Results of a tuberculosis-specific IFN-gamma assay in children at high risk for tuberculosis infection.

The specificity of the tuberculin skin test (TST) for the diagnosis of latent tuberculosis infection (LTBI) is adversely affected by bacille Calmette-Guérin (BCG) vaccination and infection with non-tuberculous mycobacteria. Interferon-gamma release assays (IGRAs) using TB-specific antigens promise higher specificity. We compared a new IGRA and TST in 184 schoolchildren at high risk for LTBI. The IGRA and TST were positive in 33.2% and 43.5% of the children, respectively (P < 0.001). If studies confirm that this difference is due to higher specificity of this IGRA, it may have an important role to play in the diagnosis of LTBI and identification of children at true risk for TB.     

Divergent effect of bacillus Calmette-Guérin (BCG) vaccination on Mycobacterium tuberculosis infection in highly related macaque species: implications for primate models in tuberculosis vaccine research

Despite the widespread use of bacillus Calmette-Guérin vaccination, Mycobacterium tuberculosis infection remains globally the leading cause of death from a single infectious disease. The complicated and often protracted dynamics of infection and disease make clinical trials to test new tuberculosis vaccines extremely complex. Preclinical selection of only the most promising candidates is therefore essential. Because macaque monkeys develop a disease very similar to humans, they have potential to provide important information in addition to small animal models. To assess the relative merits of rhesus and cynomolgus monkeys as screens for tuberculosis vaccines, we compared the efficacy of bacillus Calmette-Guérin vaccination and the course of infection in both species. Unvaccinated rhesus and cynomolgus monkeys both developed progressive disease with high levels of C-reactive protein, M. tuberculosis-specific IgG, and extensive pathology including cavitation and caseous necrosis. Bacillus Calmette-Guérin vaccination protected cynomolgus almost completely toward the development of pathology, reflected in a striking 2-log reduction in viable bacteria in the lungs compared with nonvaccinated animals. Rhesus, on the other hand, were not protected efficiently by the bacillus Calmette-Guérin. The vaccinated animals developed substantial pathology and had negligible reductions of colony-forming units in the lungs. Comparative studies in these closely related species are likely to provide insight into mechanisms involved in protection against tuberculosis.     

Partial interferon-gamma receptor signaling chain deficiency in a patient with bacille Calmette-Guérin and Mycobacterium abscessus infection

Complete deficiency of either of the two human interferon (IFN)-gamma receptor components, the ligand-binding IFN-gammaR1 chain and the signaling IFN-gammaR2 chain, is invariably associated with early-onset infection caused by bacille Calmette-Guérin vaccines and/or environmental nontuberculous mycobacteria, poor granuloma formation, and a fatal outcome in childhood. Partial IFN-gammaR1 deficiency is associated with a milder histopathologic and clinical phenotype. Cells from a 20-year-old healthy person with a history of curable infections due to bacille Calmette-Guérin and Mycobacterium abscessus and mature granulomas in childhood were investigated. There was a homozygous nucleotide substitution in IFNGR2, causing an amino acid substitution in the extracellular region of the encoded receptor. Cell surface IFN-gammaR2 were detected by flow cytometry. Cellular responses to IFN-gamma were impaired but not abolished. Transfection with the wild-type IFNGR2 gene restored full responsiveness to IFN-gamma. This is the first demonstration of partial IFN-gammaR2 deficiency in humans.     

Intravesical administration of bacillus Calmette-Guérin in patients with recurrent superficial carcinoma of the urinary bladder: report of a prospective, randomized trial

In an attempt to prevent, delay, or reduce further tumor occurrence, 88 patients with recurrent, superficial carcinoma of the urinary bladder were randomly assigned to receive either standard therapy (cystoscopy with fulguration) or standard therapy and bacillus Calmette-Guérin (BCG). BCG was administered intravesically and percutaneously once weekly for 6 weeks. No serious toxicity was seen. There were 43 evaluable patients in each of the two groups. Results in the BCG group versus the control group were as follows: reduction in the number of recurrent tumors (43 vs 27 patients [P less than 0.001] ); conversion to negative cytology (11 of 33 vs three of 34 patients [P less than 0.05] ); and tumor progression requiring cystectomy (three vs 15 patients [P less than 0.001] ). Disease-free interval (P less than 0.001), time with negative cytology (P less than 0.001), and time to progression of disease (P less than 0.003) were longer in patients treated with BCG. These results indicate that the combination of standard therapy and BCG is more effective than standard therapy alone in patients with recurrent superficial bladder tumors.     

Genetic control of resistance to experimental infection with virulent Mycobacterium tuberculosis

Over 2 billion people are estimated to be infected with virulent Mycobacterium tuberculosis, yet fewer than 10% progress to clinical tuberculosis within their lifetime. Twin studies and variations in the outcome of tuberculosis infection after exposure to similar environmental risks suggest genetic heterogeneity among individuals in their susceptibility to disease. In a mouse model of tuberculosis, we have established that resistance and susceptibility to virulent M. tuberculosis is a complex genetic trait. A new locus with a major effect on tuberculosis susceptibility, designated sst1 (susceptibility to tuberculosis 1), was mapped to a 9-centimorgan (cM) interval on mouse chromosome 1. It is located 10-19 cM distal to a previously identified gene, Nramp1, that controls the innate resistance of mice to the attenuated bacillus Calmette-Guérin vaccine strain. The phenotypic expression of the newly identified locus is distinct from that of Nramp1 in that sst1 controls progression of tuberculosis infection in a lung-specific manner. Mice segregating at the sst1 locus exhibit marked differences in the growth rates of virulent tubercle bacilli in the lungs. Lung lesions in congenic sst1-susceptible mice are characterized by extensive necrosis and unrestricted extracellular multiplication of virulent mycobacteria, whereas sst1-resistant mice develop interstitial granulomas and effectively control multiplication of the bacilli. The resistant allele of sst1, although powerful in controlling infection, is not sufficient to confer full protection against virulent M. tuberculosis, indicating that other genes located outside of the sst1 locus are likely also to be important for controlling tuberculosis infection.     

Bacillus Calmette-Guérin-induced granulomatous hepatitis in a patient with a superficial bladder carcinoma

A 71-year-old male patient with a superficial transitional cell carcinoma of the urinary bladder developed high fever and jaundice, accompanied by progressively increasing serum aminotransferase activities, 2 weeks after the fourth local instillation with an attenuated live strain of Mycobacterium bovis [bacillus Calmette-Guérin (BCG)]. A liver biopsy showed non-caseating granulomatous hepatitis. Cultures for mycobacteria were negative. Mycobacterial DNA was not detected in liver tissue using the polymerase chain reaction. Empirical treatment with rifampicin and isoniazid was started, resulting in partial recovery. After 6 months of therapy, however, serum aminotransferase activities were still twice the upper limit of normal. A second liver biopsy still demonstrated several granulomas. Only after addition of prednisolone, liver tests completely normalized. Also histologically the lesions improved dramatically. This suggests that the BCG hepatitis was at least partially caused by a hypersensitivity reaction. Our patient is the first reported case of BCG hepatitis with histological follow-up under therapy.     

Specific detection of tuberculosis infection: an interferon-gamma-based assay using new antigens

The tuberculin skin test for immunologic diagnosis of Mycobacterium tuberculosis infection has many limitations, including being confounded by bacillus Calmette-Guérin (BCG) vaccination or exposure to nontuberculous mycobacteria. M. tuberculosis-specific antigens that are absent from BCG and most nontuberculous mycobacteria have been identified. We examined the use of two of these antigens, CFP-10 and ESAT-6, in a whole blood IFN-gamma assay as a diagnostic test for tuberculosis in BCG-vaccinated individuals. Because of the lack of an accurate standard with which to compare new tests for M. tuberculosis infection, specificity of the whole blood IFN-gamma assay was estimated on the basis of data from people with no identified risk for M. tuberculosis exposure (216 BCG-vaccinated Japanese adults) and sensitivity was estimated on the basis of data from 118 patients with culture-confirmed M. tuberculosis infection who had received less than 1 week of treatment. Using a combination of CFP-10 and ESAT-6 responses, the specificity of the test for the low-risk group was 98.1% and the sensitivity for patients with M. tuberculosis infection was 89.0%. The results demonstrate that the whole blood IFN-gamma assay using CFP-10 and ESAT-6 was highly specific and sensitive for M. tuberculosis infection and was unaffected by BCG vaccination status.     

Partial purification of a serum factor that causes necrosis of tumors.

Tumor necrosis can be induced in transplanted mouse methylcholanthrene-induced sarcoma by a tumor necrosis factor in the serum of mice infected with bacillus Calmette-Guérin and given bacterial endotoxin. Sera from normal mice, endotoxin-treated mice, and mice infected with bacillus Calmette-Guérin do not contain this factor. A 20- to 30-fold purification of the serum factor has been achieved by (NH4)2SO4 fractionation, Sephadex G-100 and G-200 gel filtration, and preparative polyacrylamide electrophoresis. Tumor necrosis factor is not bacterial endotoxin. It migrates with alpha-globulins, is made up of at least four subunits, and has a molecular weight of about 150,000. The active factor is a glycoprotein that contains sialic acid and galactosamine.     

Bone Involvement in Primary Hemochromatosis and Alcoholic Cirrhosis

Biochemical indexes of bone metabolism, bone mineral density, and histomorphometry were evaluated in 14 male patients with noncholestatic cirrhosis due to primary hemochromatosis (six cases) or to chronic alcohol abuse (eight cases), and in 30 male controls of similar age. Alkaline phosphatase in alcoholic patients was significantly higher than in controls (mean +/- SD 50.4 +/- 33.7 vs 33.0 +/- 7.1 U/L, p less than 0.01), as was urinary hydroxyproline in both hemochromatotics and alcoholics (mean +/- SD, 44.3 +/- 8.4 and 40.4 +/- 16.8, respectively, vs 30.1 +/- 4.5 mg/g, p less than 0.001 and p less than 0.005). Bone mineral density was significantly lower in hemochromatotics than in alcoholics and controls (mean +/- SD, 591 +/- 90 vs 765 +/- 87 and 759 +/- 34 mg/cm2, respectively, p less than 0.005 and p less than 0.001). At bone biopsy, trabecular osteoporosis was observed in two hemochromatotics and four alcoholics, and osteomalacia was seen in another alcoholic. Overall densitometric and histomorphometric findings indicate a derangement of trabecular bone in both alcoholic and hemochromatotic cirrhosis, whereas cortical osteoporosis seems limited to hemochromatotic patients.     

Clinical reactogenicity of intradermal bacille Calmette-Guérin vaccination.

Clinical, microbiological, and immunologic responses were evaluated in volunteers vaccinated intradermally with bacille Calmette-Guérin (BCG). Most volunteers (98%) developed ulcerative lesions that drained for a mean +/- SE of 4.3 +/- 0.29 weeks. Mycobacterial DNA was detected by a polymerase chain reaction-based amplification technique in biopsy specimens from BCG ulcers 2 weeks after vaccination and in blood specimens 3 days after vaccination. Mycobacteria were cultured from ulcer drainage 2 months after vaccination, demonstrating a prolonged potential risk of contact spread of the vaccine strain. The duration of ulcer drainage was inversely correlated with prevaccination lymphoproliferative (r = -0.515; P < .002) and interferon gamma (r = -0.841; P < .002) responses specific to mycobacteria and directly correlated with postvaccination increases in lymphoproliferative (r = 0.498; P < .002) and interferon gamma (r = 0.688; P < .02) responses specific to mycobacteria. These results demonstrate the clinical reactogenicity of BCG and the potential risk of contact spread of the vaccine strain and suggest that clinical reactogenicity is a trade-off for the induction of protective mycobacterial immunity.     

Development of tuberculin reactivity and sensitization to M. scrofulaceum and M. fortuitum in children BCG-vaccinated at birth.

Since the incidence of tuberculosis is steadily declining in Finland and infections by environmental mycobacteria may be increasing, the aim of the present study was to evaluate the development of tuberculin reactivity and sensitization to environmental mycobacteria. Healthy Finnish schoolchildren aged 10.4-12.4 yrs (n=201) were tested with tuberculin purified protein derivative RT23, Mycobacterium scrofulaceum RS95 and M. fortuitum RS20 sensitins. The same children had been previously tested with the same antigens and methods at the age of 4-6 yrs in 1989. Rapid waning of tuberculin reactivity and decrease in sensitization to environmental mycobacteria were observed between 4-6 yrs. Both tuberculin and sensitin skin reaction sizes decreased significantly over the 6-yrs period. The mean tuberculin skin reaction size was 3.2 mm in diameter, which was significantly (p<0.001) smaller than the mean induration size (4.8 mm) at the age of 4-6 yrs. Similarly, the mean skin reaction sizes to M. scrofulaceum and M. fortuitum sensitins were 3.4 and 1.7 mm, respectively, which were significantly (p<0.001) smaller than 6 yrs earlier (mean 4.5 and 3.1 mm). The number of zero reactions to all antigens increased significantly during the follow-up period. Contacts with pets or farm animals were associated with larger reactions. In contrast, children suffering from allergic symptoms had smaller reactions. Contacts with mycobacteria, either with Mycobacterium tuberculosis or environmental mycobacteria, seem to be too rare to maintain tuberculin responsiveness and a high sensitivity to other mycobacteria. Different bacille Calmette-Guérin vaccine products and dosages used, the declining incidence of tuberculosis and geographical factors, which can influence environmental mycobacterial exposure, may explain the disparity between the present and previous Finnish studies.     

Dendritic cell involvement in pulmonary granuloma formation elicited by bacillus calmette-guérin in rats

Dendritic cells (DCs) are the most potent antigen-presenting cells that play a central role in initiating the primary immune response. However, their role in granulomatous inflammation has not been well studied. The aim of the present study was to elucidate the role of DCs in granuloma formation. Using a rat model of bacillus Calmette-Guérin (BCG)-elicited pulmonary granulomas, we investigated the distribution of DCs in the granulomas by immunohistochemistry with a rat-DC-specific monoclonal antibody, OX62. We found numerous large, pleiomorphic OX62(+) cells accumulating at the borders of the pulmonary granulomas. The OX62(+) cells isolated from the granulomatous lung showed intense surface expression of major histocompatibility complex class II, B7-1, and B7-2, and a lack of T cell- and monocyte/macrophage-specific markers. Their ultrastructural morphology was characteristic of DCs. Functionally, they had potent capacity to stimulate allogeneic T cells as well as purified protein derivative-specific syngeneic T cells in the absence of exogenous peptides. Based on these findings, the OX62(+) cells infiltrating the granulomas were considered to be DCs expressing BCG-derived peptides. These results indicate that DCs contribute to pulmonary granuloma formation elicited by BCG by means of their potent antigen-presenting function, providing a novel insight into DC function in T cell-mediated granulomatous immune responses.     

QuantiFERON®-TB Gold In-Tube test can be used for screening latent tuberculosis before biological treatment in a Bacille Calmette-Guérin (BCG)-vaccinated country: the HUR-BIO single-center real-life results

Clinical Rheumatology - The Turkish population is vaccinated with Bacille Calmette-Guérin (BCG), and the BCG vaccination decreases the specificity of the tuberculin skin test (TST). The...     

Prevalence and mechanisms of hyperhomocysteinemia in chronic alcoholics

BACKGROUND: Homocysteine (Hcy) is formed as an intermediary in methionine metabolism. Impairment of Hcy remethylation or transulfuration leads to hyperhomocysteinemia, which is considered as a risk factor for atherosclerotic vascular disease and stroke in chronic alcoholics. The aim of the study was to investigate the prevalence of hyperhomocysteinemia in chronic alcoholics and the influence of alcohol consumption, vitamin deficiencies and liver damage on the plasma levels of Hcy. METHODS: 228 chronic alcoholic patients consecutively admitted for detoxication, classified according to clinical and biochemical data in normal liver (n = 117), and in mild to moderate liver disease (n = 111), and 49 healthy controls were studied. Blood levels of Hcy, vitamin B6, vitamin B12 and folate were measured. RESULTS: Plasma Hcy was significantly higher in chronic alcoholics than in controls (9.66 +/- 8.1 vs. 6.93 +/- 2.33 mumol/liter, p < 0.025). Furthermore, plasma Hcy levels were significantly higher in chronic alcoholics with liver injury (12.17 +/- 10.14 mumol/liter) than in those with normal liver and in controls (p < 0.001). The prevalence of hyperhomocysteinemia was also significantly higher in alcoholics with liver damage than in those with normal liver and in controls (29.7%, 5.1%, and 2%, respectively, p < 0.001). Serum folate values were lower in chronic alcoholics than in controls (4.7 +/- 2.6 vs. 7.6 +/- 2.4 nmol/liter, p < 0.001). The lowest values of folate were found in alcoholics with liver disease, especially in those with hyperhomocysteinemia, with a negative correlation between the two parameters. CONCLUSIONS: Moderate hyperhomocysteinemia is common in chronic alcoholics, mainly in those with liver damage, suggesting that, although folate deficiencies may have a contributory role, liver impairment, through changes in methionine metabolism, is the most important mechanism for the elevated plasma Hcy found in these patients.     

Avoiding the effect of BCG vaccination in detecting Mycobacterium tuberculosis infection with a blood test.

Bacille Calmette-Guérin (BCG) vaccination can confound tuberculin skin test (TST) reactions in the diagnosis of latent tuberculosis infection (LTBI). The TST was compared with a Mycobacterium tuberculosis (MTB)-specific enzyme-linked immunospot (ELISPOT) assay during an outbreak of MTB infection at a police academy in Germany. Participants were grouped according to their risk of LTBI in close (n = 36) or occasional (n = 333) contacts to the index case. For the TST, the positive response rate was 53% (19 out of 36) among close and 16% (52 out of 333) among occasional contacts. In total, 56 TST-positive contacts (56 out of 71 = 78.9%) and 27 TST-negative controls (27 out of 298 = 9.1%) underwent ELISPOT testing. The odds ratio (OR) of a positive test result across the two groups was 29.2 (95% confidence interval (CI) 3.5-245.0) for the ELISPOT and 19.7 (95% CI 2.0-190.2) for the TST with a 5 mm cut-off. Of 369 contacts, 158 (42.8%) had previously received BCG vaccination. The overall agreement between the TST and the ELISPOT was low, and positive TST reactions were confounded by BCG vaccination (OR 4.8 (95% CI 1.3-18.0)). In contrast, use of a 10-mm induration cut-off for the TST among occasional contacts showed strong agreement between TST and ELISPOT in nonvaccinated persons. In bacille Calmette-Guérin-vaccinated individuals, the Mycobacterium tuberculosis-specific enzyme-linked immunospot assay is a better indicator for the risk of latent tuberculosis infection than the tuberculin skin test.     

Body composition changes induced by chronic ethanol abuse: evaluation by dual energy X-ray absorptiometry

OBJECTIVE: Nutritional disorders in alcoholics remain one of the most relevant medical problems in Western societies. As ethanol can supply >50% of the dietary energy in alcoholics, body composition alterations may easily occur. The aim of the present study was to evaluate the influence of chronic alcohol consumption on body composition in alcoholics compared to healthy social drinkers. METHODS: A total of 34 alcoholics defined according to DSM III R criteria, aged 41.6 +/- 9.3 yr and with a body mass index (BMI) 23.8 +/- 3.2 kg/m2, were consecutively enrolled in the study. In addition, 43 healthy male social drinkers were used as controls. Body composition was assessed using dual energy x-ray absorptiometry (DXA), and dietary habits were determined by a 3-day food diary. RESULTS: Mean daily alcohol intake was 194 +/- 62.4 g/day in alcoholics and 35.7 +/- 5.2 in healthy subjects (p < 0.0001). Body weight did not differ between alcoholics and controls (70.1 +/- 9.9 vs 71.8 +/- 6.4 kg). Alcoholics had a lower percent body fat (PBF) than control subjects (18.7 +/- 3.7 vs 23.9 +/- 3.9%; p < 0.01), as well as a lower fat mass content (13.4 +/- 3.8 vs 17.0 +/- 3.7 kg; p < 0.01). BMI was highly correlated with PBF in the patient population studied (R = 0.79; p < 0.0001). Significantly higher waist-to-hip ratios were found in alcoholics than in healthy subjects (p < 0.01). No correlation was found between dose of ethanol or duration of alcohol abuse and any of the variables examined. CONCLUSIONS: Alcoholics showed a reduced fat mass and a good preservation of lean body mass with respect to control subjects, and duration of alcohol use and alcohol dose did not seem to influence body composition. These data suggest that, unlike control subjects, alcoholics cannot store the calories provided by ethanol as fat deposits.     

Detection of active tuberculosis infection by T cell responses to early-secreted antigenic target 6-kDa protein and culture filtrate protein 10

The purified protein derivative (PPD) skin test has no predictive value for tuberculosis (TB) in Mycobacterium bovis bacillus Calmette-Guérin (BCG)-vaccinated individuals because of cross-reactive responses to nonspecific constituents of PPD. T cell responses to early-secreted antigenic target 6-kDa protein (ESAT-6) and the newly identified culture filtrate protein 10 (CFP-10), 2 proteins specifically expressed by M. tuberculosis (MTB) but not by BCG strains, were evaluated. Most TB patients responded to ESAT-6 (92%) or CFP-10 (89%). A minority of BCG-vaccinated individuals responded to both ESAT-6 and CFP-10, their history being consistent with latent infection with MTB in the presence of protective immunity. No responses were found in PPD-negative controls. The sensitivity and specificity of the assay were 84% and 100%, respectively, at a cutoff of 300 pg of interferon-gamma/mL. These data indicate that ESAT-6 and CFP-10 are promising antigens for highly specific immunodiagnosis of TB, even in BCG-vaccinated individuals.     

Combined-modality treatment of inoperable lung cancer (i.v. immunotherapy, chemotherapy, and radiotherapy)

Patients with inoperable non-small cell carcinoma of the bronchus were treated by iv methanol extraction residue of bacillus Calmette-Guérin (MER) followed by combination chemotherapy with methotrexate, doxorubicin, cyclophosphamide, and lomustine (MACC). Radiotherapy was added in patients with localized disease. MER was given iv in four weekly courses. The dose ranged between 0.1 and 0.8 mg/m2/course and was determined according to the skin reactivity to MER. None of the 19 patients treated with iv MER had an objective tumor response during immunotherapy. Five of 16 evaluable patients receiving MACC (31%) achieved partial response. The overall median survival for all 17 patients treated was 11 months according to the protocol. During immunotherapy the following side effects were observed: all patients had fever and chills and most had malaise, nausea and/or vomiting, and headache. Transient abnormalities of liver function tests were found in six patients, and in one patient roentgenographic changes suggestive of lung granulomas were seen. The side effects were more severe during the first course than in the further treatments. Side effects of the MACC combination were comparable to those observed in previously reported studies. Immunological monitoring performed during immunotherapy revealed that iv MER had an unfavorable effect on the immune system. Following four courses there was a decrease in the in vivo skin reactivity to MER and to five recall antigens. Twenty-four hours after the initiation of iv MER there was a tendency for decrease in the lymphoproliferation to phytohemagglutinin, an increase in the indices of the adherent cell suppressor system and the prostaglandin-related suppressor system, and an increase in the percentage of adherent mononuclear cells. No consistent changes in serum lysozyme were found. A transient increase in the total number of peripheral blood leukocytes and a decrease in the number of lymphocytes were noticed 24 hours after iv MER. Taking into account the results of the immunological studies and the overall therapeutic results, we do not feel justified in continuing to use this combined modality treatment.     

The nonredundant roles of two 4'-phosphopantetheinyl transferases in vital processes of Mycobacteria

Mycobacterium tuberculosis contains >20 enzymes that require activation by transfer of the 4'-phosphopantetheine moiety of CoA onto a conserved serine residue, a posttranslational modification catalyzed by 4'-phosphopantetheinyl transferases (PPTases). The modified proteins are involved in key metabolic processes such as cell envelope biogenesis and the production of virulence factors. We show that two PPTases conserved in all Mycobacterium spp. and in related genera activate two different subsets of proteins and are not functionally redundant. One enzyme, AcpS, activates the two fatty acid synthase systems of mycobacteria, whereas the other PPTase, PptT, acts on type-I polyketide synthases and nonribosomal peptide synthases, both of which are involved in the biosynthesis of virulence factors. We demonstrate that both PPTases are essential for Mycobacterium smegmatis viability and that PptT is required for the survival of Mycobacterium bovis bacillus Calmette-Guérin. These enzymes are thus central to the biology of mycobacteria and for mycobacterial pathogenesis and represent promising targets for new antituberculosis drugs.