CD4+CD25+ T regs with acetylated FoxP3 are associated with immune suppression in human leprosy.

Leprosy is a chronic human disease that results from infection of Mycobacterium leprae. T reg cells have been shown to have important implications in various diseases. However, in leprosy, it is still unclear whether T regs can mediate immune suppression during progression of the disease. In the present study, we have proposed the putative mechanism leading to high proportion of T reg cells and investigated its significance in human leprosy. High levels of TGF-ß followed by adaptation of FoxP3(+) naive and memory (CD4(+)CD45RA(+)/RO(+)) T cells were observed as the principal underlying factors leading to higher generation of T reg cells during disease progression. Furthermore, TGF-ß was found to be associated with increased phosphorylation-mediated-nuclear-import of SMAD3 and NFAT towards BL/LL pole to facilitate FoxP3 expression in these cells, the same as justified after using nuclear inhibitors of SMAD3 (SIS3) and NFAT (cyclosporin A) in CD4(+)CD25(+) cells in the presence of TGF-ß and IL-2. Interestingly, low ubiquitination of FoxP3 in T reg cells of BL/LL patients was revealed to be a major driving force in conferring stability to FoxP3 which in turn is linked to suppressive potential of T regs. The present study has also pinpointed the presence of CD4(+)CD25(+)IL-10(+) sub class of T regs (Tr1) in leprosy.

IL-10 production from dendritic cells is associated with DC SIGN in human leprosy.

The defective antigen presenting ability of antigen presenting cells (APCs) modulates host cytokines and co-stimulatory signals that may lead to severity of leprosy. In the present study, we sought to evaluate the phenotypic features of APCs along with whether DC SIGN (DC-specific intercellular adhesion molecule-grabbing nonintegrin) influences IL-10 production while moving from tuberculoid (BT/TT) to lepromatous (BL/LL) pole in leprosy pathogenesis. The study revealed an increased expression of DC SIGN on CD11c⁺ cells from BL/LL patients and an impaired form of CD83 (∼50 kDa). However, the cells after treatment with GM-CSF+IL-4+ManLAM showed an increased expression of similar form of CD83 on DCs. Upon treatment with ManLAM, DCs were found to show increased nuclear presence of NF-κB, thus leading to higher IL-10 production. High IL-10 production from ManLAM treated PBMCs further suggested the role of DC SIGN in subverting the DCs function towards BL/LL pole of leprosy. Anti-DC SIGN treatment resulting in restricted nuclear ingression of NF-κB as well as its acetylation along with enhanced T cell proliferation validated our findings. In conclusion, Mycobacterium leprae component triggers DC SIGN on DCs to induce production of IL-10 by modulating intracellular signalling pathway at the level of transcription factor NF-κB towards BL/LL pole of disease.

C-type lectin DC-SIGN modulates Toll-like receptor signaling via Raf-1 kinase-dependent acetylation of transcription factor NF-kappaB.

Adaptive immune responses by dendritic cells (DCs) are critically controlled by Toll-like receptor (TLR) function. Little is known about modulation of TLR-specific signaling by other pathogen receptors. Here, we have identified a molecular signaling pathway induced by the C-type lectin DC-SIGN that modulates TLR signaling at the level of the transcription factor NF-kappaB. We demonstrated that pathogens trigger DC-SIGN on human DCs to activate the serine and threonine kinase Raf-1, which subsequently leads to acetylation of the NF-kappaB subunit p65, but only after TLR-induced activation of NF-kappaB. Acetylation of p65 both prolonged and increased IL10 transcription to enhance anti-inflammatory cytokine responses. We demonstrated that different pathogens such as Mycobacterium tuberculosis, M. leprae, Candida albicans, measles virus, and human immunodeficiency virus-1 interacted with DC-SIGN to activate the Raf-1-acetylation-dependent signaling pathway to modulate signaling by different TLRs. Thus, this pathway is involved in regulation of adaptive immunity by DCs to bacterial, fungal, and viral pathogens.

Ribosomal protein L7 included in tuberculin purified protein derivative (PPD) is a major heat-resistant protein inducing strong delayed-type hypersensitivity.

The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis. It consists of more than 100 denatured proteins in a culture filtrate of a heated culture of Mycobacterium tuberculosis. In two-dimensional electrophoretic analysis of PPDs from M. tuberculosis and M. bovis BCG, most proteins were diffusely separated and could not be seen as spots because of denaturation, whereas a few proteins showed relatively clear spots, indicating heat resistance. Two such proteins corresponded to ribosomal proteins L7 and L12. The mixture of these proteins L7/L2 induced a strong delayed-type hypersensitivity reaction. Another protein showing a clear spot was a GroES analogue, but this did not induce delayed-type hypersensitivity. There were a few other unidentified proteins. It is well known that L7 and L12 are encoded by the same gene and that they differ from each other only by an acetylic post-translational modification that occurs at the N-terminus of L12 converting it to L7 in Escherichia coli. L12, but not L7, was found in two-dimensional electrophoresis of BCG ribosomes, although we found two proteins corresponding to L7 and L12 in PPDs and a native culture filtrate of BCG. We compared the delayed-type hypersensitivity reaction elicited by L7/L12 derived from a culture filtrate of BCG and L12 derived from BCG ribosomes. L7/L12 from the culture filtrate could induce delayed-type hypersensitivity, but L12 from ribosomes could not, indicating that L7 was attributable to the induction of delayed-type hypersensitivity. The activity of L7/L12 was heat resistant. Neither glycosylation nor phosphorylation of L7/L12 from a culture filtrate could be detected. The acetylation at N-terminal of L12 was essential for the delayed-type hypersensitivity activity.

Identification of antibodies directed against O-acetylated sialic acids in visceral leishmaniasis: its diagnostic and prognostic role.

A significantly increased O-acetylated sialic acid (O-AcSA) binding fraction was purified from serum of visceral leishmaniasis (VL) patients by affinity chromatography on immobilized bovine submaxillary mucin (BSM) and found to be immunoglobulin in origin. The serodiagnostic and prognostic potential of BSM as a capture antigen was established by ELISA with no cross reactivity with coendemic diseases like malaria, tuberculosis, leprosy, chagas disease and cutaneous leishmaniasis; however, a strong cross reactivity was present with trypanosomiasis patients. In 56 clinically diagnosed VL patients, the BSM-ELISA was compared with diagnosis by microscopy using Giemsa stained tissue smears and direct ELISA using crude parasite antigen (parasite-ELISA); 49/56(87.5%) and 5/56(9.0%) were positive and negative respectively by all 3 methods. The BSM-ELISA failed to diagnose 2/56(3.5%) patients which were biopsy and parasite-ELISA positive. The prognostic potential of the BSM-ELISA in 18 longitudinally monitored VL patients before and after conventional antimonial treatment showed a significant decrease in anti O-AcSA titres in drug responsive patients whereas anti O-AcSA levels persisted in drug unresponsive patients. The IgG subclass distribution of antibodies directed against O-AcSA showed increased IgG2 levels in VL patients as compared to healthy controls. The BSM-based ELISA holds great promise as a serodiagnostic and prognostic assay for VL.

Determination of the structure of exochelin MN, the extracellular siderophore from Mycobacterium neoaurum.

BACKGROUND: Siderophores are compounds produced by bacteria to acquire iron. Exochelin MN, the extracellular siderophore from Mycobacterium neoaurum, is of particular interest because it has been shown to transport iron into M. leprae, which is responsible for the disease leprosy. Exochelins from other species cannot mediate iron transport in M. leprae, suggesting a specific uptake mechanism involving exochelin MN. We set out to determine the structure of exochelin MN and identify the features of the molecule that may account for this specificity. RESULTS: The structure of exochelin MN was elucidated by a combination of techniques including nuclear magnetic resonance, mass spectrometry, derivatization and gas chromatography. Exochelin MN is a peptide, containing the unusual amino acid beta-hydroxyhistidine and an unusual N-methyl group. The peptide coordinates iron(III) octahedrally using its two cis-hydroxamate groups plus the hydroxyl and imidazole nitrogen of the beta-hydroxyhistidine. The three-dimensional structure of the hexadentate exochelin/gallium complex was deduced from NMR data. CONCLUSIONS: Exochelin MN has some structural features in common with other siderophores, but has a unique three-dimensional structure, which is presumably important for its specific activity in M. leprae. Exochelin MN may be a target for drug design in the fight against infection with this pathogen.

Modulation of chromatin folding by histone acetylation.

A homogeneous oligonucleosome complex was prepared by reconstitution of highly hyperacetylated histone octamers onto a linear DNA template consisting of 12 tandemly arranged 208-base pair fragments of the 5 S rRNA gene from the sea urchin Lytechinus variegatus. The ionic strength-dependent folding of this oligonucleosome assembly was monitored by sedimentation velocity and electron microscopy. Both types of analysis indicate that under ionic conditions resembling those found in the physiological range and in the absence of histone H1, the acetylated oligonucleosome complexes remain in an extended conformation in contrast to their nonacetylated counterparts. The implications of this finding in the context of a multistate model of chromatin folding (Hansen, J. C., and Ausio, J. (1992) TIBS 197, 187-191) as well as its biological relevance are discussed.

Structural definition of acylated phosphatidylinositol mannosides from Mycobacterium tuberculosis: definition of a common anchor for lipomannan and lipoarabinomannan.

Based on chemical analysis, we have previously concluded that the biologically important lipoarabinomannan (LAM) and lipomannan (LM) from Mycobacterium are multiglycosylated forms of the phosphatidylinositol mannosides (PIMs), the characteristic cell envelope mannophosphoinositides of mycobacteria. Using definitive analytical techniques, we have now re-examined the reported multiacylated nature of PIMs in order to gain a better insight into their possible roles as biosynthetic precursors of LM and LAM. High-sensitivity fast atom bombardment-mass spectrometry analyses of the perdeuteroacetyl and permethyl derivatives of PIMs from Mycobacterium tuberculosis and Mycobacterium leprae enabled us to define the exact fatty acyl compositions of the multiacylated, heterogeneous PIM families, notably the dimannoside (PIM2) and the hexamannoside (PIM6). Specifically, in conjunction with other chemical and gas chromatography-mass spectrometry (GC-MS) analyses, the additional C16 fatty acyl substituent on PIM2 and its lyso form were defined as attached to the C6 position of mannose. We also present evidence for triacylated mannophosphoinositide as a common lipid anchor for both LM and LAM, and further postulate that acylation of PIM2 may constitute a key regulatory step in their biosynthesis.

Influence of the rapid acetylator phenotype on the emergence of DDS resistant Mycobacterium leprae

Determinou-se o fenótipo acetilador da insoniazida de 21 hansenianos suspeitos de terem desenolvido resistência à DDS. A resitência do Mycobacterium leprae desses pacientes foi investigada por intermédio de testes no coxim plantar de camundongos da estirpe BALB/c. Nossos resultados indicam que o surgimento de resistência à DDS no M. leprae dos acetiladores rápidos é mais provável do que nos bacilos dos acetiladores lentos. Parece plausível sugerir que os acetiladores rápidos recebem maiores doses de DDS do que os lentos, visto que a concentraçäo bacilar nas lesöes de pacientes com M. leprae sensível à DDS nos acetiladores rápidos foi mais de 17 vezes maior do que nos lentos

Acetylation phenotype and genotype in aboriginal leprosy patients from the north-west region of Western Australia.

N-Acetyltransferases (NAT1, NAT2) play an important role in biotransformation of a number of drugs and carcinogens. A polymorphism in the metabolism of such compounds by NAT2 has been known for many years but it is only recently that the underlying molecular genetics has been elucidated. In the present study, we have correlated acetylation phenotype and genotype in a group of 49 Australian Aborigines (26 males and 23 females; mean age = 50.5 yr) from the Derby region of Western Australia. Phenotype was determined using caffeine and genotype by an allele-specific polymerase chain reaction. The percentages of slow and rapid phenotypes were 36.7 and 63.3%, respectively, while the distribution of alleles for the NAT2 gene was 41% for the wildtype and 2, 17 and 40% for the M1, M2 and M3 mutations, respectively. This is the highest proportion of M3 mutations reported for any ethnic population. The observed genotype proportions were not significantly different from those predicted by the Hardy-Weinberg Law (chi 2 = 1.07, p > 0.05). Phenotype was predictable from genotype in 100% of patients. At the time of study, 29 of the Aborigines were receiving acedapsone intramuscularly for control of leprosy. Plasma dapsone concentrations in these patients were similar for both slow (n = 11) and rapid (n = 18) acetylators, suggesting that phenotype is unlikely to influence treatment outcome. The data show that Aborigines have a similar phenotype distribution to that of some Asian populations, but that there are differences in the frequencies of the M1, M2 and M3 mutant alleles.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation polymorphism and leprosy.

The sulfones are the drug of choice in the treatment of leprosy, with dapsone as the clear favorite. The major route for dapsone metabolism leading to its inactivation and excretion is via acetylation by hepatic N-acetyl transferase (NAT), as is the case with isoniazid (INH) and sulfamethazine (SMZ). The enzyme is known to exhibit genetic polymorphism. The object of the present study is mainly to determine the incidence of acetylator phenotype in a population of leprosy patients with a view to evaluating the degree of association, if any, between phenotype and the disease. Obviously a knowledge of the incidence of the phenotypes may provide a valuable contribution to the institution of more rational and successful therapy. In the normal or control subjects, as well as in the leprosy patients, the frequency distribution histograms of the percentage acetylsulfamethazine in urine and serum samples are bimodal, and this indicates the existence of a genetic polymorphism. Based on the bimodality, individuals were classified as either "rapid" or "slow" acetylators, and the incidence of the slow acetylator phenotype of about 51% was observed in the leprosy population. This gives a relatively high incidence of the allele controlling the slow acetylator (q = 0.73). Although there is evidence that the mean percentage of SMZ acetylated in leprosy patients of the slow acetylator phenotype is significantly higher than that observed for the same phenotype in the controls (t = 4.86, P less than 0.02), statistical analyses show that there is no association between the slow acetylator phenotype and the disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of a novel glucosamine-containing phosphoglycolipid from Deinococcus radiodurans.

The structure of a major novel lipid from Deinococcus radiodurans has been determined to be 2'-O-(1,2-diacyl-sn-glycero-3-phospho)-3'-O-(alpha-N-acetylglucosaminyl) -N- glyceroyl alkylamine. The lipid was shown to contain a phosphatidic acid backbone by digestion with phospholipase A2 and by hydrolysis with hydrofluoric acid. Using a combination of chemical and NMR spectroscopic techniques, the structure of this lipid was elucidated and compared with that of a similar phosphoglycolipid reported earlier (Anderson, R., and Hansen, K. (1985) J. Biol. Chem. 260, 12219-12223) in which galactose was found in place of N-acetylglucosamine. The fatty acid compositions of the two lipids were similar.

Chemical synthesis of an artificial antigen containing the trisaccharide hapten of Mycobacterium leprae.

The trisaccharide allyl O-(3,4-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl-al pha-L- rhamnopyranosyl)-(1----2)-3-O-methyl-alpha-L-rhamnopyranoside was synthesized from partially methylated monosaccharide derivatives. Condensation of 1,4-di-O-acetyl-2,3-di-O-methyl-alpha-L-rhamnopyranose promoted by boron trifluoride etherate with the appropriate alcohol proceeded stereoselectively and with very high yields. Selective deacetylation and glycosylation with 2,4-di-O-acetyl-3,6-di-O-methyl-alpha-D-glucopyranosyl bromide led to a trisaccharide. The acrylamide copolymers of mono-, di-, and tri-saccharide were compared in their ability to specifically bind antibodies from leprosy patients.

Influence of acetylator phenotype of the leprosy patient on the emergence of dapsone resistant leprosy.

The half time of disappearance of dapsone and monoacetyl dapsone and the acetylator phenotype of the leprosy patients who harboured dapsone sensitive and dapsone resistant M. leprae was assessed in 27 subjects. Sixteen patients were rapid acetylators, five were slow and six were intermediate acetylators. The mean T 1 1/2 lives of dapsone (30.26 +/- 11.0) and monoacetyl dapsone (31.11 +/- 12.0) were also studied in the above patients. The percentage of different acetylators in both resistant and sensitive groups were similar showing no correlation between the emergence of drug resistance and the phenotype of the patient. The mean time of disappearance of DDS and MAD in the different acetylators did not show significant difference. The ratios of MAD/DDS in an individual at 3, 6 or 24 hours after the dose were similar. The mean T 1 1/2 lives of DDS and MAD in resistant and sensitive patients also showed no difference. Neither T 1 1/2 lives of DDS or MAD nor the acetylator phenotype seem to influence the emergence of dapsone resistance.

Associaçäo entre fenótipo acetilador de INH e índices morfológicos e baciloscópicos em pacientes hansenianos virchovianos / Association between the INH acetylator phenotype and morphological and bacilluscopic rates in virchowian leprosy leprous patients

Procuramos, neste trabalho, relacionar o fenótipo acetilador de isoniazida com índices morfológicos, baciloscópicos e tempo de duraçäo de tratamento em 2 grupos de pacientes hansenianos, virchovianos e branqueados, sendo que o primeiro deles apresenta suspeita clínica de sulfono resistencia. Säo todos pacientes do sexo masculino, internados e ou controlados no Hospital Lauro de Souza Lima, Bauru, Estado de Säo Paulo. O nosso objetivo principal foi pesquisar através de exames simples, na correlaçäo proposta acima, evidência de algum fator que pudesse ajudar numa investigaçäo ampla com pacientes virchovianos, fator este que sugerisse predisposiçäo para sulfono resistência

Intra-adipose administration of monoacetyldapsone to healthy volunteers.

The pharmacokinetics of intra-adiposely administered monoacetyldapsone (particle size less than 20 micron) were investigated in 11 male and 11 female healthy volunteers. Dapsone and monoacetyldapsone concentrations in serum were determined by high-pressure liquid chromatography (HPLC). Injection of 1175 mg monoacetyldapsone, which is equivalent to 1000 mg dapsone, resulted in dapsone concentration/time profiles in all the volunteers characterized by peak concentrations ranging from 0.14 to 0.85 mg/l, and by averaged dapsone concentrations after 28, 42, and 56 days of 0.33, 0.19, and 0.10 mg/l, respectively. Areas under the curves ranged from 6.7 to 25.3 mg X day/l. Detectable concentrations (greater than 6 ng/ml) of dapsone were achieved for 56 days in most of the subjects. An estimation of the mean concentration after repeated injection every 4 weeks ranged from 0.24 to 0.90 mg/l. No differences in dapsone concentration/time course were detectable between men and women or between rapid and slow acetylators. The injection was generally well tolerated by the subjects. This, combined with the excellent sustained release properties, makes it a promising injection. In the future, it might contribute to combat noncompliance among leprosy patients, which is believed to be one of the main causes of dapsone resistance.

Survey of the human acetylator polymorphism in spontaneous disorders.

There is ample evidence that the human acetylator phenotypes are associated with drug induced phenomena. It is principally the slow acetylators who exhibit toxic adverse effects because of their relative inability to detoxify the original drug compounds. In rare instances, however, it is the rapid acetylators who are at a disadvantage. In the matter of association of spontaneous disease with either acetylator phenotype, there are two groups of disorders to consider. First, disorders in which carcinogenic amines are known to be an aetiological factor. This is because these amines are substrates for the polymorphic N-acetyltransferase activity and hence there is a possible rational basis for searching for an association. Secondly, other disorders where searches for associations are based more on hunches. In the first group there is a definite statistical association between cancer of the bladder and the slow acetylator phenotype. In prevalence studies the slow phenotype is 39% more associated with bladder cancer than is the rapid phenotype. On the basis of the evidence now available it is not possible to say whether this association is because slow acetylators develop the disease more frequently or whether they survive longer. In the second group the relevant studies show (1) a greatly increased prevalence of slow acetylators in Gilbert's disease; (2) a confirmed association between the rapid acetylator phenotype and diabetes; (3) a possible association between the rapid acetylator phenotype and breast cancer; (4) a possible association between the slow acetylator phenotype and leprosy in Chinese patients; (5) an earlier age of onset of thyrotoxicosis (Graves' disease) in slow acetylators than in rapid acetylators; (6) no evidence of an association between either phenotype and spontaneous systemic lupus erythematosus.