Genetic polymorphisms of the tumor necrosis factor and lymphotoxin alpha in type 2 diabetes.

OBJECTIVE: To investigate the frequency of the single-base change polymorphic variants identified in tumor necrosis factor (TNF) gene (-308 G/A) and lymphotoxin alpha (LTA) (+252 G/A) in patients with type 2 diabetes (T2D). METHODS: A prospective study in a Mexican-mestizo population of 51 patients with T2D and 48 healthy subjects was carried out. We took a peripheral blood sample from each individual for identification of the polymorphic genotypes by polymerase chain reaction. RESULTS: The genotype distribution in T2D was: TNF alpha homozygous 0%; TNFG/A heterozygous 20%; TNFG homozygous 80%. CONCLUSIONS: In regards to the TNF -308 G/A genotype, we found a significant difference (p = 0.012) with a bigger frequency in the group of patients. The health controls showed a higher frequency of TNF -308 G/G genotype (p = 0.034).

Functional state analysis of phagocytic cells of patients with type 2 diabetes and pulmonary tuberculosis.

BACKGROUND: The phagocytic function in pulmonary tuberculosis (PTB) and Type 2 diabetes (T2D) has been explored mainly in macrophages but not in polymorphonuclears (PMN). The purpose of this study was to determine the functional status of PMN leukocytes in patients with pulmonary tuberculosis (PTB), Type 2 diabetes (T2D), and in patients with both diseases. METHODS: An observational, prospective, and comparative study was carried out. 30 ambulatory patients with T2D, 10 with PTB undergoing treatment and 10 patients with PTB and T2D, and 44 healthy subjects were studied. PMN leukocytes were separated, the capacity of these cells to produce hydrogen peroxide and to reduce nitroblue tetrazolium (NBT) in response to stimulus with the phorbolic ester of myristic acid (PMA) was measured; and the capacity of PMN leukocytes to adhere to surfaces was determined. RESULTS: Concerning the test for adherence, on comparing healthy subjects with patients with T2D+PTB, we observed a clear decrease in cellular adherence in the group of patients with both diseases; it was statistically significant (p = 0.007).With regard to phagocytic function, we observed that in NBT reduction as well as in hydrogen peroxide production, statistically significant differences were not obtained on comparing healthy subjects with any of the three groups of patients. CONCLUSIONS: We observed a clear decrease in cellular adherence when both diseases co-exist. These results could indicate the need for the co-existence of T2D and TB to cause deterioration in the cells' adherence activity. The microtechniques employed permit the evaluation in a practical manner of certain phagocytic-activity expressions.

The use of the microplate alamar blue assay (MABA) to assess the susceptibility of Mycobacterium lepraemurium to anti-leprosy and other drugs.

Although murine leprosy is no longer a common illness, our understanding of the biology of this disease is incomplete. One particular example of this concerns the etiologic agent Mycobacterium lepraemurium (MLM). MLM is a fastidious microorganism that is difficult to grow in axenic media; in a way, this has hampered attempts to thoroughly study its physiological and metabolic characteristics. MLM is an obligate intracellular bacillus that invades macrophages and replicates profusely with a generation time that oscillates between 0.5 and 11 days. In the present study, we have successfully maintained MLM alive for more than 12 days in vitro, providing us with an opportunity to study its susceptibility to several anti-leprosy agents and other drugs. To achieve this, we used a fluorescence reduction assay of alamar blue (a resazurin) in a microplate format (microplate-alamar-blue-assay; MABA), which is a highly sensitive, practical, and inexpensive method for assaying cell viability. We found that MLM was highly susceptible to clofazimine and rifampicin and was less susceptible to streptomycin, thiacetazone, kanamycin, dapsone, and ethionamide, in that order. MLM was not susceptible to four plant triterpenoids (oleanolic acid, neolignan-c, sitosterol, and ursolic acid) for which bactericidal activity has been reported in M. tuberculosis. Because the MABA has high sensitivity, it can be used to monitor the activity of microorganisms that are difficult to cultivate (such as M. lepraemurium), in response to various drugs, thus offering a method to complement the study of murine leprosy, about which many questions remain unanswered.

Fate of Mycobacterium tuberculosis in peroxidase-loaded resting murine macrophages.

BACKGROUND: Myeloperoxidase (MPO), in the presence of hydrogen peroxide and a halide represent an efficient microbicidal mechanism of phagocytic cells. MPO is abundant in neutrophils which also respond to infection by producing large amounts of reactive oxygen species (ROS). MPO, ROS and halide constitute a very toxic antimicrobial system (called the Klebanoff system or KS). Resting mature macrophages do not contain granular MPO and thus are unable to kill pathogenic mycobacteria and some other microorganisms by this system. EXPERIMENTAL: Under the hypothesis that transforming macrophages into peroxidase-positive (PO(+)) cells, these cells would be able to kill Mycobacterium tuberculosis, in this study, mature macrophages were loaded with exogenous peroxidase and were tested for their capacity to kill the Mycobacterium in the presence or in the absence of hydrogen peroxide. RESULTS: It was found that PO-loaded macrophages eagerly ingest M. tuberculosis, but do not show a significant mycobactericidal activity on this microorganism despite that it is highly susceptible to the Klebanoff system in vitro. Failure of PO-loaded macrophages to kill M. tuberculosis may obey either to an inappropriate location of the exogenous PO in these cells or more likely, to the presence of efficient detoxifying mechanisms in the bacteria. On the contrary, MPO-loaded or unloaded macrophages efficiently killed Listeria monocytogenes. CONCLUSION: The lack of granular MPO in mature macrophages, and the predilection of mycobacteria to infect these cells are two situations that favor the development of tuberculosis and related diseases, such as leprosy and Buruli ulcer.

Polymorphisms in tumor necrosis factor and lymphotoxin A in tuberculosis without and with response to treatment.

This study compared the frequency of the genetic polymorphisms of tumor necrosis factor (TNF) in pulmonary tuberculosis without and with response to treatment. We carried out an observational, prospective, comparative study. Three groups were studied: healthy subjects, responders, and non-responders to directly observed treatment short-course. We took a peripheral blood sample for identification of polymorphic genotypes TNF -308G/A and lymphotoxin A (LTA) +252G/A by polymerase chain reaction, and their later digestion with the Nco1 restriction enzyme. We studied a total of 138 subjects: 42 (non-responders) and 48 in each of the remaining groups. Healthy subjects had significantly high frequency of the LTA +252A allele compared to groups of patients and could be related with protection from the disease. Patients had higher frequency of the non-polymorphic allele LTA +252G than healthy subjects. With regard to LTA +252G/A genotype, we did find a significant difference with a greater frequency in the group of patients. The LTA +252G/A genotype was associated with impaired response to treatment.