr. 3, March 2013

Rheinwald JG, Green H.  (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells.  Cell. Nov;6(3):331-43




Evidence for a regulatory loop between IFNγ and IL-33 in skin inflammation

Atopic dermatitis (AD) is characterized by a delicate cutaneous micromilieu of various cytokines and chemokines. Among those, interleukin-33 (IL-33), a member of the IL-1 cytokine family, has recently gained much attention due to its role in Th2 responses and damage- induced inflammation. Understanding the role of IL-33 in inflammatory diseases such as AD and allergic asthma might lead to novel treatments. Therefore, Seltmann and coworkers (2013) investigated the expression and secretion of IL-33 in resident skin cells and its impact on CD4+ T cells. The authors showed that keratinocytes and dermal fibroblasts differ in their regulation of IL-33. While in fibroblasts, TNFα, and IL-1β were the strongest inducers, IFNγ is clearly the key regulator of IL-33 in keratinocytes. Notably, keratinocytes from AD patients showed stronger responses, suggesting disturbed IL-33 regulation in these cells. Interestingly, secreted IL-33 acts on T cells and increases their production of IFNγ. The latter effect is substantially enhanced by the presence of IL-12, which is expressed in chronic AD lesions. These results suggest that IL-33 and IFNγ are closely interlinked in epidermal inflammation in AD. IFNγ induces IL-33 in keratinocytes and IL-33 acts on activated T cells to further increase the release of IFNγ, thereby driving a loop of skin inflammation towards chronic responses. In this respect, IL-33 can be linked to other IL-1 family members such as IL-1 and IL-18 and might serve as a novel target for future therapies.

Selected by J. Schauber, Munich, Germany

Seltmann J, Werfel T, Wittmann M. Evidence for a regulatory loop between IFN-γ and IL-33 in skin inflammation. Exp Dermatol. 2013 Feb; 22(2): 102-7. doi: 10.1111/exd.12076.




The Sox21 gene plays an important role for the binding of lipids on hair

Sox genes encode a family of transcription factors and are defined as containing the high mobility group box of a gene involved in sex determination called SRY, which resides on the Y-chromosome. Knocking out the Sox21 gene leads to a down-regulation of some proteins in the mouse hair cuticle, leading to improper cuticle formation. The outermost surface of the cuticle is naturally very hydrophobic due to a monomolecular layer of fatty acids, of which 18-methyleicosanoic acid (18-MEA) is a major and uniquely important component. The fatty acids are bound to the underlying protein layer through thioester linkages. Kawaminami et al (2013) now show that after knocking out the Sox21 gene the overall level and distribution of 18-MEA on the surface of hair remain largely unchanged, while a virtually comprehensive disruption of its covalent attachment occurs. Furthermore, they show that other lipids are in individual cases subject to significant changes in their concentration levels due to the knock-out. Since the bound lipids on the surface of hair fibres are known to impart beneficial properties to hair, such as hydrophobicity, low friction, protection against penetration of chemicals, and retardation of bacterial and fungal growth, they are of special importance in hair cosmetic science.

Selected by F.J. Wortmann, Manchester, United Kingdom

Kawaminami S, Breakspear S, Saga Y, Noecker B, Masukawa Y, Tsuchiya M, Oguri M, Inoue Y, Ishikawa K, Okamoto M. Deletion of the Sox21 gene drastically affects hair lipids. Exp Dermatol. 2012 Dec; 21(12): 968-70. doi: 10.1111/exd.12050



This quiz relates to the Research Techniques Made Simple article “FLUORESCENCE IN SITU HYBRIDIZATION (FISH)” published in the May 2013 issue of the Journal of Investigative Dermatology.






1. What does FISH detect?

a. Protein structure abnormalities
b. Specific chromosome copy number aberrations
c. Presence of specific antigens
d. Presence of complement



2. Where does the FISH probe localize to?

a. Golgi apparatus
b. Cytoplasm
c. Cell membrane
d. Nucleus
3. What is the FISH probe composed of?

a. Proteins
b. Lipids
c. Carbohydrates
d. Nucleic acids



4. What is the maximum number of FISH probes that can be used in a single experiment?

a. Two
b. Three
c. Four
d. Five





1.         The correct answer is b:  Specific chromosome copy number aberrations

2.         The correct answer is d:  Nucleus

3.         The correct answer is d:  Nucleic acids

4.         The correct answer is c:  Four