Development of bioprocess for the production of purified protein derivative with Brazilian strains of Mycobacterium tuberculosis for diagnosis use.

Tuberculin, a purified protein derivative (PPD), when diluted in adequate concentration it is utilized for an early detection and to control tuberculosis. In Brazil, the PPD is imported and distributed by the Health System in all Brazilian regions. This, in addition to eventual delay in delivery caused by the legal import process, makes difficult the access of the product to poor communities from distant places as Brazil is a geographically a large country. Thus, indigenous production of PPD would be very beneficial for the society. This work was undertaken with a view to initiate studies towards the development of an indigenous technology for PPD production, using the strains of Mycobacterium tuberculosis isolated from patients during the course of disease from several regions of Brazil. The strain selection criteria for PPD production were the sequencing of three immunodominant proteins and the genetic differentiation by DNA fingerprinting and grouped by UPGMA program, the capacity of protein production in liquid medium, and finally the intradermal injection tests used on animal model. Compared to gold standard, the PPD showed similar indurations when tested individually, and better results were obtained when products were combined in pool. These strategies are discussed in detail in this research.

Genetic analysis of Mycobacterium avium complex strains used for producing purified protein derivatives.

For over a century, purified protein derivatives (PPD) have been used to detect mycobacterial infections in humans and livestock. Among these, reagents to detect infections by Mycobacterium avium complex organisms have been produced, but the utility of these reagents has not been clearly established due in part to limited biologic and immunologic standardization. Because there is little information about the strains used to produce these reagents (avian PPD, intracellulare PPD, scrofulaceum PPD, and Johnin), we have performed genetic characterizations of strains used to produce these products. Sequence analysis of 16S rRNA and the hsp65 gene provided results concordant with species designations provided for M. avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum organisms. For M. avium strains, comparative genomic hybridization was performed on a whole-genome DNA microarray, revealing one novel 7.9-kilobase genomic deletion in certain Johnin-producing strains, in addition to genomic variability inherent to the particular M. avium subspecies. Our findings indicate that considerable genomic differences exist between organisms used for reagents and the infecting organism being studied. These results serve as a baseline for potency studies of different preparations and should aid in comparative studies of newly discovered antigens for the diagnosis of infection and disease by M. avium complex organisms.

Phenotypical and functional analysis of memory and effector human CD8 T cells specific for mycobacterial antigens.

Mycobacterium tuberculosis infects one-third of the global population and claims two million lives every year. Because memory CD8 T cells exhibit a high heterogeneity in terms of phenotype and functional characteristic, we investigated the frequency, phenotype, and functional properties of Ag85A epitope-specific HLA-A*0201 CD8 T cells in children affected by tuberculosis (TB) before and 4 mo after chemotherapy and healthy contact children. Using Ag85A peptide/HLA-A*0201 pentamer, we found a low frequency of blood peptide-specific CD8 T cells in tuberculous children before therapy, which consistently increased after therapy to levels detected in healthy contacts. Ex vivo analysis of the expression of CD45RA and CCR7 surface markers indicated a skewed representation of Ag85A epitope-specific CD8 T cells during active TB, with a predominance of T central memory cells and a decrease of terminally differentiated T cells, which was reversed after therapy. Accordingly, pentamer-specific CD8 T cells from tuberculous patients produced low levels of IFN-gamma and had low expression of perforin, which recovered after therapy. The finding of an elevated frequency of pentamer-specific CD8 T cells with T effector memory and terminally differentiated phenotypes in the cerebrospinal fluid of a child with tuberculous meningitis strongly indicates compartmentalization of such CD8 effectors at the site of disease. Our study represents the first characterization of Ag-specific memory and effector CD8 T cells during TB and may help to understand the type of immune response that vaccine candidates should stimulate to achieve protection.

Trace elements incorporated into the culture medium of Mycobacterium tuberculosis promote the presence of tuberculoprotein C in the preparation of purified protein derivatives.

Two purified protein derivatives (PPDs) were prepared from Mycobacterium tuberculosis, H37Ra. One of the PPDs was prepared from the culture filtrate of organisms grown on Proskauer-Beck medium to which trace elements had been added. The other PPD was prepared from the culture filtrate of organisms grown on the same medium but without trace elements, and was used as the control. Comparative skin reactions in sensitized rabbits showed that the PPD prepared from organisms grown in the presence of trace elements was less potent than the control. Since it has long been recognized that of the many tuberculoproteins present in PPD, the C protein (a 44 to 66 kDa protein) was always the least potent fraction when tested in equivalent concentrations in both serological and skin test assays, the possibility existed that the PPD obtained from organisms grown in trace element medium had more of the C protein complex than the control. Comparative studies of these two PPDs in terms of their chemical composition, skin test responses, and electrophoretic profiles obtained by SDS-polyacrylamide gel electrophoresis provide support for this assumption.

Preparation of 14C-labeled tuberculin purified protein derivative.

(14)C-labeled tuberculin purified protein derivative ((14)C-tuberculin PPD) has been prepared from culture filtrates of Mycobacterium tuberculosis var. hominis grown in a culture medium containing uniformly labeled (14)C-amino acids. With a mixture of (14)C-amino acids (an acid hydrolysate of (14)C-Chlorella protein) in the medium, the recoveries of (14)C in the final product were higher than with (14)C-labeled-l-glutamic acid. (14)C-tuberculin PPD was separated into tuberculoprotein and nucleic acid by paper electrophoresis. The specific radioactivity of tuberculoprotein was substantially greater than that of the nucleic acid. (14)C-tuberculin PPD is advocated as a means to measure the adsorption of tuberculin to glass or other surfaces. It could also prove useful as a means to study the structure and mode of action of tuberculin.

Correlação tuberculina-lepromina / Correlation tuberculin-lepromin

There existe no mutual relation between positiveness to Mantoux test and to that of Mitsuda among lepromatous patients. This fact constitutes, just like many others in the field of leprosy, an open question to further investigations.