Melanocyte spheroids are formed by repetitive long-term trypsinization.

BACKGROUND: Autologous melanocyte transplantation plays an important role in the treatment of vitiligo. OBJECTIVE: Previous studies have indicated that, compared with melanocytes growing in monolayers, melanocyte spheroids have a better survival in growth factor- and serum-deprived conditions. METHODS: Melanocyte spheroids were obtained from human epidermis by repetitive long-term trypsinization and maintained an aggregated morphology for a short period in certain conditions. RESULTS: Melanocyte spheroids were capable of growing into normal dendritic melanocytes in monolayer when they were harvested and reinoculated in 24-well plates. Immunohistochemical analysis of the melanocyte spheroids revealed that they were positive for HMB45, a melanosome-specific marker. No melanomas occurred when melanocyte spheroids were transplanted into mice. CONCLUSION: Our study provides a promising approach for melanocyte transplantation to treat vitiligo.

Deciphering the proteomic profile of Mycobacterium leprae cell envelope.

The complete sequence of the Mycobacterium leprae genome, an obligate intracellular pathogen, shows a dramatic reduction of functional genes, with a coding capacity of less than 50%. Despite this massive gene decay, the leprosy bacillus has managed to preserve a minimal gene set, most of it shared with Mycobacterium tuberculosis, allowing its survival in the host with ensuing pathological manifestations. Thus, the identification of proteins that are actually expressed in vivo by M. leprae is of high significance in understanding obligate, intracellular mycobacterial pathogenesis. In this study, a high-throughput proteomic approach was undertaken resulting in the identification of 218 new M. leprae proteins. Of these, 60 were in the soluble/cytosol fraction, 98 in the membrane and 104 in the cell wall. Although several proteins were identified in more than one subcellular fraction, the majority were unique to one. As expected, a high percentage of these included enzymes responsible for lipid biosynthesis and degradation, biosynthesis of the major components of the mycobacterial cell envelope, proteins involved in transportation across lipid barriers, and lipoproteins and transmembrane proteins with unknown functions. The data presented in this study contribute to our understanding of the in vivo composition and physiology of the mycobacterial cell envelope, a compartment known to play a major role in bacterial pathogenesis.

Distribution of genes encoding the trypsin-dependent lantibiotic ruminococcin A among bacteria isolated from human fecal microbiota.

Fourteen bacterial strains capable of producing a trypsin-dependent antimicrobial substance active against Clostridium perfringens were isolated from human fecal samples of various origins (from healthy adults and children, as well as from adults with chronic pouchitis). Identification of these strains showed that they belonged to Ruminococcus gnavus, Clostridium nexile, and Ruminococcus hansenii species or to new operational taxonomic units, all from the Clostridium coccoides phylogenetic group. In hybridization experiments with a probe specific for the structural gene encoding the trypsin-dependent lantibiotic ruminococcin A (RumA) produced by R. gnavus, seven strains gave a positive response. All of them harbored three highly conserved copies of rumA-like genes. The deduced peptide sequence was identical to or showed one amino acid difference from the hypothetical precursor of RumA. Our results indicate that the rumA-like genes have been disseminated among R. gnavus and phylogenetically related strains that can make up a significant part of the human fecal microbiota.

CD4+ T-cell responses to recombinant hsp65 and hsp18 of M. leprae and their trypsin-digested fragments in leprosy: diversity in HLA-DR restriction.

Mycobacterium leprae heat-shock proteins hsp65 and hsp18 have received immense attention as major T-cell target antigens in leprosy. Both of these hsps and their tryptic fragments were characterized for their ability to stimulate CD4+ T cells derived from polar leprosy cases and healthy contacts. The optimal digestion of hsps with trypsin yielded four fragments of hsp65--TDB65-1 (24 kDa), TDB65-2 (18 kDa), TDB65-3 (17 kDa), TDB65-4 (14 kDa)-- and three of hsp18--TDB18-1 (10 kDa), TDB18-2 (5 kDa), TDB18-3 (3 kDa). While all of these tryptic fragments and undigested hsps triggered CD4+ T cells from tuberculoid (TT) leprosy patients and healthy contacts (SI > 2), only two fragments--TDB65-2 and TDB18-3--were found to be stimulatory in anergic lepromatous (LL) leprosy patients (SI = 5.27 and 3.0, respectively). Blocking studies using allele-specific anti-DR monoclonal antibodies revealed multiple HLA-Dr restriction, with DR2 providing the strongest restriction in both TT as well as LL leprosy. These findings indicate that M. leprae hsps and their trypsin-digested fragments are promiscuous and recognizable in the context of diverse HLA alleles, of which DR2 is the most efficient restriction element. The 18-kDa fragment of hsp65 and the 3-kDa fragment of hsp18 are the most versatile fragments that could elicit in vitro proliferation in both polar forms of leprosy.

[Elimination of contaminants in a homogenate of nude-mouse footpad experimentally infected with Mycobacterium leprae].

For elimination of contaminants in a homogenate of nude-mouse footpad infected with M.leprae, the pretreatment with trypsin was tested. The results obtained indicated that the final sterile product was satisfactorily obtained, if the pretreatment with trypsin 0.05%, 37 degrees C, 60min to the homogenate was carried out before the treatment with sodium hydroxide 1%, 37 degrees C, 15min. This procedure might contribute to make a suspension of partially purified M.leprae from the infected tissues, because both the treatments with trypsin and sodium hydroxide could eliminate the infected tissues surrounding M.leprae as well as any contaminants, except other mycobacteria, present in a tissue homogenate.

Modulation of the stability of rabbit skeletal muscle myosin light chain kinase through the calmodulin-binding domain.

The binding of Ca2+(4).calmodulin (CaM) to rabbit skeletal muscle myosin light chain kinase (MLCK) is required for expression of the enzyme's activity. While both MLCK and CaM were stable at 30 degrees C, their complex was not. The binding of CaM to MLCK resulted in a time- and temperature-dependent inactivation that reflected an intrinsic instability of the complex. Separation of the components of the inactive complex yielded functional CaM, but catalytically inert MLCK, indicating that the site of the inactivating event was confined to MLCK. The behavior of proteolytic fragments further localized this event to the C-terminal 60% of the 603-residue protein. Changes in the tryptophan fluorescence and proteolytic susceptibility of MLCK-CaM indicated that a conformational change accompanied, and thus may have caused, inactivation. Substrates protected against inactivation, as did millimolar concentrations of Mg2+, Mn2+, and Ca2+. These metals appeared to bind to a site on MLCK distinct from that which recognized Mg2+.ATP. A proteolytic fragment of MLCK lacking the ability to bind CaM, C beta 35 (residues 255-584; Edelman, A. M., Takio, K., Blumenthal, D. K., Hansen, R. S., Walsh, K. A., Titani, K., and Krebs, E. G. (1985) J. Biol. Chem. 260, 11275-11285), was unstable at 30 degrees C, whereas a similar fragment which does bind CaM, T beta 40 (residues 236-595; Edelman, A. M., Takio, K., Blumenthal, D. K., Hansen, R. S., Walsh, K. A., Titani, K., and Krebs, E. (1985) J. Biol. Chem. 260, 11275-11285), was unstable only when CaM was bound.

An electron microscopic study of alterations in the morphology and permeability of purified Mycobacterium leprae.

This communication reports the association of changes in ultrastructure of Mycobacterium leprae with alterations in its permeability. To study morphologic changes of the organisms under different conditions (of temperature and exposure to NaOH and trypsin), ultrathin sections of the bacteria were cut and examined in an electron microscope. In the untreated bacilli and those washed with trypsin, the cytoplasmic membrane and the cell wall (peptidoglycan layer) remained intact; dapsone showed little effect on diphenoloxidase of the bacteria. M. leprae is unique among mycobacteria in possessing an unusual form of the enzyme diphenoloxidase. The antileprosy drug dapsone is a potent inhibitor of the enzyme, but it does not readily penetrate the bacteria where the cell envelope remains intact. The cell wall of M. leprae exposed to -80 degrees C or washed with NaOH was partially detached from the cell membrane; dapsone readily penetrated these organisms and inhibited the bacterial enzyme. In the above preparations, the cytoplasmic membrane appeared undamaged and the bacteria remained viable, as evidenced by multiplication in mouse foot pads. At 50 degrees C, the peptidoglycan layer became completely separated from the membrane and the cytoplasm was partially denatured. These organisms were permeable to dapsone, but were no longer viable. At 100 degrees C, the structural organization of the bacilli was completely destroyed, and of course, they lost their enzyme activity as well as viability. Evidently, the intact cell wall layer mediates the exclusion of dapsone from M. leprae, and there is no correlation between its viability and permeability. The ultrathin sections also reveal the internal organization and cytoplasmic inclusions of M. leprae, as never before seen.

Immunologic unresponsiveness in leprosy is mediated by modulation of E-receptor.

By using an indirect immunofluorescence technique with OKT3 and OKT11 monoclonal antibodies, the percentage of CD2 positive cells was found to be reduced in the peripheral blood of bacterial index positive lepromatous leprosy patients; however, in these patients, CD3 positive cells were at the normal level. Further CD2 positive cells attained the normal proportion in lepromatous patients when mycobacterial load was reduced. Both CD2 and CD3 receptors were expressed at the normal level in tuberculoid leprosy patients. Prior treatment of peripheral blood mononuclear cells from healthy controls with Mycobacterium leprae significantly decreased the percentage of CD2 but not CD3 positive cells. Such a modulation of CD2 on T cells also resulted in blocking the lymphoproliferative response induced by mitogen and antigen. These results suggest that there is a strong correlation between CD2 modulation and immunologic unresponsiveness in leprosy.

Trypsin-treated and coomassie blue-stained epimastigote antigen in a microagglutination test for Chagas' disease.

A microagglutination test using trypsin-treated and Coomassie blue-stained Trypanosoma cruzi epimastigote antigen was adapted for the diagnosis of Chagas' disease. When incorporated in the test, 2-mercaptoethanol treatment of chagasic sera had no influence on antibody titer. In contrast, titers in sera from patients with visceral leishmaniasis, African trypanosomiasis, and autoimmune disorders, subjected to similar treatment, showed remarkable decline. Accordingly, a lower cut-off point for Chagas' disease serological negativity could be taken resulting in a higher sensitivity (95.6%); the specificity was 94.7%. Similar specificities were obtained with Leishmania donovani chagasi and L. d. donovani antigens applied to homologous visceral leishmaniasis and heterologous Chagas' sera. Of 316 nonchagasic sera, only 3 with leptospirosis and 1 with leprosy showed seropositive titers prior to and after 2-mercaptoethanol treatment.

Temperature-induced changes in viability, diphenoloxidase and permeability of Mycobacterium leprae.

Among mycobacteria secretion of the enzyme diphenoloxidase has been established as a property of Mycobacterium leprae. The antileprosy drug dapsone (DDS), which completely inhibits the enzyme from plant and mammalian sources, does not readily penetrate intact M. leprae. When the drug is complexed with polylysine, it easily permeates the bacteria and produces 100% inhibition of its diphenoloxidase, suggesting a permeability barrier of the cytoplasmic membrane of M. leprae to dapsone. In this study: (1) when the organisms, purified from fresh tissues of experimentally infected armadillos, were treated with dilute alkali or exposed to warmer temperatures, DDS penetrated the bacteria and inhibited the diphenoloxidase. Washing with trypsin had no effect. Dapsone easily permeated the bacilli, purified from tissues stored at 0 degrees C or at -80 degrees C. (2) Diphenoloxidase of freshly-prepared M. leprae was stimulated when the bacteria were exposed to 50 degrees C for 10 min; at 60 degrees C the activity decreased, and at 100 degrees C the enzyme was completely inactivated. When the enzyme was assayed at temperatures below 37 degrees C, the activity was considerably lower, indicating that M. leprae may not be a psychrophilic organism in this respect. (3) The bacteria exposed to 50 degrees C failed to multiply in mouse footpads. M. leprae remained viable in tissues stored at 0 degrees C or -80 degrees C; but when the bacteria purified from these tissues were frozen, they lost their viability. On the other hand, the organisms separated from fresh tissues remained viable when frozen at -80 degrees C. The inhibition of diphenoloxidase of M. leprae by dapsone could serve as an indirect method to assess the integrity of the bacterial cell membrane and to predict whether the bacteria would retain their viability on freezing.

Antigen specific macrophage-lymphocyte interaction in lepromatous leprosy.

Peripheral blood derived macrophages from lepromatous leprosy patients were unable to interact with lymphocytes in the presence of M. leprae. This lack of interaction is probably not associated with membrane HLA-Dr antigens since trypsin and colchicine restored M. leprae induced depression in the latter but were unable to bring about a positive interaction. Two possible defects exist therefore in the lepromatous macrophage. These are an innate inability to process and present M. leprae antigens to lymphocytes and an induced inability to express some membrane receptors, an event detrimental to the normal functioning of a macrophage.

Alterations in the membrane of macrophages from leprosy patients.

Macrophage cultures pulsed with viable Mycobacterium leprae were assessed for erythrocyte rosetting in three groups of individuals, i.e., normal subjects, and tuberculoid and lepromatous patients. Of these, only the lepromatous group showed a reduction in rosetting ability after infection with M. leprae. The specificity of such a reduction pattern was confirmed by using various mycobacteria to infect the macrophages. A threshold effect was noted in all three groups. Although a reduction was obtained in the amount of rosetting of macrophages from lepromatous patients with 10(4) acid-fast bacilli per culture, tuberculoid and normal macrophages resisted such an effect with as large a dose as 20 X 10(6) to 30 X 10(6) and 30 X 10(6) bacilli per culture, respectively. The M. leprae-caused alterations in macrophages from lepromatous patients were reversible by treatment with trypsin and colchicine. Cytochalasin B and Tween 80 were unable to alter the pattern. Treatment of cells with neuraminidase was inconclusive since it enhanced rosetting values of both control and infected cultures. These manipulations were significant in elucidating the target point of the host (macrophage) and parasite (M. leprae) interaction and in delineation of the external and internal effects upon the macrophages. Both M. leprae and macrophages were participants in Fc reduction, as treatment of the former with rifampicin and of the latter with cyclocheximide significantly augmented the rosetting ability. In conclusion, it appears that M. leprae, upon entering a lepromatous macrophage, initiates the production of a protein which acts via the microtubules to alter membrane topography. It is possible that the altered membrane prevents effective macrophage-lymphocyte interaction. This could be one of the mechanisms by which cell-mediated immunity is suppressed in lepromatous leprosy.

Permeability of Mycobacterium leprae to dapsone: alteration by purification procedures.

Permeability of Mycobacterium leprae to dapsone in vitro was determined by the ability of the drug to inhibit o-diphenoloxidase of the bacilli. Dapsone showed little effect on the enzyme activity of the intact organisms. When the M. leprae preparations were washed with trypsin, NaOH, or acetone and ether, DDS penetrated the bacillus to inhibit its o-diphenoloxidase. The method might be useful in studying the utilization of added metabolites by purified M. leprae suspensions.

Mycobacterium leprae and phenoloxidase activity.

Our earlier studies indicated that the enzyme o-diphenoloxidase was absent in Mycobacterium leprae separated from depromatous human tissues. At that time the bacilli were not available from any other source. The existence or absence of this enzyme in M. leprae recovered from infected armadillo tissues were reinvestigated. The intact cells which were metabolically active, failed to oxidize DOPA. Likewise, DOPA and its derivatives were not oxidized by the enzymatically active cell-free preparations from M. leprae. Upon incubation of DOPA for more than 2 h with whole cell suspensions or particulate fractions, there was no development of colour with an absorption maximum of 540 nm as has been reported for an intermediate of DOPA oxidation. However, DOPA and several phenolic compounds were very actively oxidized by mushroom tyrosinase. The results suggested that M. leprae is deficient in o-diphenoloxidase, and this enzyme is not an intrinsic characteristic of this mycobacterium.

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.

Separation of M. leprae from human leproma and the development of a cytoplasmic skin test antigen from purified bacilli.

A successful method for purification of M. leprae from human leproma without subjection to heat has been developed. The "floater" phenomenon has been described which consists of bacillary tendency to float in the supernate when bacilli which are not autoclaved are separated from tissues by enzymatic digestion. A method for preparing cytoplasmic fractions from purified M. leprae has been developed for the production of a skin test antigen for leprosy. The cytoplasmic fraction of M. leprae elicited positive skin test responses in people with tuberculoid leprosy and negative responses in lepromatous leprosy. Cytoplasmic preparations from purified M. leprae had little cross-reactive relationship with the organism BCG. The small particulate fraction elicited positive reactions in PPD-S negative as well as BCG vaccinated individuals.