[Show abstract][Hide abstract] ABSTRACT: Jorge Lobo's disease (JLD) is a chronic infection that affects the skin and subcutaneous tissues. Its etiologic agent is the fungus Lacazia loboi. Lesions are classified as localized, multifocal, or disseminated, depending on their location. Early diagnosis and the surgical removal of lesions are the best therapeutic options currently available for JLD. The few studies that evaluate the immunological response of JLD patients show a predominance of Th2 response, as well as a high frequency of TGF-β and IL-10 positive cells in the lesions; however, the overall immunological status of the lesions in terms of their T cell phenotype has yet to be determined. Therefore, the objective of this study was to evaluate the pattern of Th1, Th2, Th17 and regulatory T cell (Treg) markers mRNA in JLD patients by means of real-time PCR. Biopsies of JLD lesions (N = 102) were classified according to their clinical and histopathological features and then analyzed using real-time PCR in order to determine the expression levels of TGF-β1, FoxP3, CTLA4, IKZF2, IL-10, T-bet, IFN-γ, GATA3, IL-4, IL-5, IL-13, IL-33, RORC, IL-17A, IL-17F, and IL-22 and to compare these levels to those of healthy control skin (N = 12). The results showed an increased expression of FoxP3, CTLA4, TGF-β1, IL-10, T-bet, IL-17F, and IL-17A in lesions, while GATA3 and IL-4 levels were found to be lower in diseased skin than in the control group. When the clinical forms were compared, TGF-β1 was found to be highly expressed in patients with a single localized lesion while IL-5 and IL-17A levels were higher in patients with multiple/disseminated lesions. These results demonstrate the occurrence of mixed T helper responses and suggest the dominance of regulatory T cell activity, which could inhibit Th-dependent protective responses to intracellular fungi such as L. loboi. Therefore, Tregs may play a key role in JLD pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: Leprosy, an infectious disease caused by Mycobacterium leprae, affects millions of people worldwide. However, little is known regarding its molecular pathophysiological mechanisms. In this study, a comprehensive assessment of human mRNA was performed on leprosy skin lesions by using DNA chip microarrays, which included the entire spectrum of the disease along with its reactional states. Sixty-six samples from leprotic lesions (10TT, 10BT, 10BB, 10BL, 4LL, 14R1, and 10R2) and nine skin biopsies from healthy individuals were used as controls (CC) (ages ranged from 06 to 83 years, 48 were male and 29 female). The evaluation identified 1580 differentially expressed mRNAs [Fold Change (FC) ≥ 2.0, p ≤ 0.05] in diseased lesions vs. healthy controls. Some of these genes were observed in all forms of the disease (CD2, CD27, chit1, FA2H, FAM26F, GZMB, MMP9, SLAMF7, UBD) and others were exclusive to reactional forms (Type “1” reaction: GPNMB, IL1B, MICAL2, FOXQ1; Type “2” reaction: AKR1B10, FAM180B, FOXQ1, NNMT, NR1D1, PTX3, TNFRSF25). In literature, these mRNAs have been associated with numerous pathophysiological processes and signaling pathways and are present in a large number of diseases. The role of these mRNAs maybe studied in the context of developing new diagnostic markers and therapeutic targets for leprosy.
Full-text · Article · Nov 2015 · Frontiers in Genetics
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate the effects of the protein-calorie malnutrition in BALB/c isogenic mice infected with Lacazia loboi, employing nutritional and histopathological parameters. Four groups were composed: G1: inoculated with restricted diet, G2: not inoculated with restricted diet, G3: inoculated with regular diet, G4: not inoculated with regular diet. Once malnutrition had been imposed, the animals were inoculated intradermally in the footpad and after four months, were sacrificed for the excision of the footpad, liver and spleen. The infection did not exert great influence on the body weight of the mice. The weight of the liver and spleen showed reduction in the undernourished groups when compared to the nourished groups. The macroscopic lesions, viability index and total number of fungi found in the footpads of the infected mice were increased in G3 when compared to G1. Regarding the histopathological analysis of the footpad, a global cellularity increase in the composition of the granuloma was observed in G3 when compared to G1, with large numbers of macrophages and multinucleated giant cells, discrete numbers of lymphocytes were present in G3 and an increase was observed in G1. The results suggest that there is considerable interaction between Jorge Lobo's disease and nutrition.
Full-text · Article · Oct 2015 · Revista do Instituto de Medicina Tropical de São Paulo
[Show abstract][Hide abstract] ABSTRACT: We synthesized a series of novel dapsone-thalidomide hybrids (3a-i) by molecular hybridization and evaluated their potential for the treatment of type 2 leprosy reactions. All of the compounds had analgesic properties. Compounds 3c and 3h were the most active antinociceptive compounds and reduced acetic acid-induced abdominal constrictions by 49.8% and 39.1%, respectively. The hybrid compounds also reduced tumor necrosis factor-α levels in lipopolysaccharide-stimulated L929 cells. Compound 3i was the most active compound; at concentrations of 15.62 and 125μM, compound 3i decreased tumor necrosis factor-α levels by 86.33% and 87.80%, respectively. In nude mice infected with Mycobacterium leprae in vivo, compound 3i did not reduce the number of bacilli compared with controls. Compound 3i did not have mutagenic effects in Salmonella typhimurium strains TA100 and TA102, with or without metabolic activation (S9 mixture). Our results indicate that compound 3i is a novel lead compound for the treatment of type 2 leprosy reactions.
No preview · Article · May 2014 · Bioorganic & Medicinal Chemistry Letters
[Show abstract][Hide abstract] ABSTRACT: Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae
in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1nu). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.
Preview · Article · Mar 2014 · Journal of Visualized Experiments
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis and lymphangiogenesis are the processes of neovascularization that evolve from preexisting blood and lymphatic vessels. There are few studies on angiogenesis and none on lymphangiogenesis in leprosy. Thus, the role of neovascularization in the pathophysiological mechanisms of the disease was studied across the spectrum of leprosy, its reactional states and its residual lesions.
Seventy-six biopsies of leprosy skin lesions and seven healthy controls were selected. Fifty-five serum samples were used for the detection of CD105 by ELISA. Histological sections were stained with antibodies against CD31 (blood and lymphatic vessels), D2-40/podoplanin (lymphatic vessels), and CD105/endoglin (neovessels). Microvessels were counted in 100 high-power fields (400x) and the number of vessels was evaluated in relation to the extension of the inflammatory infiltrate (0-3), to the bacillary index (0-6) and to the clinical forms. Angiogenesis, as marked by CD31 and CD105, was observed across the leprosy spectrum, compared with the controls. Additionally, there was a positive correlation between these markers with extension of the infiltrate (p <0.0001). For D2/40, lymphangiogenesis was observed in the tuberculoid form (p <0.0001). There was no statistical significance for values of CD105 detected in plasma by ELISA.
Angiogenesis is present across the spectrum of leprosy and in its reactional forms. The increase in the number of vessels, as detected by CD31 and CD105 staining, is related to the extension of the inflammatory infiltrate. Samples from reactional lesions have a higher number of CD31+ and CD105+ stained vessels, which indicates their involvement in the pathophysiological mechanisms of the reactional states. The regression of lesions is accompanied by the regression of neovascularization. Drugs inhibiting angiogenesis may be relevant in the treatment of leprosy, in addition to multidrugtherapy, and in the prevention of the development of reactions.
[Show abstract][Hide abstract] ABSTRACT: Objectives:
To investigate podoplanin expression in epithelial odontogenic tumours with and without ectomesenchyme and verify the association between its immunoexpression and proliferative activity in keratocystic odontogenic tumours (KCOTS) and orthokeratinized odontogenic cysts (OOCs).
Eight ameloblastomas, nine adenomatoid odontogenic tumours, twenty KCOTS, five OOC, one calcifying epithelial odontogenic tumour, two ameloblastic fibromas, four ameloblastic fibro-odontomas and five calcifying cystic odontogenic tumours were immunohistochemically analysed with anti-podoplanin antibody. For KCOTS and OOC, the cell proliferation index was determined with Ki-67 immunostaining and compared by Spearman correlation coefficient.
Podoplanin was expressed in the peripheral odontogenic epithelium of most tumours. Ectomesenchyme was negative, except for odontoblasts. KCOTS exhibited positive podoplanin expression while in OOC it was absent/weak. There was statistically significant correlation (p=0.006) between podoplanin expression and cellular proliferation index of KCOTS and OOC.
Podoplanin seems to be related to the proliferative activity of KCOTS and may have a role in the process of local invasion of odontogenic tumours with and without ectomesenchyme.
[Show abstract][Hide abstract] ABSTRACT: Studies investigating the immunopathological aspects of Jorge Lobo's disease have shown that the inflammatory infiltrate consists
mainly of histiocytes and multinucleated giant cells involving numerous yeast-like cells of Lacazia loboi, with the T lymphocytes more common than B lymphocytes and plasma cells. The quantification of cytokines in peripheral blood
mononuclear cells culture supernatant has revealed alterations in the cytokines profile, characterized by predominance of
a Th2 profile. In view of these findings and of the role of cytokines in cell interactions, the objective of the present study
was to investigate the presence of the cytokines IL-10, TGF-ß1 and TNF-α, as well as iNOS enzyme in granulomas induced by
L. loboi. Histological sections obtained from skin lesions of 16 patients were analyzed by immunohistochemistry for the presence of
these cytokines and iNOS. The results showed that TGF-ß1 was the cytokine most frequently expressed by cells present in the
inflammatory infiltrate, followed by IL-10. There was a minimum to discrete positivity of cells expressing TNF-α and iNOS.
The results suggest that the presence of immunosuppressive cytokines in skin lesions of patients with the mycosis might be
responsible for the lack of containment of the pathogen as demonstrated by the presence of numerous fungi in the granuloma.
Full-text · Article · Jul 2011 · Medical mycology: official publication of the International Society for Human and Animal Mycology
[Show abstract][Hide abstract] ABSTRACT: We investigated the in vitro and skin lesions production of cytokines in non-treated borderline tuberculoid (BT) and borderline lepromatous (BL) patients.
Seven untreated, non-reactional BT patients and 12 untreated, non-reactional BL patients were studied. Levels of the cytokines IFN-gamma, IL-10, TGF-beta1 and TNF-alpha were measured in supernantant of peripheral blood mononuclear cells (PBMC) cultures, stimulated with specific M. leprae antigen (sonicated and whole). The cytokines iNOS, IL-10 and TGF-beta1 were detected by immunohistochemistry in skin biopsies.
BT patients produced higher levels of IFN-gamma than BL patients; iNOS expression in skin lesions was also higher in BT patients. TGF-beta1 was detected in more cells in BL patients; IL-10 expression was similar in both groups. There was a negative correlation between iNOS and TGF-beta1 expression in skin biopsies, positive correlation between TGF-beta1 in skin lesions and bacillary index, as well as positive correlation between iNOS detected in skin biopsies and PBMC IFN-gamma production.
The BT patients had a mainly a Th1-profile of cytokines in their skin lesions and BL patients had a Th2 profile.
[Show abstract][Hide abstract] ABSTRACT: Possible drug resistance in Mycobacterium leprae strains from Venezuela and three other South American countries was surveyed by molecular methods. None of the 230 strains
from new leprosy cases exhibited drug resistance-associated mutations. However, two of the three strains from relapsed cases
contained dapsone resistance mutations, and one strain also harbored a rifampin resistance mutation. Single nucleotide polymorphism
analysis of these strains revealed five subtypes: 3I (73.8%), 4P (11.6%), 1D (6.9%), 4N (6%), and 4O (1.7%).
Full-text · Article · Mar 2011 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: Jorge Lobo's disease is a cutaneous and subcutaneous mycosis that affects patients in the Amazon region. The number of patients is relatively small, but the real situation of the disease as public health problem is not known, because Jorge Lobo's disease is not a notifiable disease. This study aims to report the clinical evolution in patients affected and to determine the prevalence and areas of occurrence of the disease. A retrospective study was carried out based on the analysis of the clinical records, which included a collection of photographs of patients in the Department of Sanitary Dermatology, in Rio Branco, and patients seen in the interior of the state. In a decade, in Rio Branco, 249 cases of the disease were reported, 30 were females and 219 males. Of these patients, 153 had localized lesions, 94 of them were on one ear, 55 had multifocal lesions and 41 had disseminated lesions. The average time between the onset of symptoms and diagnosis was 19 years. The average age at the time of diagnosis was 53 years, and ages ranged from 14 to 96 years.
Preview · Article · Oct 2010 · Revista do Instituto de Medicina Tropical de São Paulo
[Show abstract][Hide abstract] ABSTRACT: Apoptosis eliminates pathogen-infected cells. Its modulation can influence the course of infections, permitting the survival of intracellular pathogens. In leprosy, which presents several clinical manifestations related to bacillary burden and host immune status, the mechanisms responsible for the persistence of the bacillus are unknown. Few studies have focused on apoptosis over the disease spectrum and as a defense mechanism against Mycobacterium leprae. We evaluated apoptosis using terminal transferase dUTP nick end labeling and the expression of Bcl-2 by immunohistochemistry in skin lesions from 11 tuberculoid and 15 lepromatous leprosy patients. Each specimen was evaluated by determining the number of positive cells in 10 fields at × 400 magnification. We observed a higher number of apoptotic cells in tuberculoid lesions in comparison with lepromatous leprosy (42.5 cells per 10 fields vs. 11.5 cells per 10 fields, P<0.0001). Expression of Bcl-2, conversely, was larger in lepromatous than in tuberculoid samples (172.0 cells per 10 fields vs. 17.7 cells per 10 fields, P<0.0001). These observations suggest modulation of apoptosis in leprosy, primarily in lepromatous patients, for which the decrease in cell death could support M. leprae survival and contribute to the success of infection. Conversely, in tuberculoid patients, apoptosis could contribute to reducing propagation of the bacillus.
[Show abstract][Hide abstract] ABSTRACT: Procedures involving the use of Mycobacterium leprae and Lacazia loboi, uncultivated organisms, depend on the collection of material from the lesions of patients or experimental animals. This study compared fine-needle aspiration (FNA) and skin biopsy methods for obtaining bacilli and fungal cells to experimentally infect animals.
Lepromas from one armadillo and one enlarged footpad of a mouse previously inoculated with L. loboi were submitted to FNA and biopsy. Materials collected were processed for inoculation in mice.
Acid-fast bacilli (AFB) collected by two FNA procedures yielded 7.2×10(7) and 5.3×10(6) AFB/ml and biopsies yielded 1.58×10(8) and 3.5×10(8) AFB/ml from each leproma. Yeast-like cells of L. loboi collected by FNA yielded 1.0×10(6) fungal cells/ml and biopsy 1.0×10(7) fungal cells/ml. After 8 months, inoculated animals were sacrificed and the inoculated footpads submitted to histopathological examination and counting of AFB and fungal cells. The results obtained by the two methods were comparable for both microorganisms.
Biopsy may be replaced by FNA during harvesting of material for different purposes, especially for experimental inoculation of mice in leprosy and Jorge Lobo's disease, with the advantage of FNA being a simpler, less invasive, and less costly method.
Full-text · Article · Feb 2010 · International journal of infectious diseases: IJID: official publication of the International Society for Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Lacazia loboi is a geographically restricted, uncultivated fungal pathogen of humans and dolphins. Previous investigations using 18S small unit rDNA, chitin synthase 2 and gp43 DNA sequences positioned L. loboi as a close relative of Paracoccidioides brasiliensis. However, given the few individuals of L. loboi studied and the high degree of genetic variation observed in P. brasiliensis, the existence of L. loboi as an independent species has been questioned. To investigate the phylogenetic position of this species, we conducted a phylogenetic analysis using 20 L. loboi collections (L. loboi was obtained from proven cases of lacaziosis and 14 collections were maintained in mice, the others were analyzed from DNA taken directly from infected human tissue.). L. loboi DNA sequence was compared to that from 17 P. brasiliensis strains that represented the known variation in this species, and outgroup taxa in the Onygenales (Ajellomyces and Coccidioides species). Our analyses used DNA sequence from ITS rRNA, and partial coding sequences of chitin synthase 4, ADP-ribosylation factor, and gp43. Nucleotide variation among strains of L. loboi was minor but numerous nucleotide mismatches and multiple gaps were found for these gene regions among members in the Ajellomycetaceae, including P. brasiliensis. Phylogenies inferred using neighbor-joining, maximum parsimony and Bayesian analyses showed no significant conflict and depicted L. loboi as a well-supported, monophyletic group that was sister to the Paracoccidioides clade. These results argue for maintaining L. loboi as a taxon independent from Paracoccidioides within the Ajellomycetaceae.
Full-text · Article · Jun 2009 · Mycological Research
[Show abstract][Hide abstract] ABSTRACT: Jorge Lobo's disease (Lacaziosis) is a subcutaneous infection of humans living in the Amazon region of Latin America, and in dolphins inhabiting the east coastal areas of the United States. The disease mainly affects people from rural areas living or working in close contact with vegetation and aquatic environments. Most patients refer having developed lesions after accidental trauma with plant thorns or insect bites. Inter-human transmission has never been confirmed suggesting that Lacazia loboi is acquired from environmental propagules.
We report the case of a 41-year-old woman from São Paulo, Brazil, a non-endemic area of Jorge Lobo's disease, with L. loboi skin infection most likely accidentally acquired while manipulating experimentally infected mice in the laboratory.
Because many patients with Jorge Lobo's disease do not recall accidental skin trauma before their infections, the possibility of accidentally acquired Jorge Lobo's disease through unnoticed broken skin should be considered during the clinical investigation of nodular skin diseases in people who have contact with the fungus or who live in endemic areas. This is the second report of animal to human transmission of this disease.
Full-text · Article · Feb 2009 · Journal of Medical Case Reports
[Show abstract][Hide abstract] ABSTRACT: Infectious and parasitic diseases have always challenged man. Although many of them are typically seen in some areas of the world and can be adequately managed by just improving socioeconomic status and sanitary conditions, they are still quite prevalent and may sometimes be seen outside their original geographical areas. Human migration due to different reasons, tourism, blood transfusion and solid organ transplantation has created new concerns for health professionals all over the world. If not for diagnostic purposes, at least these tropical and infectious diseases should be largely known because their epidemiology, pathogenesis, host/parasite interaction, inflammatory and reparative responses are quite interesting and teach us about human biology. Curiosity is inherent to pathology practice and so we are compelled to look for things other than tumours or degenerative diseases. This review focuses on infectious and parasitic diseases found in a developing country and brings up-to-date information on diseases caused by viruses (dengue, yellow fever), bacteria (typhoid fever, leprosy), parasites (Chagas' disease, cutaneous and visceral leishmaniasis, amoebiasis, Capillaria hepatica, schistosomiasis, cysticercosis) and caused by fungi (paracoccidioidomycosis, cryptococcosis, histoplasmosis) that may be useful for pathologists when facing somewhat strange cases from developing countries.
[Show abstract][Hide abstract] ABSTRACT: Antibodies in the sera of patients with lacaziosis recognized an approximately 193-kDa antigen and other Lacazia loboi antigens. Paracoccidioides brasiliensis gp43 antigen was detected by all evaluated sera, but they failed to detect a protein with the same molecular mass in L. loboi extracts. This study is the first to examine the humoral response to L. loboi antigens by using multiple host sera.
Full-text · Article · Feb 2008 · Clinical and vaccine Immunology: CVI
[Show abstract][Hide abstract] ABSTRACT: Since there are no studies evaluating the participation of the complement system (CS) in Jorge Lobo's disease and its activity on the fungus Lacazia loboi, we carried out the present investigation. Fungal cells with a viability index of 48% were obtained from the footpads of BALB/c mice and incubated with a pool of inactivated serum from patients with the mycosis or with sterile saline for 30 min at 37 masculineC. Next, the tubes were incubated for 2 h with a pool of noninactivated AB+ serum, inactivated serum, serum diluted in EGTA-MgCl2, and serum diluted in EDTA. The viability of L. loboi was evaluated and the fungal suspension was cytocentrifuged. The slides were submitted to immunofluorescence staining using human anti-C3 antibody. The results revealed that 98% of the fungi activated the CS by the alternative pathway and no significant difference in L. loboi viability was observed after CS activation. In parallel, frozen histological sections from 11 patients were analyzed regarding the presence of C3 and IgG by immunofluorescence staining. C3 and IgG deposits were observed in the fungal wall of 100% and 91% of the lesions evaluated, respectively. The results suggest that the CS and immunoglobulins may contribute to the defense mechanisms of the host against L. loboi.
Full-text · Article · Apr 2007 · Revista do Instituto de Medicina Tropical de São Paulo
[Show abstract][Hide abstract] ABSTRACT: Jorge Lobo's disease is a chronic cutaneous-subcutaneous mycosis caused by the fungus Lacazia loboi. The mechanisms involved in susceptibility and resistance to infection by this pathogen, especially genetic background, are yet to be understood. The objective of the present study was to evaluate the phenotypic distribution of the Duffy blood group in Jorge Lobo's disease patients and calculate its gene frequencies. Twenty one patients were analyzed, among them 13 Caucasoid and 8 from mixed ethnical backgrounds (Afro-Brazilians) originating from the State of Acre. The control group consisted of 44 healthy individuals from the city of Rio Branco (AC). The Duffy phenotype was defined by the presence of antigens in erythrocytes as visualized by the agglutination reaction of erythrocytes in the presence of anti-Fya and anti-Fyb antisera incubated at 37°C by the human antiglobulin test. Results showed that the phenotypic distribution of the Duffy blood group system in patients was significantly different from controls, with the frequency of individuals with phenotype Fy(a-b-) being very high. Patients presented remarkably higher frequencies of the allele FY and ower frequencies of the allele FYA compared to controls. Frequencies of the allele FYB (not including non-expressed alleles) were similar in both groups. The comparison between Duffy blood group distribution with clinical forms of the disease suggests predominance of the phenotype Fy(a-b+) in patients with the localized form of Jorge Lobo's disease, followed by phenotype Fy(a-b-) and absence of phenotype (a+b-). On the other hand, we observed a higher incidence of phenotype Fy(a+b-) in patients with the disseminated form of the disease. The multifocal forms were less expressive. Therefore, we may infer that the negative Duffy phenotype is probably more prevalent in the region studied. There is scarce data available on phenotype distributions, it is necessary to develop similar studies in populations affected by Jorge Lobo's disease in order to improve knowledge on the biological role of Duffy antigens. Rev. bras. hematol. hemoter. 2006;28(4):264-268.
Full-text · Article · Dec 2006 · Revista Brasileira de Hematologia e Hemoterapia