Protection conferred by heterologous vaccination against tuberculosis is dependent on the ratio of CD4(+)/CD4(+) Foxp3(+) cells

CD4(+) Foxp3(+) regulatory T cells inhibit the production of interferon-?, which is the major mediator of protection against Mycobacterium tuberculosis infection. In this study, we evaluated whether the protection conferred by three different vaccines against tuberculosis was associated with the number of spleen and lung regulatory T cells. We observed that after homologous immunization with the 65 000 molecular weight heat-shock protein (hsp 65) DNA vaccine, there was a significantly higher number of spleen CD4(+) Foxp3(+) cells compared with non-immunized mice. Heterologous immunization using bacillus Calmette Guerin (BCG) to prime and DNA-hsp 65 to boost (BCG/DNA-hsp 65) or BCG to prime and culture filtrate proteins (CFP)-CpG to boost (BCG/CFP-CpG) induced a significantly higher ratio of spleen CD4(+)/CD4(+) Foxp3(+) cells compared with non-immunized mice. In addition, the protection conferred by either the BCG/DNA-hsp 65 or the BCG/CFP-CpG vaccines was significant compared with the DNA-hsp 65 vaccine. Despite the higher ratio of spleen CD4(+)/CD4(+) Foxp3(+) cells found in BCG/DNA-hsp 65-immunized or BCG/CFP-CpG-immunized mice, the lungs of both groups of mice were better preserved than those of DNA-hsp 65-immunized mice. These results confirm the protective efficacy of BCG/DNA-hsp 65 and BCG/CFP-CpG heterologous prime-boost vaccines and the DNA-hsp 65 homologous vaccine. Additionally, the prime-boost regimens assayed here represent a promising strategy for the development of new vaccines to protect against tuberculosis because they probably induce a proper ratio of CD4(+) and regulatory (CD4(+) Foxp3(+)) cells during the immunization regimen. In this study, this ratio was associated with a reduced number of regulatory cells and no injury to the lungs.

Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage

Abstract Background Vaccination of neonates is generally difficult due to the immaturity of the immune system and consequent higher susceptibility to tolerance induction. Genetic immunization has been described as an alternative to trigger a stronger immune response in neonates, including significant Th1 polarization. In this investigation we analysed the potential use of a genetic vaccine containing the heat shock protein (hsp65) from Mycobacterium leprae (pVAXhsp65) against tuberculosis (TB) in neonate mice. Aspects as antigen production, genomic integration and immunogenicity were evaluated. Methods Hsp65 message and genomic integration were evaluated by RT-PCR and Southern blot, respectively. Immunogenicity of pVAXhsp65 alone or combined with BCG was analysed by specific induction of antibodies and cytokines, both quantified by ELISA. Results This DNA vaccine was transcribed by muscular cells of neonate mice without integration into the cellular genome. Even though this vaccine was not strongly immunogenic when entirely administered (three doses) during early animal's life, it was not tolerogenic. In addition, pVAXhsp65 and BCG were equally able to prime newborn mice for a strong and mixed immune response (Th1 + Th2) to pVAXhsp65 boosters administered later, at the adult life. Conclusion These results suggest that pVAXhsp65 can be safely used as a priming stimulus in neonate animals in prime-boost similar strategies to control TB. However, priming with BCG or pVAXhsp65, directed the ensuing immune response triggered by an heterologous or homologous booster, to a mixed Th1/Th2 pattern of response. Measures as introduction of IL-12 or GM-CSF genes in the vaccine construct or even IL-4 neutralization, are probably required to increase the priming towards Th1 polarization to ensure control of tuberculosis infection.

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

Abstract Background A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses. Methods To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity. Results It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes. Conclusion Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy.

B cells Can Modulate the CD8 Memory T Cell after DNA Vaccination Against Experimental Tuberculosis

Abstract Background Although B cells are important as antigen presenting cells (APC) during the immune response, their role in DNA vaccination models is unknown. Methods In this study in vitro and in vivo experiments were performed to evaluate the ability of B cells to protect mice against Mycobacterium tuberculosis challenge. Results In vitro and in vivo studies showed that B cells efficiently present antigens after naked plasmid pcDNA3 encoding M. leprae 65-kDa heat shock protein (pcDNA3-Hsp65) internalization and protect B knock-out (BKO) mice against Mycobacterium tuberculosis infection. pcDNA3-Hsp65-transfected B cells adoptively transferred into BKO mice rescued the memory phenotypes and reduced the number of CFU compared to wild-type mice. Conclusions These data not only suggest that B cells play an important role in the induction of CD8 T cells but also that they improve bacterial clearance in DNA vaccine model.

A study of multidrug-resistant tuberculosis in risk groups in the city of Santos, Sao Paulo, Brazil

Monitoring the extent of and trends in multidrug-resistant tuberculosis (MDR-TB) is a priority of the Brazilian National Tuberculosis Control Programme. The current study aimed to estimate the incidence of MDR-TB, describe the profile of TB drug resistance in risk groups and examine whether screening for MDR-TB adhered to the recommended guidelines. A descriptive study that examined diagnosed cases of pulmonary TB was conducted in the city of Santos, Brazil, between 2000-2004. Of the 2,176 pulmonary TB cases studied, 671 (30.8%) met the criteria for drug sensitivity testing and, of these cases, 31.7% (213/671) were tested. Among the tested cases, 9.4% were resistant to one anti-TB drug and 15% were MDR. MDR was observed in 11.6% of 86 new TB cases and 17.3% of 127 previously treated cases. The average annual incidence of MDR-TB was 1.9 per 100,000 inhabitants-years. The extent of known MDR-TB in the city of Santos is high, though likely to be underestimated. Our study therefore indicates an inadequate adherence to the guidelines for MDR-TB screening and suggests the necessity of alternative strategies of MDR-TB surveillance.

DELAY IN THE DIAGNOSIS OF TUBERCULOSIS IN PRISONS: THE EXPERIENCE OF INCARCERATED PATIENTS

This study analyzed the causes of delay in the diagnosis of tuberculosis in the prison system, according to the experience of incarcerated patients. The theoretical and methodological framework of the French school of discourse analysis was used, which seeks to comprehend the processes of meaning production, in the relationship of language with ideology and the development of subjects in their positions. Semi-directed interviews were conducted with seven incarcerated tuberculosis patients in a hospital of Joao Pessoa, Paraiba, Brazil, between August and October 2009. The delay in the diagnosis of tuberculosis was related to the naturalization of the lack of care for the prisoner, to the interpretation of the prison as a place of death and suffering and to the deprivation of the right to health for the detainees as a result of their position in the asymmetric power relationships and ideological effects.

CARING FOR TUBERCULOSIS PATIENTS IN THE FAMILY HEALTH STRATEGY: THE NURSES' PERCEPTIONS

The objective of this study was to analyze the nurses' perceptions regarding tuberculosis control, according to the theoretical axis of comprehensive healthcare and the concepts of attachment and teamwork. This qualitative study involved 13 nurses from the Family Health Strategy of a priority city in the metropolitan region of Joao Pessoa, Paraiba, Brazil. Data were collected in focal groups and subjected to thematic content analysis. Factors that strengthen tuberculosis control were: supervised treatment, free medication and the provision of supplies. Weakening factors were: worker's turnover, the lack of encouragement for patients and incipient educational actions. The factors that, according to the nurses, weaken tuberculosis patient care should be reviewed by administrators, workers, users and educators with a view to redefining healthcare activities that strengthen attachment, comprehensive healthcare and teamwork.

Assessment of tuberculosis treatment accessibility for patients co-infected or not with the human immunodeficiency virus

This study aimed to evaluate accessibility to treatment for people with TB co-infected or not with HIV. This cross-sectional study addressed issues regarding accessibility to treatment in a city in the interior of Sao Paulo state, Brazil. The instrument Primary Care Assessment Tool was utilized with 95 people. To evaluate access to treatment, Student's t test was used. The mean scores of variables were analyzed separately and compared between two groups (people with TB co-infected with HIV and people with TB not co-infected with HIV). Mean scores showed that HIV co-infected people presented greater difficulties in gaining access than those not co-infected. Professionals visited co-infected people more often when compared to those not co-infected; the co-infected people almost never accessed treatment for their disease in the Health Unit nearest their home. There is, therefore, the need for greater integration and communication between the programs for treatment of Tuberculosis and STD/AIDS.

Epidemiological aspects of tuberculosis in children and adolescents in Rio de Janeiro

Objective: To describe the epidemiological aspects of childhood tuberculosis (TB) in a Brazilian reference hospital. Methods: This was a retrospective study (1999-2008) of 473 subjects (0-14 year olds) with confirmed TB, or with clinical improvement by the fourth month of treatment under the unit's care, including the review of medical records, monitoring reports and notifications by the TB unit. Results: Among 473 TB cases included in the study, positive tuberculin skin test was observed in 52%, history of contact with a patient with pulmonary tuberculosis in 66%, mostly intra-household, and with the father/stepfather most commonly involved; and disseminated TB in 22%. The result of HIV testing was obtained in 265 (56%) cases, being positive in 45 (17%). The diagnosis of TB was confirmed in 31% of cases, most frequently in children older than 5 years, with negative tuberculin skin test, and in disseminated forms. Of the 65 cultures positive for TB performed in the study, drug sensitivity testing to anti-TB drugs was done in 30 (46%) clinical samples, among which 10 (33%) were resistant to one or more anti-TB drugs, and 2 (0.8%) were multi-drug-resistant. Among patients with confirmed pulmonary TB, 31% did not meet the criteria for starting anti-TB treatment according to the scores of the Ministry of Health (<= 25 points). Conclusion: The high proportion of drug-resistant TB and co-infection with HIV identified in this study highlight the necessity to carry out additional studies in order to evaluate the impact of TB control activities on childhood TB.

Dropouts or Discontinuity of the Treatment of Tuberculosis in Rio Branco, Acre

This research investigates the factors related to the discontinuity of the treatment of tuberculosis in Rio Branco-Acre. To the fulfillment of this research, a contribution to ethnography has been adopted, for the successful apprehension of the reality to be studied. The research has been developed in two mapping steps: on the first, a search for information on the SINAN (System of National Injuries Notification - Sistema de Notificacao de Agravos Nacionais) and on the Municipal Coordination of the Tuberculosis Program databases was taken; the second aimed to record facts from the observation of the care given on a health care unit, which serves assistance to tuberculosis carriers in treatment, and the interviews of the elected subjects. On the analysis of the observations, narrative and interview collection, it was observed that the professionals of health services label some people as auspicious to discontinue the treatment and don't consider the different ways of life on the approach of their patients, complicating the formation of the bond and favoring the discontinuity. It was also identified people treating for tuberculosis that had many ways of dealing with the limitations generated by the disease, such as restrictions for the work, among others, and people that discontinued the treatment took in consideration their system of beliefs and values, as well as the perception of health/disease, due to the feeling of cure when the treatment was interrupted.

Pleural tuberculosis: is radiological evidence of pulmonary-associated disease related to the exacerbation of the inflammatory response?

OBJECTIVE: Pleural tuberculosis is the most frequently occurring form of extra pulmonary disease in adults. In up to 40% of cases, the lung parenchyma is concomitantly involved, which can have an epidemiological impact. This study aims to evaluate the pleural and systemic inflammatory response of patients with pleural or pleuropulmonary tuberculosis. METHODS: A prospective study of 39 patients with confirmed pleural tuberculosis. After thoracentesis, a high resolution chest tomography was performed to evaluate the pulmonary involvement. Of the 39 patients, 20 exhibited only pleural effusion, and high resolution chest tomography revealed active associated-pulmonary disease in 19 patients. The total protein, lactic dehydrogenase, adenosine deaminase, vascular endothelial growth factor, interleukin-8, tumor necrosis factor-alpha, and transforming growth factor-beta(1) levels were quantified in the patient serum and pleural fluid. RESULTS: All of the effusions were exudates with high levels of adenosine deaminase. The levels of vascular endothelial growth factor and transforming growth factor-beta(1) were increased in the blood and pleural fluid of all of the patients with pleural tuberculosis, with no differences between the two forms of tuberculosis. The tumor necrosis factor-alpha levels were significantly higher in the pleural fluid of the patients with the pleuropulmonary form of tuberculosis. The interleukin-8 levels were high in the pleural fluid of all of the patients, without any differences between the forms of tuberculosis. CONCLUSION: Tumor necrosis factor-alpha was the single cytokine that significantly increased in the pleural fluid of the patients with pulmonary involvement. However, an overlap in the results does not permit us to suggest that cytokine is a biological marker of concomitant parenchymal involvement. Although high resolution chest tomography can be useful in identifying these patients, the investigation of fast acid bacilli and cultures for M. tuberculosis in the sputum is recommended for all patients who are diagnosed with pleural tuberculosis.

Differentiating between tuberculosis-related and lymphoma-related lymphocytic pleural effusions by measuring clinical and laboratory variables: Is it possible?

Objective: To describe clinical and laboratory characteristics in patients with tuberculosis-related or lymphoma-related lymphocytic pleural effusions, in order to identify the variables that might contribute to differentiating between these diseases. Methods: This was a retrospective study involving 159 adult HIV-negative patients with tuberculosis-related or lymphoma-related lymphocytic effusions (130 and 29 patients, respectively), treated between October of 2008 and March of 2010 at the Pleural Diseases Outpatient Clinic of the University of Sao Paulo School of Medicine Hospital das Clinicas Heart Institute, in the city of Sao Paulo, Brazil. Results: Mean age and the mean duration of symptoms were lower in the tuberculosis group than in the lymphoma group. The levels of proteins, albumin, cholesterol, amylase, and adenosine deaminase (ADA) in pleural fluid, as well as the serum levels of proteins, albumin, and amylase, were higher in the tuberculosis group, whereas serum cholesterol and triglycerides were higher in the lymphoma group. Pleural fluid leukocyte and lymphocyte counts were higher in the tuberculosis group. Of the tuberculosis group patients, none showed malignant cells; however, 4 showed atypical lymphocytes. Among the lymphoma group patients, cytology for neoplastic cells was positive, suspicious, and negative in 51.8%, 24.1%, and 24.1%, respectively. Immunophenotyping of pleural fluid was conclusive in most of the lymphoma patients. Conclusions: Our results demonstrate clinical and laboratory similarities among the patients with tuberculosis or lymphoma. Although protein and ADA levels in pleural fluid tended to be higher in the tuberculosis group than in the lymphoma group, even these variables showed an overlap. However, none of the tuberculosis group patients had pleural fluid ADA levels below the 40-U/L cut-off point.

Equity in health: tuberculosis in the Bolivian immigrant community of Sao Paulo, Brazil

Objectives To analyse the profile of tuberculosis (TB) among Bolivian immigrants, investigate the impact that this population has on the trends of TB and assess equity in access to TB treatment, in the city of Sao Paulo, Brazil. Methods Descriptive study of the epidemiological profile of TB in four city districts with large Bolivian populations, comparing cases among Brazilians and Bolivians, during the 19982008 period was carried out. We used logistic regression to adjust the treatment outcome for potential confounders. Results We identified 2056 new TB cases: 65.7% in Brazilians, 32.1% in Bolivians and 2.2% among other nationalities. Although TB incidence remained stable (high) over the study period, the annual proportion of cases among Bolivians increased from 15.0% to 53.0%. In comparison with the Brazilians, the Bolivians were younger (median age, 24 vs. 40 years; P < 0.0001) and presented a lower unemployment rate (3.1%vs. 11.6%; P < 0.0001), a lower rate of HIV co-infection (1.5%vs. 28.5%; P < 0.001), a higher proportion of individuals receiving supervised treatment (81.5%vs. 62.0%; P < 0.0001) and a higher proportion of cures (71.6%vs. 63.2%; P < 0.0001). After having been adjusted for potential confounder, cure after treatment was not associated with nationality. Conclusions Bolivian immigrants influenced the incidence but not the trends of TB among Brazilians in the study area. We found no significant differences between Bolivians and Brazilians regarding healthcare access or treatment outcome. Guaranteed universal health care access for all, including undocumented individuals, contributes to health equity. Specific intervention strategies are warranted for immigrants with tuberculosis.

The coordination of care for tuberculosis control

This descriptive epidemiological study analyzed the coordination of tuberculosis (TB) patient care in primary healthcare services according to 23 patients, 16 professionals, and 17 administrators from Ribeirao Preto, Sao Paulo, using an instrument adapted to evaluate TB. According to the informants, the coordination of healthcare provided to patients under the treatment of the Tuberculosis Control Program team was considered satisfactory; however, when there is a need to refer the patient to other care units there are weak points in the coordination of healthcare, which include: interruption of communication flow; and patients' incipient participation in the care process, with a need to increase the sense of responsibility for patient care and encourage patients to become active agents in the process.