Effects of keratinocyte-derived and fibroblast-derived exosomes on human epidermal melanocytes.

BACKGROUND: Exosomes have been demonstrated to carry proteins, membrane lipids, mRNAs and microRNAs which can be transferred to surrounding cells and regulate the functions of those recipient cells. OBJECTIVES: The objective of the study was to investigate the effects of exosomes released by keratinocytes and fibroblasts on the proliferation, tyrosinase activity and melanogenesis of melanocytes. METHODS: Melanocytes, keratinocytes and fibroblasts obtained from human foreskin were cultured and exosomes secreted by keratinocytes and fibroblasts were harvested from the culture supernatants by ultracentrifugation. Each exosome fraction was divided into two parts; one part was subjected to high-throughput sequencing using an Illumina HiSeq sequencer to characterize the microRNA expression profiles, while the other part was labeled with the fluorescent dye PKH67 and was then co-cultivated with epidermal melanocytes. RESULTS: High-throughput sequencing analysis showed 168 differentially expressed microRNA within exosomes derived from keratinocytes and from fibroblasts, 97 of those being up-regulated with the other 71 down-regulated. Gene ontology analysis showed that the target genes responsible for these differentially expressed microRNAs were mainly enriched in the protein-binding region of molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that target genes regulated by differentially expressed microRNA were mainly involved in mitogen-activated protein kinase (MAPK) signaling pathway, Ras signaling pathway, cAMP signaling pathway and Wnt signaling pathway. Keratinocyte-derived exosomes were taken up by melanocytes co-cultured with them and promoted the proliferation, tyrosinase activity and melanin synthesis of those melanocytes. However, fibroblast-derived exosomes had no similar effects on melanocytes. CONCLUSION: Keratinocyte-derived exosomes but not fibroblast-derived exosomes were taken up by melanocytes in co-culture and significantly stimulated their proliferation, tyrosinase activity and melanin synthesis. Those different effects may be mainly due to the differential expression of microRNAs in exosomes derived from the different types of cells. LIMITATIONS: Electron microscopy of the obtained exosomes and in-depth study of apparently differentially expressed microRNAs were not performed.

Development of melanocye-keratinocyte co-culture model for controls and vitiligo to assess regulators of pigmentation and melanocytes.

BACKGROUND: There is a need to develop an in vitro skin models which can be used as alternative system for research and testing pharmacological products in place of laboratory animals. Therefore to study the biology and pathophysiology of pigmentation and vitiligo, reliable in vitro skin pigmentation models are required. AIM: In this study, we used primary cultured melanocytes and keratinocytes to prepare the skin co-culture model in control and vitiligo patients. METHODS: The skin grafts were taken from control and patients of vitiligo. In vitro co-culture was prepared after culturing primary melanocytes and keratinocytes. Co- cultures were treated with melanogenic stimulators and inhibitors and after that tyrosinase assay, MTT assay and melanin content assay were performed. RESULTS: Melanocytes and keratinocytes were successfully cultured from control and vitiligo patients and after that co-culture models were prepared. After treatment of co-culture model with melanogenic stimulator we found that tyrosinase activity, cell proliferation and melanin content increased whereas after treatment with melanogenic inhibitor, tyrosinase activity, cell proliferation and melanin content decreased. We also found some differences in the control co-culture model and vitiligo co-culture model. CONCLUSION: We successfully constructed in vitro co-culture pigmentation model for control and vitiligo patients using primary cultured melanocytes and keratinocytes. The use of primary melanocytes and keratinocytes is more appropriate over the use of transformed cells. The only limitation of these models is that these can be used for screening small numbers of compounds.

Unique metabolic properties of Mycobacterium leprae.

My first contact with Dr. Dharmendra was through correspondence. While working for Ph.D., I wrote to him that a section in his book "Notes on Leprosy" was ambiguous. Instead of ignoring the letter, he replied, agreeing to clarify it in the revised edition. I went to work at Carville at the invitation of Dr. Kirchheimer, who had seen my Ph.D. thesis. Dr. Dharmendra visited Carville to receive the Damien-Dutton award and stayed there for a few days. Carville is an isolated place with no public transportation. I used to take him for afternoon drives to the countryside around Carville. He published some of our papers in Leprosy in India and later in Indian Journal of Leprosy. He was very prompt in acknowledging receipt of manuscripts and suggesting any changes to be made. He also reprinted in the Journal several of our papers published elsewhere, and also a lecture I gave at a meeting of the Japanese Leprosy Association. During one of my visits to India, Dr. M. C. Vaidya had arranged a talk by me at the All India Institute of Medical Sciences, New Delhi. At the invitation of Dr. Dharmendra, I visited him in his home. We used to exchange new year cards and letters. He wrote to me about his eye infection and consequent loss of sight in one eye. He asked me to write an editorial for an issue of Indian Journal of Leprosy (January 1989). The last time I met him was during the International Leprosy Congress held in New Delhi.

A possible metabolic role for o-diphenoloxidase in Mycobacterium leprae.

Among mycobacteria, Mycobacterium leprae is unique in its ability to oxidize a variety of diphenols to quinones in vitro. What physiologic role o-diphenoloxidase has in the organism remained unknown. Reducing substrates like NADPH, NADH and ascorbic acid reacted with the quinone formed from dopa (3,4-dihydroxyphenylalanine); the substrates were oxidized and the quinone was reduced back to diphenol in the process. Since the quinone undergoes reversible oxidation-reduction, diphenoloxidase might serve as an alternative respiratory mechanism in M. leprae for the utilization of other substrates, as has been reported in plants.

Immunological properties of M. leprae culture isolates ICRC bacilli: hypothesis on relationship between M. leprae and ML-culture isolates.

The M. leprae-culture isolate, being a culture, is identified by biochemical criteria. Majority belong to either M. avium-intracellulare-scrofulacieum complex, or corynobacterium group. The identification is also determined by serological methods using soluble sonicates. Skin tests with heat-killed suspension often give positive Mitsuda response and hence the isolates are discarded as non-M. leprae. The data on ICRC strains show that Dharmendra type antigen prepared from ICRC bacilli compares very well with lepromin in lepromatous patients. The CMI/DTH tests in mice by FPE and LMI technique have experimentally demonstrated that ICRC bacillus is the first cultivable mycobacterium that can sensitize mice against lepromin. These data also brought out antigenic differences between the ICRC strains. The clinical trial on ICRC vaccine has shown that the ICRC bacilli is immunogenic in Man. The biochemical data on ICRC strain show that they possess DOPA-Oxidase and can express biochemical character of 9/10 similarity index with M. leprae with appropriate substrates in the medium. Thus, the ICRC strains possess both M. leprae and M. avium characters. An hypothesis is proposed to explain these observations--The 'Janus face' of M. leprae--by proposing a relationship between M. leprae and its culture isolates, e.g. ICRC bacilli (1). The M. leprae-culture isolate may by a recombinent of M. leprae with M. avium background (2). The ML-isolate may express the CMI related antigenic determinants corresponding to that of M. leprae in the biopsy (3). The CMI related antigens of whole bacilli in the ML-culture isolate may be inducing type as against only eliciting antigen expressed by M. leprae (4). Recombination may provide immunogenicity as well as 'in vitro' growth potential to the ML-culture isolate (5). Absence or failure to express inducing antigen by M. leprae maybe alternate biological escape mechanism from immune surveillance for survival in vivo, with or without suppressor mechanisms.

Dopa-oxidase activity on ICRC bacilli.

Presence of O-phenoloxidase is regarded as M. leprae specific character. This enzyme activity was found to be present in ICRC bacilli, Strain C-44. Though this strain is cultivable 'in vitro', the expression of DOPA-Oxidase activity strongly suggests that it carries M. leprae genome. The ICRC bacilli, therefore, may thus from a group of M. leprae culture isolates, distinct from other known cultivable mycobacteria which do not possess this enzyme activity.

Utilization of indole compounds by Cryptococcus neoformans to produce a melanin-like pigment.

Several indoles served as substrates for the phenoloxidase of Cryptococcus neoformans and resulted in the production of a melanin-like pigment. In general, a higher percentage of C. neoformans var. neoformans (A and D serotypes) isolates could produce pigment from indoles than could those of var. gattii (B and C serotypes). Only compounds with a hydroxyl or an amino group on the phenyl ring produced pigment; methoxy, nitro, methyl, and fluorine substituents on the phenyl ring were inactive, as was a hydroxyl group at the 2 position on the indole ring. The phenoloxidase of C. neoformans thus appears to differ from that found in Mycobacterium leprae, which cannot use a hydroxyindole, desoxyfructo-5-hydroxytryptamine, as a substrate. In addition, C. neoformans differs from M. leprae in that desoxyfructo-5-hydroxytryptamine does not inhibit the uptake of dihydroxyphenylalanine into the cell.

Growth of Mycobacterium leprae in a redox system.

Mycobacteria recovered from human lepromatous nodules and presumably M. leprae, have been grown in a medium that ensured a minimal oxygen tension at initiation of growth, and an increasing availability of oxygen as bacillary growth increased requiring marginal increments in oxygen tension. This physicochemical environment was achieved by the addition of strong biological reductants in the medium, and a combination of partial vacuum and alkaline pyrogallol in the culture vessel. In addition, n-tetradecane, a straight chain hydrocarbon, and lipids like Cholesterol and Lecithin, all three substances mixed in the aqueous medium as liposomes, were added and found to be useful. Menadione, or Vitamin K3, added to the medium considerably improved growth efficiency. Growth occurred initially as non-acid fast coccoids and bacilli that gradually changed to acid-fast bacilli and globi, and cell-wall deficient, spherical L-form elements. Appearance of growth in any form was perceptible within 1 to 2 weeks and optimal growth as acid fast bacilli took upto 3 months. Both the acid fast and non-acid fast bacilli could not be grown in conventional media, but the non-acid fast coccoids could be readily isolated from these cultures in a specially enriched liquid medium. The problem of harvesting of the growth free of lipid-hydrocarbon substances has still not been solved, as also an optimum oxidation-reduction potential. The growth is transferable.

Oxidation of 3,4-dihydroxyphenylalanine by connective tissue constituents. Identification of Mycobacterium leprae not related to phenolase activity.

The oxidation of 3,4-dihydroxyphenylalanine (DOPA) was studied by spectrophotometric methods at pH 6.8. In the presence of L- or D-DOPA, a color development occurred in the presence of the following substances as measured by increase in absorption both at 540 nm and 480 nm: hyaluronic acid, trypsinized human skin and umbilical cord extract, trypsin treated rat tissue from subcutaneous rat leproma, trypsin treated M. lepraemurium isolated from rat lepromata, and trypsinized M. leprae isolated from non-treated lepromatous leprosy cases. Normal human skin and connective tissue extract and nontrypsinized connective tissue of rat leprosy granuloma did not oxidize DOPA. While the trypsin-treated partially purified M. leprae suspension oxidized DOPA at both wave-lengths, the hyaluronidase-treated same suspension of M. leprae failed to oxidize these phenolic compounds. Mushroom tyrosinase oxidized D-DOPA, L-DOPA, epinephrine and norepinephrine at 480 nm. Hyaluronic acid also oxidized epinephrine and norepinephrine at both wave-lengths. Since it is known that M. leprae in the human host is closely associated with the presence of the acid mucopolysaccharides of the skin, and since acid mucopolysaccharides and skin constituents strongly oxidized DOPA, and since the hyaluronidase treated M. leprae failed to oxidize DOPA, it became evident that hyaluronic acid and not M. leprae is responsible for DOPA oxidation, and phenolase activity is not associated with the metabolism of M. leprae. Evidence is presented that DOPA is not a unique characteristic of the human leprosy bacillus. For instance, trypsin-treated murine leprosy bacilli from the rat strongly oxidized DOPA. The reaction of DOPA oxidation, therefore, must be rejected as a test for the identification of M. leprae. The obtained results confirmed the pertinent findings of Skinsnes and his co-workers.

A non-acid-fast coccoid precursor--possible cultivable phase of Mycobacterium leprae.

A non-acid-fast coccoid organism isolated from human leproma, and skin and nasal smears of leprosy patients shows tendency to revert to an acid-fast mycobacterial form during test tube passages. One of these coccoid isolates gave strong DOPA oxidase activity. There is also preliminary evidence of mycobacterial conversion from these coccoids in intraperitoneally inoculated mice. The possibility that these non-acid-fast coccoids could be a cultivable precursor phase of M. leprae has been raised and discussed.

Hyaluronic acid and yeast extract do not oxidize DOPA.

Hyaluronic acid and yeast extract, separately and in combination, were tested for their ability to oxidize dopa to pigment. Two types of hyaluronic acid and yeast extract were used. The activities were assayed both spectrophotometrically and by oxygen-uptake determinations. The results were uniformly negative. The biological role of hyaluronic acid is discussed. Use of pigment formation from phenolic compounds as an identification test of another organism is pointed out.

Melanocytic activity in leprosy lesions with special reference to cellular infiltrate.

To assess the correlation, if any, between the clinical hypopigmentation in leprosy affected skin and the inflammatory cellular infiltrate in d rmis, skin tissue sections from the maculo-anaesthetic and tuberculoid lesion of 50 cases were studied with Dopa and H & E stains. The results are indicative of (i) proportionate lack of Dopa oxidase activity in the hypopigmented leprosy lesions in commensuration with the relative degree of clinical hypopigmentation; (ii) cellular infiltrate is not related with the clinical hypopigmentation or dopa oxidase activity.