Urogenital tuberculosis in a patient with AIDS: an unusual presentation

Background A 38-year-old man with AIDS presented to hospital with a 3-month history of fevers, bilateral lumbar pain, dysuria and increased urinary frequency. Six years earlier he had received 6 months` treatment for pulmonary tuberculosis. At presentation, he was on antiretroviral therapy with a combination of efavirenz, stavudine and lamivudine. Investigations Physical examination, evaluation of HIV viral load, CD4 count, measurement of serum hemoglobin concentration, white blood cell count, urinalysis, urine culture for usual pathogens, direct smear and urine culture for Mycobacterium tuberculosis, chest radiography, abdominal CT, measurement of serum creatinine concentration and estimated creatinine clearance. Diagnosis Urogenital tuberculosis. Management The patient`s symptoms and radiological abnormalities persisted despite antibiotic therapy for presumed bacterial infection. After urine culture had confirmed M. tuberculosis infection, he was administered pharmacological treatment comprising isoniazid, rifampin, pyrazinamide and ethambutol for 2 months, with isoniazid and rifampin given for a further 7 months. His symptoms improved within a few days of initiating treatment. Six months after treatment started, CT revealed a nonfunctioning right kidney and a functional left kidney with areas of scarring. The patient refused right nephrectomy, and completed his pharmacological treatment. No evidence of disease recurrence was observed during 2 years of follow-up.

Enhancement of diagnostic efficiency by a gamma interferon release assay for pulmonary tuberculosis

This study was designed to examine the use of the QuantiFERON-TB Gold assay as an aid in the diagnosis of active pulmonary tuberculosis (TB) in Brazilian patients. Using the receiver operating characteristic curve, the cutoff was adjusted to >= 0.20 IU/ml. The sensitivity increased to 86%, with 100% specificity. All TB patients with negative sputum smear microscopy and negative culture results were positive using this test.

Using BCG, MPT-51 and Ag85 as antigens in an indirect ELISA for the diagnosis of bovine tuberculosis

This study evaluated the Mycobacterium tuberculosis protein antigen MPT-51, the trimeric antigen 85 (Ag85) complex, and Bacillus Calmette-Guerin (BCG) in an indirect ELISA to diagnose bovine tuberculosis (TB) from serum samples. Serum was collected from 208 intra-dermal tuberculin test (ITT)-positive and 54 ITT-negative animals from a region where bovine TB is endemic. Using the Ag85 and BCG antigens, the indirect ELISA was able to discriminate ITT-positive from ITT-negative animals. This level of discrimination was not achieved when using the MPT-51 antigen. The highest sensitivity (Se) and specificity (Sp) of the test was found when BCG was used (Se, 82%; Sp, 91%). Further work in different epidemiological settings and with larger numbers of animals will be required to validate these findings. (C) 2009 Elsevier Ltd. All rights reserved.

A subunit vaccine based on biodegradable microspheres carrying rHsp65 protein and KLK protects BALB/c mice against tuberculosis infection

Of the hundreds of new tuberculosis ( TB) vaccine candidates some have therapeutic value in addition to their prophylactic properties. This is the case for the DNA vaccine encoding heat-shock protein 65 (DNAhsp65) from Mycobacterium leprae. However, there are concerns about the use of DNA vaccines in certain populations such as newborns and pregnant women. Thus, the optimization of vaccination strategies that circumvent this limitation is a priority. This study evaluated the efficacy of a single dose subunit vaccine based on recombinant Hsp65 protein against infection with M. tuberculosis H37Rv. The Hsp65 protein in this study was either associated or not with immunostimulants, and was encapsulated in biodegradable PLGA microspheres. Our results demonstrate that the protein was entrapped in microspheres of adequate diameter to be engulfed by phagocytes. Mice vaccinated with a single dose of Hsp65-microspheres or Hsp65 + CpG-microspheres developed both humoral and cellular-specific immune responses. However, they did not protect mice against challenge with M. tuberculosis. By contrast, Hsp65+KLK-microspheres induced specific immune responses that reduced bacilli loads and minimized lung parenchyma damage. These data suggest that a subunit vaccine based on recombinant protein Hsp65 is feasible.

Experimental tuberculosis: Designing a better model to test vaccines against tuberculosis

Experimental models of infection are good tools for establishing immunological parameters that have an effect on the host-pathogen relationship and also for designing new vaccines and immune therapies. In this work, we evaluated the evolution of experimental tuberculosis in mice infected with increasing bacterial doses or via distinct routes. We showed that mice infected with low bacterial doses by the intratracheal route were able to develop a progressive infection that was proportional to the inoculum size. In the initial phase of disease, mice developed a specific Th1-driven immune response independent of inoculum concentration. However, in the late phase, mice infected with higher concentrations exhibited a mixed Th1/Th2 response, while mice infected with lower concentrations sustained the Th1 pattern. Significant IL-10 concentrations and a more preeminent T regulatory cell recruitment were also detected at 70 days post-infection with high bacterial doses. These results suggest that mice infected with higher concentrations of bacilli developed an immune response similar to the pattern described for human tuberculosis wherein patients with progressive tuberculosis exhibit a down modulation of IFN-gamma production accompanied by increased levels of IL-4. Thus, these data indicate that the experimental model is important in evaluating the protective efficacy of new vaccines and therapies against tuberculosis. (C) 2010 Elsevier Ltd. All rights reserved.

Neonatal BCG Immunization Followed by DNAhsp65 Boosters: Highly Immunogenic but not Protective Against Tuberculosis - a Paradoxical Effect of the Vector?

A new tuberculosis vaccine is urgently needed. Prime-boost strategies are considered very promising and the inclusion of BCG is highly desirable. In this investigation, we tested the protective efficacy of BCG delivered in the neonatal period followed by boosters in the adult phase with a DNA vaccine containing the hsp65 gene from Mycobacterium leprae (pVAXhsp65). Immune responses were characterized by serum anti-hsp65 antibody levels and IFN-gamma and IL-5 production by the spleen. Amounts of these cytokines were also determined in lung homogenates. Protective efficacy was established by the number of colony-forming units (CFU) and histopathological analysis of the lungs after challenge with Mycobacterium tuberculosis. Immunization with BCG alone triggered a significant reduction of CFU in the lungs and also clearly preserved the pulmonary parenchyma. BCG priming also increased the immunogenicity of pVAXhsp65. However, boosters with pVAXhsp65 or the empty vector abolished the protective efficacy of BCG. Also, higher IL-5 levels were produced by spleen and lungs after DNA boosters. These results demonstrated that neonatal BCG immunization followed by DNAhsp65 boosters is highly immunogenic but is not protective against tuberculosis.

Mycobacterium tuberculosis Culture Filtrate Proteins plus CpG Oligodeoxynucleotides Confer Protection to Mycobacterium bovis BCG-Primed Mice by Inhibiting Interleukin-4 Secretion

Culture filtrate proteins (CFP) are potential targets for tuberculosis vaccine development. We previously showed that despite the high level of gamma interferon (IFN-gamma) production elicited by homologous immunization with CFP plus CpG oligodeoxynucleotides (CFP/CpG), we did not observe protection when these mice were challenged with Mycobacterium tuberculosis. In order to use the IFN-gamma-inducing ability of CFP antigens, in this study we evaluated a prime-boost heterologous immunization based on CFP/CpG to boost Mycobacterium bovis BCG vaccination in order to find an immunization schedule that could induce protection. Heterologous BCG-CFP/CpG immunization provided significant protection against experimental tuberculosis, and this protection was sustained during the late phase of infection and was even better than that conferred by a single BCG immunization. The protection was associated with high levels of antigen-specific IFN-gamma and interleukin-17 (IL-17) and low IL-4 production. The deleterious role of IL-4 was confirmed when IL-4 knockout mice vaccinated with CFP/CpG showed consistent protection similar to that elicited by BCG-CFP/CpG heterologous immunization. These findings show that a single dose of CFP/CpG can represent a new strategy to boost the protection conferred by BCG vaccination. Moreover, different immunological parameters, such as IFN-gamma and IL-17 and tightly regulated IL-4 secretion, seem to contribute to the efficacy of this tuberculosis vaccine.

In vitro uptake and antimycobacterial activity of liposomal usnic acid formulation

The cellular uptake and antimycobacterial activity of usnic acid (UA) and usnic acid-loaded liposomes (UA-LIPOs) were assessed on J774 macrophages. The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of UA and UA-LIPO against Mycobacterium tuberculosis were determined. Concentrations required to inhibit 50% of cell proliferation (IC(50)) were 22.5 (+/- 0.60) and 12.5 (+/- 0.26) mu g/ml, for UA and UA-LIPO, respectively. The MICs of UA and UA-LIPO were 6.5 and 5.8 mu g/mL, respectively. The MBC of UA-LIPO was twice as low (16 mu g/mL) as that of UA (32 mu g/mL). An improvement in the intracellular uptake of UA-LIPO was found (21.6 x 10(4) +/- 28.3 x 10(2) c.p.s), in comparison with UA (9.5 x 10(4) +/- 11.4 x 10(2) c.p.s). In addition, UA-LIPO remains much longer inside macrophages (30 hours). All data obtained from the encapsulation of usnic acid into liposomes as a drug delivery system (DDS) indicate a strong interaction between UA-liposomes and J774 macrophages, thereby facilitating UA penetration into cells. Considering such a process as ruling the Mycobacterium-transfection by magrophages, we could state that associating UA with this DDS leads to an improvement in its antimycobacterial activity.

Host genetic background affects regulatory T-cell activity that influences the magnitude of cellular immune response against Mycobacterium tuberculosis

Using two mouse strains with different abilities to generate interferon (IFN)-gamma production after Mycobacterium tuberculosis infection, we tested the hypothesis that the frequency and activity of regulatory T (Treg) cells are influenced by genetic background. Our results demonstrated that the suppressive activity of spleen Treg cells from infected or uninfected BALB/c mice was enhanced, inhibiting IFN-gamma and interleukin (IL)-2 production. Infected C57BL/6 mice exhibited a decrease in the frequency of lung Treg cells and an increased ratio CD4(+):CD4(+)Foxp3(+) cells compared with infected BALB/c mice and uninfected C57BL/6 mice. Moreover, infected C57BL/6 mice also had a decrease in the immunosuppressive capacity of spleen Treg cells, higher lung IFN-gamma and IL-17 production, and restricted the infection better than BALB/c mice. Adoptive transfer of BALB/c Treg cells into BALB/c mice induced an increase in bacterial colony-forming unit (CFU) counts. Furthermore, BALB/c mice treated with anti-CD25 antibody exhibited lung CFU counts significantly lower than mice treated with irrelevant antibody. Our results show that in BALB/c mice, the Treg cells have a stronger influence than that in C57BL/6 mice. These data suggest that BALB/c and C57BL/6 mice may use some different mechanisms to control M. tuberculosis infection. Therefore, the role of Treg cells should be explored during the development of immune modulators, both from the perspective of the pathogen and the host. Immunology and Cell Biology (2011) 89, 526-534; doi:10.1038/icb.2010.116; published online 19 October 2010

Investigation of the in vivo activity of CYP3A in Brazilian volunteers: comparison of midazolam and omeprazole as drug markers

Objective This study compares midazolam with omeprazole as marker drugs for the evaluation of CYP3A activity in nine healthy self-reported white Brazilian volunteers. Methods Omeprazole was also used to evaluate the CYP2C19 phenotype. The volunteers received p.o. 20 mg omeprazole, and blood samples were collected 3.5 h after drug administration. After a washout period of 10 days, the volunteers received p.o. 15 mg midazolam maleate, and serial blood samples were collected up to 6 h after administration of the drug. CYP2C19 was genotyped for the allelic variants CYP2C19*1, CYP2C19*2, CYP2C19*3, and CYP2C19*17. Analysis of omeprazole, hydroxyomeprazole, omeprazole sulfone, and midazolam in plasma was carried out by LC-MS/MS. Results The volunteers genotyped as CYP2C19*1*17, CYP2C19*17*17, CYP2C19*1*1 (n=8), or CYP2C19*17*2 (n=1) presented a median hydroxylation index (omeprazole/hydroxyomeprazole) of 1.35, indicating that all of them were extensive metabolizers of CYP2C19. The volunteers (n=9) presented a 0.12 log of the omeprazole/sulfone ratio and a median oral clearance of midazolam of 17.89 ml min(-1) kg(-1), suggesting normal CYP3A activity. Conclusions Orthogonal regression analysis between midazolam clearance and log of the plasma concentrations of the omeprazole/omeprazole sulfone ratio (R=-0.7544, P < 0.05) suggests that both midazolam and omeprazole can be used as markers of CYP3A activity in the population investigated.

T allele of-344C/T polymorphism in aldosterone synthase gene is not associated with resistant hypertension

Resistant hypertension (RH) is the maintenance of elevated blood pressure concurrent with the use of three different antihypertensive drugs, one of which is a diuretic. The Renin-Angiotensin-Aldosterone System plays a major role in volume-dependent hypertension. Therefore, its components are interesting targets for genetic association studies. This work focused on the -344 C/T polymorphism in the CYP11b2 gene, which encodes aldosterone synthase. This work evaluates the association between T allele and resistance to anti-hypertensive treatment. Genotyping analysis included 88 subjects with RH, 142 who were responsive to anti-hypertensive treatment and 110 subjects as a control group. Plasmatic concentrations of aldosterone, renin and cortisol, carotid intima-media thickness and carotid-femoral pulse wave velocity were assessed in a smaller subset of hypertensive patients. An association was found between T allele and hypertension (P < 0.005), but there was no difference in allele frequencies between both hypertensive groups. There was no difference in plasmatic parameters either, in remodeling indicators between the genotypic groups.

The influence of glutathione modulators on the course of Leishmania major infection in susceptible and resistant mice

Glutathione (GSH) has an important dual role in parasite-host relationship in Leishmania major infection. Our previous studies showed that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, participate in the protection of Leishmania against the toxic effect of nitrogen-derived reactive species. On the other hand, GSH also is very important to the modulation of the effective immune response, inducting NO production and leishmanicidal activity of macrophages. In the present study, we investigated the role of host GSH during the course of L. major infection, analysing the size of footpad lesions and parasite load from mice treated with two GSH modulators, N-acethyl-L-cysteine (NAC) and buthionine sulphoximine (BSO). Resistant mice treated with BSO, which depletes GSH develop exacerbated lesions, but only harbour higher parasite load in their lesions 2 weeks post-infection. Although the NAC treatment does not affect the footpad lesions development in susceptible BALB/c mice, it significantly reduced the tissue parasitism in the lesions throughout the course of infection. Interestingly, the treatment with BSO did not change the course of L. major infection on susceptible mice when compared with nontreated mice. These results suggest that GSH is an important antioxidant modulator during anti-Leishmania immune response in vivo.