Development of an immunochromatographic lateral flow dipstick for the detection of Mycobacterium tuberculosis 16 kDa antigen (Mtb-strip).

Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium that causes tuberculosis (TB). This contagious disease remains a severe health problem in the world. The disease is transmitted via inhalation of airborne droplets carrying Mtb from TB patients. Early detection of the disease is vital to prevent transmission of the infection to people in close contact with the patients. To date, there is a need of a simple, rapid, sensitive and specific diagnostic test for TB. Previous studies showed the potential of Mtb 16 kDa antigen (Ag16) in TB diagnosis. In this study, lateral flow immunoassay, also called simple strip immunoassay or immunochromatographic test (ICT) for detection of Ag16 was developed (Mtb-strip) and assessed as a potential rapid TB diagnosis method. A monoclonal antibody against Ag16 was optimized as the capturing and detection antibody on the Mtb-strip. Parameters affecting the performance of the Mtb-strip were also optimized before a complete prototype was developed. Analytical sensitivity showed that Mtb-strip was capable to detect as low as 125 ng of purified Ag16. The analytical sensitivity of Mtb-strip suggests its potential usefulness in different clinical applications.

Tuberculosis vaccine candidates based on mycobacterial cell envelope components.

Even after decades searching for a new and more effective vaccine against tuberculosis, the scientific community is still pursuing this goal due to the complexity of its causative agent, Mycobacterium tuberculosis (Mtb). Mtb is a microorganism with a robust variety of survival mechanisms that allow it to remain in the host for years. The structure and nature of the Mtb envelope play a leading role in its resistance and survival. Mtb has a perfect machinery that allows it to modulate the immune response in its favor and to adapt to the host's environmental conditions in order to remain alive until the moment to reactivate its normal growing state. Mtb cell envelope protein, carbohydrate and lipid components have been the subject of interest for developing new vaccines because most of them are responsible for the pathogenicity and virulence of the bacteria. Many indirect evidences, mainly derived from the use of monoclonal antibodies, support the potential protective role of Mtb envelope components. Subunit and DNA vaccines, lipid extracts, liposomes and membrane vesicle formulations are some examples of technologies used, with encouraging results, to evaluate the potential of these antigens in the protective response against Mtb.

Sequence comparison of six human microRNAs genes between tuberculosis patients and healthy individuals.

OBJECTIVE/BACKGROUND: MicroRNAs (miRNAs) play an important role in diseases development. Therefore, human miRNAs may be able to inhibit the survival of Mycobacterium tuberculosis (Mtb) in the human host by targeting critical genes of the pathogen. Mutations within miRNAs can alter their target selection, thereby preventing them from inhibiting Mtb genes, thus increasing host susceptibility to the disease. METHODS: This study was undertaken to investigate the genetic association of pulmonary tuberculosis (TB) with six human miRNAs genes, namely, hsa-miR-370, hsa-miR-520d, hsa-miR-154, hsa-miR-497, hsa-miR-758, and hsa-miR-593, which have been predicted to interact with Mtb genes. The objective of the study was to determine the possible sequence variation of selected miRNA genes that are potentially associated with the inhibition of critical Mtb genes in TB patients. RESULTS: The study did not show differences in the sequences compared with healthy individuals without antecedents of TB. CONCLUSION: This result could have been influenced by the sample size and the selection of miRNA genes, which need to be addressed in future studies.

Experimental model of graft vs. host disease in non-immunosuppressed F1 (CBA/J x C57BL/6) mice.

The experimental model of graft vs. host disease (GvHD) has a potential use in the evaluation of different manipulation procedures of the immune system applicable to development of vaccines. In the present study an experimental model of GvHD in F1 (CBA/J x C57BL/6) mice by means of the parenteral inoculation of spleen lymphoid cells from parental male CBA/J to 10-day-old animals (experimental group) was developed. Animals inoculated with Medium 199 (n = 42) (Medium 199 group), or with splenic lymphoid cells either from the hybrids (n = 16) (F1 group), or from mice of the inbred strain Balb/c (n = 10) (Balb/c group) were used as controls. In all groups body and spleen weights, relative spleen index (RSI), and spleen index (SI) were determined. Additionally, histopathologic and morphometric studies were done in the spleens of the animals studied. Significant increases in body and spleen weights, RSI, and lymphocytic perimeter and area were associated with distinctive splenic GvHD lesions found in the experimental group. The experimental SI value was higher than twice the SI value of any of the control groups. We conclude that ours is a useful model of GvHD with many potential applications in the field of vaccine production.

Sequential study of an experimental model of graft vs. host disease (GvHD) in non-immunosuppressed F1 (CBA/J x C57BL/6) mice.

In order to determine the optimal day for the evaluation of an experimental model of GvHD in F1 mice and the histopathologic evolution of the lesions in different organs, we studied 10-day-old F1 (CBA/J x C57BL/6) mice inoculated with splenic lymphoid cells of the male parental CBA/J strain (n = 42) that were sacrificed between 1 and 14 days postinoculation. The evolution of the relative spleen index (RSI) and the histopathologic lesions in different organs were also determined. F1 mice inoculated with Medium 199 were used as controls. Significant RSI increases (p < 0.0001) were found in the experimental group between 2 and 14 days postinoculation, with a peak at the eighth day, associated with the most severe histopathologic lesions in the organs studied. We suggest the eighth day as the optimal time for evaluation of this experimental model.

Histopathologic and humoral study of Balb/c mice inoculated with BCG by different routes.

With the aim of determining the distribution and humoral immunogenicity of the bacillus Calmette-Guérin (BCG) administered by the oral (O), intravenous (IV) and subcutaneous (SC) routes, we studied 54 male Balb/c mice weighing 17-22 g that had been inoculated with BCG (10(6) CFU) by the O (n = 18), IV (n = 18) and SC (n = 18) routes. At weekly intervals we determined the distribution of the microorganism using histopathological techniques including Ziehl-Neelsen staining. Serum samples of the same animals were analyzed by ELISA and Western blot to determine the antibody response to the microorganism. In all groups, distinctive histopathologic lesions harboring the microorganism were found. Using the SC route the lesions were located at the inoculation site, whereas there was systemic dissemination with the O and IV routes, being more prominent with the latter. Anti-BCG antibodies were detected by ELISA in all groups; this response was more intense in the IV group, followed by the SC and O groups. In the Western blot analysis, reactivity against multiple bands and the predominant recognition of a 65 kd band in all groups was observed.

Study of KIR genes in tuberculosis patients.

A total of 97 patients with tuberculosis (TB) and 51 controls from Xalapa, Veracruz, Mexico, were studied for the presence and absence of killer cell immunoglobulin-like receptor (KIR) genes. The number of patients with either KIR2DL1 or KIR2DL3 differed significantly compared with the controls. However, only the difference in KIR2DL3 remained significant after correction for the number of factors analysed. We also found KIR2DS2 with its presumed C1 group ligand less prevalent in TB patients than in the control group, but this result lost significance after correction.

Specific cellular and humoral immune response in Balb/c mice immunised with an expression genomic library of Trypanosoma cruzi.

An expression genomic library of Trypanosoma cruzi (T. cruzi) constructed using pcDNA3 plasmid was used for the immunisation (25 micrograms) of Balb/c mice. Expression of T. cruzi antigens in the muscle of inoculated mice was detected by indirect immunofluorescence 7 days after immunisation. Specific IgG antibodies were significatively increased (P < 0.05) in animals that were reimmunized with 50 micrograms of the genomic library. An antigen specific lymphoproliferative response was detected in one animal of the group inoculated with one dose of the library.

[Specific humoral response in Balb/c mice inoculated with a genome library of expression of Trypanosoma cruzi]. / Respuesta humoral específica en ratones Balb/c inoculados con una librería genómica de expresión de Trypanosoma cruzi.

A genomic expression library of Trypanosoma cruzi (T. cruzi) was made using plasmid pcDNA3 as a vector, with which male mice from the Balb/c isogenic line were intramuscullary inoculated. It was used a positive control group that was administered soluble antigens of T. cruzi. Other 2 groups received genomic and plasmid DNA, respectively. One group was not immunized. Weekly blood samples were obtained from all the animals until the fourth week and 2 weeks after reimmunization to study the response of specific antibodies against the microorganism antigens by an indirect immunoenzymatic assay (ELISA). It was observed a significant increase of specific antibodies in the animals reimmunized with 50 micrograms of the library, as well as in the group immunized with soluble antigens of T. cruzi.