Immunophenotyping of peripheral blood mononuclear cells and intracellular detection of IL-2, IFNγ and IL-4 by flow cytometry in patients with actinomycetoma by Nocardia brasiliensis and Actinomadura madurae. Findings in six patients

Mycetoma is a chronic, granulomatous, progressive inflammatory disease;it usually involves the subcutaneous tissue after a traumatic inoculation of the causative organism. Mycetoma may be caused by true fungi or bacteria (actinomycetes) and hence it is classified as eumycetoma and actinomycetoma respectively. Mycetoma immunological studies have fre- quently addressed humoral aspects. Few reports have addressed the role of cellular immunity in humans, for this reason, we were interested in finding differences in the circulating population of mononuclear cells or in the ability of this pathology to produce cytokines after mitogen stimulation. In this study, immunophenotyping of peripheral blood mononuclear cells (PBMN) and intracellular detection by flow cytometry of IL-2, IFNγ and IL-4 in patients with actino- mycetoma by Nocardia brasiliensis and Actino- madura madurae were evaluated. We investi- gated the expression of T-cells (CD3, CD4 and CD8), B-cells (CD19), monocytes (CD14), and natural killer cells (CD16 and CD56) markers. CD69 and CD25 were used to monitor individual activated cell subsets. The percentage values of the cells were calculated. Our results indicated that PBMC from patients with mycetoma show similar percentages of circulating cells when compared with healthy donors. The expression of IL-2 receptor (IL-2R) by mitogen activation was similar in these two groups. These findings suggest that circulating lymphocytes are not affected by this pathology. Intracellular IL-4 was increased only in patients with mycetoma by N. brasiliensis, suggesting a TH2 profile;this ob- servation has been reported by other authors.

Stimulation of the Protective Response to Actinomycetoma by Nocardia brasiliensis in Mice Treated with Candida albicans Antigens

Actinomycetoma is a chronic subcutaneous lesion caused by the pathogenic bacterium Nocardia (N.) brasiliensis. Yeast antigens of Candida (C.) albicans increase the interferon (IFN)-γ and TCD4+ cells levels that enhance the phagocytic killing of N. brasiliensis, able to survive inside phagocytes and to grow in clustered colonies that form grains. The aim of this work was to test the effect of a crude protein extract of C. albicans on the levels of IFN-γ producing TCD4+ cells and on the outcome of actinomycetoma lesion. Five BALB/c mice with N. brasiliensis infection at left hind footpad were treated four times every other day with C. albicans crude protein extract (CPE). Five uninfected mice treated with CPE or infected mice treated with sterile phosphate-saline buffer were included as positive and negative control groups, respectively. Footpad thickness was recorded in all groups. Once the treatments were finished, single cell suspensions from blood and spleen were prepared for assessing the amount (%) of IFN-γ producing TCD4+ cells by cytofluorometry;presence of TCD4+ and IFN-γ in footpad sections was detected by immunofluorescence and immunohistochemistry, respectively. By comparison with the negative control group, infected mice treated with CPE had lower footpad thickness, higher percentage of blood and spleen IFN-γ producing TCD4+ cells as well as in situ presence of IFN-γ and TCD4+ cells. These findings showed that CPE from C. albicans displayed an immunoadjuvant activity that enhanced the presence of IFN-γ producing TCD4+ cells and IFN-γ for the resolution of N. brasiliensis actinomycetoma in mice.

Detection of β-Glucuronidase Activity within Actinomadura madurae Grains of Human Actinomycetoma

Actinomycetoma syndrome by Actinomadura (A.) madurae is characterized by a subcutaneous chronic lesion that affects fascia, muscle and bone. A. madurae produces colonies that form grains of less than 1 mm in diameter. Grains are surrounded and infiltrated by neutrophils involved in the grain disruption by enzymes like β-glucuronidase released after the neutrophil degranulation. The aim of this work was to evaluate the polysaccharide degradation of grains treated with β-glucuronidase and to detect the presence and activity of β-glucuronidase within the A. madurae grains. Actinomadura madura grains from patients infected were processed to quantify the total content of polysaccharide with the phenol-sulfuric acid reaction. Grains were treated with β-glucuronidase at different conditions to evaluate the optimal polysaccharide degradation. Grains were analyzed to detect the enzyme by using anti-human β-glucuronidase antibody while enzymatic activity was assessed by evaluating the release of reduced sugars and by in situ enzymatic activity. Optimal degradation of polysaccharide in the grains treated with β-glucuronidase was found with 300 units/ml of enzyme and 24 hr of incubation at 37°C. Presence and activity of β-glucuronidase enzyme within the grains were detected. Results suggested that β-glucuronidase present within A. madurae grain resulted from degranulated neutrophils surrounding and/or infiltrated within the grain.

Activation and IL-1B secretion of human peripheral phagocytes infected with Actinomadura madurae,Nocardia asteroides and Candida albicans

Objective: To evaluate the ability of Actinomadura madurae(A. madurae) and Nocardia asteroids(N. asteroides), using Candida albicans(C. albicans) as prototypic control, to elicit the activation and IL-1β secretion of blood phagocytic cells from healthy donors. Methods: Microcopic evaluation of phagocytosis/activation, cell viability and spectrophotometric quantitation of endocytosis/activation, were assessed by using formazan blue test in human blood phagocytes infected with C. albicans, A. madurae or N. asteroides treated with either normal human serum(NHS) or with decomplemented NHS. Interlukin-1β from culture supernatants of infected polymorphonuclear was tested by ELISA kit assay. Results: Microscopic assay showed that phagocytosis and activation of adherent mononuclear phagocytes were greater with C. albicans followed by A. madurae and then by N. asteroides. Spectrophotometric assay in polymornuclear phagocytes infected with NHS-treated pathogens indicated that activation was similarly higher by C. albicans and A. madurae and lower by N. asteroides. Kinetic assays in infected polymorphonuclear cells showed that viability was decreased by C. albicans and N. asteroides or unaffected with A. madurae. Levels of IL-1β at 8 h of incubation were higher with C. albicans followed by A. madurae whereas lower levels were found with N. asteroides. Conclusions: The extent of cell-viability and activation as well IL-1β secretion may be related with the virulence of C. albicans and N. asteroides and other parameters remain to be explored for assesing the virulence of A. madurae.

Mycetoma at a tertiary care hospital in Saudi Arabia: correlation of histopathological and clinical findings

Objective: To present the histopathological and clinical correlation of mycetoma among patients attending King Abdulaziz University Hospital between 1998-2013.Methods: The data of all histopathologically diagnosed mycetomas in the period between January 1998 and January 2013 were collected through a computerized database search of the anatomic pathology archives at King Abdulaziz University Hospital. The collected data were analysed. Identification of species were performed for five patients using 16 S ribosomal DNA and internal transcribed spacer 2.Results: There were 19 patients with mycetoma with an average age of 44.26 years and male: female ratio of 4:1. Actinomycetoma were 63.15% and eumycetoma were 36.84%. All patients presented with the classic lesions; presenting as painless subcutaneous mass, sinuses and discharge containing grains. The swellings were of slow evolution, with preferential foot localization. Species specification performed for samples from five patients with active lesions revealed species of Actinomyces israelii and Madurella mycetomatis in respective cases.Conclusions: Actinomycetoma is more common than eumycetoma in this region. The fact that one of the patients with eumycetoma was a Saudi national raises the possibility of an indigenous species similar to Maduraella mycetomatis to be further explored for characteristics and pathogenesis. The disease has to be prioritized again and more robust and quick molecular diagnostic tools should be made available in order to save patients form disfiguring amputations.