Decreased IFN- gamma and increased IL-4 production by human CD8(+) T cells in response to Mycobacterium tuberculosis in tuberculosis patients.

To investigate the role of MHC class I restricted CD8(+) T cells in host defense to M. tuberculosis, peripheral blood mononuclear cells (PBMC) from healthy BCG-vaccinated donors and untreated pulmonary tuberculosis (TB) patients in The Gambia were stimulated for 6 days with M. bovis BCG or M. tuberculosis and the CD8(+) T cell response analyzed. Intracellular FACS analysis of cytokine production by CD8(+) T cells showed that IFN- gamma and TNF- alpha production were greatly reduced in TB patients compared to healthy controls. IL-4-producing CD8(+) T cells were detected in TB patients, a phenotype absent in controls. Collectively, these data suggest that an alteration in the type 1/type 2 cytokine balance occurs in CD8(+) T cells during clinical tuberculosis, and that this may provide a surrogate marker for disease.

Priming by DNA immunization augments protective efficacy of Mycobacterium bovis Bacille Calmette-Guerin against tuberculosis.

Sequential immunization with mycobacterial antigen Ag85B-expressing DNA and Mycobacterium bovis bacille Calmette-Guerin (BCG) was more effective than BCG immunization in protecting against Mycobacterium tuberculosis infection. Depletion of the CD8(+) T cells in the immunized mice impaired protection in their spleens, indicating that this improved efficacy was partially mediated by CD8(+) T cells.

Human CD8+ CTL specific for the mycobacterial major secreted antigen 85A.

The role of CD8(+) CTL in protection against tuberculosis in human disease is unclear. In this study, we stimulated the peripheral blood mononuclear cells of bacillus Calmette-Guérin (BCG)-vaccinated individuals with live Mycobacterium bovis BCG bacilli to establish short-term cell lines and then purified the CD8(+) T cells. A highly sensitive enzyme-linked immunospot (ELISPOT) assay for single cell IFN-gamma release was used to screen CD8(+) T cells with overlapping peptides spanning the mycobacterial major secreted protein, Ag85A. Three peptides consistently induced a high frequency of IFN-gamma responsive CD8(+) T cells, and two HLA-A*0201 binding motifs, P(48-56) and P(242-250), were revealed within the core sequences. CD8(+) T cells responding to the 9-mer epitopes were visualized within fresh blood by ELISPOT using free peptide or by binding of HLA-A*0201 tetrameric complexes. The class I-restricted CD8(+) T cells were potent CTL effector cells that efficiently lysed an HLA-A2-matched monocyte cell line pulsed with peptide as well as autologous macrophages infected with Mycobacterium tuberculosis or recombinant vaccinia virus expressing the whole Ag85A protein. Tetramer assays revealed a 6-fold higher frequency of peptide-specific T cells than IFN-gamma ELISPOT assays, indicating functional heterogeneity within the CD8(+) T cell population. These results demonstrate a previously unrecognized, MHC class I-restricted, CD8(+) CTL response to a major secreted Ag of mycobacteria and supports the use of Ag85A as a candidate vaccine against tuberculosis.

Human CD8(+) T cells specific for Mycobacterium tuberculosis secreted antigens in tuberculosis patients and healthy BCG-vaccinated controls in The Gambia.

Intracellular flow cytometry analysis of perforin production by CD8(+) T cells showed levels were greatly reduced in tuberculosis (TB) patients compared to healthy controls. Reduced cytotoxic-T-lymphocyte activity was also obtained with CD8(+) T cells from TB patients compared to healthy controls in The Gambia. A change in antigen recognition was noted between the two groups of donors: in addition to recognition of Ag85A and Ag85B, as seen in healthy donors, a prominent ESAT-6 response was found in TB patients.

Measurements of body image in clinical weight loss participants with and without binge-eating traits.

This study measured body image disturbances of individuals in a residential weight loss program who were identified as having binge-eating disorder (BED) traits. The study population (N=97) was a convenience sample of 74 men (76%, mean age=51.0) and 23 women (24%, mean age=49.6) in the program who completed the Eating Questionnaire-Revised (EQ-R), the Attitudes About Weight and Dieting (AAWD), the Physical Appearance State and Trait Anxiety Scale (PASTAS): State Version, and the Contour Drawing Rating Scale (CDR). Fifty-five individuals reported having binge traits (57%) while 42 (43%) had no binge traits. Individuals with the binge traits had a significantly higher BMI than nonbinge trait individuals (P=.008). The binge trait group scored higher on the total AAWD (P=.004), the AAWD factor "Fear of Fat" (P=.002), the total PASTA (P=.001), and the PASTA factor "Weight" (P=.001) than the nonbinge trait group. Logistic regression analysis indicated that the odds of having a binge trait were 1.44 times more likely for a person at a given score on the PASTA subscale Weight vs. a person at a score of 5 units less. Feelings of being unable to control eating among individuals seeking weight control is associated with several characteristics related to body image. Individuals showing greater concern about weight and dieting and specifically greater fears of becoming fat were more likely to have a problem with binge eating than those without these concerns. The results of this study suggest that a negative body image is an important factor to consider when treating individuals indicating binge-eating traits.

Vaccinia expression of Mycobacterium tuberculosis-secreted proteins: tissue plasminogen activator signal sequence enhances expression and immunogenicity of M. tuberculosis Ag85.

There is increasing evidence to implicate a role for CD8(+) T cells in protective immunity against tuberculosis. Recombinant vaccinia (rVV) expressing Mycobacterium tuberculosis (MTB) proteins can be used both as tools to dissect CD8(+) T-cell responses and, in attenuated form, as candidate vaccines capable of inducing a balanced CD4(+)/CD8(+) T-cell response. A panel of rVV was constructed to express four immunodominant secreted proteins of MTB: 85A, 85B and 85C and ESAT-6. A parallel group of rVV was constructed to include the heterologous eukaryotic tissue plasminogen activator (tPA) signal sequence to assess if this would enhance expression and immunogenicity. Clear expression was obtained for 85A, 85B and ESAT-6 and the addition of tPA resulted in N-glycosylation and a 4-10-fold increase in expression. Female C57BL/6 mice were immunised using the rVV-Ag85 constructs, and interleukin-2 and gamma-interferon were assayed using a co-culture of immune splenocytes and recall antigen. There was a marked increase in cytokine production in mice immunised with the tPA-containing constructs. We report the first data demonstrating enhanced immunogenicity of rVV using a tPA signal sequence, which has significant implications for future vaccine design.

Characterization of human Mycobacterium bovis bacille Calmette-Guérin-reactive CD8+ T cells.

Gamma interferon (IFN-gamma)-secreting CD4+ T cells have long been established as an essential component of the protective immune response against Mycobacterium tuberculosis. It is now becoming evident from studies with the murine model of tuberculosis that an important role also exists for major histocompatibility complex (MHC) class I-restricted CD8+ T cells. These cells are capable of acting as both IFN-gamma secretors and cytotoxic T lymphocyte (CTL) effectors; however, their exact role in immunity against tuberculosis remains unclear. This study demonstrates the presence of Mycobacterium bovis BCG-reactive CD8+ T cells in healthy BCG-vaccinated donors and that these CD8+ T cells are potent cytokine producers as well as cytotoxic effector cells. Using FACScan analysis, we have shown that restimulation with live M. bovis BCG induced more CD8+-T-cell activation than the soluble antigen purified protein derivative and that these cells are actively producing the type 1 cytokines IFN-gamma and tumor necrosis factor alpha (TNF-alpha). These CD8+ T cells also contain the cytolytic granule perforin and are capable of acting as potent CTLs against M. bovis BCG-infected macrophages. The mycobacterial antigens 85A and B (Ag85A and Ag85B, respectively), and to a lesser extent the 19- and 38-kDa proteins, are major antigenic targets for these mycobacterium-specific CD8+ T cells, while whole-M. bovis BCG activated effector cells from these BCG-vaccinated donors, as expected, failed to recognize the 6-kDa ESAT-6 protein. The use of metabolic inhibitors and blocking antibodies revealed that the CD8+ T cells recognize antigen processed and presented via the classical MHC class I pathway. These data suggest that CD8+ T cells may play a critical role in the human immune response to tuberculosis infection.

B-cell epitopes and quantification of the ESAT-6 protein of Mycobacterium tuberculosis.

ESAT-6 is an important T-cell antigen recognized by protective T cells in animal models of infection with Mycobacterium tuberculosis. In an enzyme-linked immunosorbent assay (ELISA) with overlapping peptides spanning the sequence of ESAT-6, monoclonal antibody HYB76-8 reacted with two peptides in the N-terminal region of the molecule. Assays with synthetic truncated peptides allowed a precise mapping of the epitope to the residues EQQWNFAGIEAAA at positions 3 to 15. Hydrophilicity plots revealed one hydrophilic area at the N terminus and two additional areas further along the polypeptide chain. Antipeptide antibodies were generated by immunization with synthetic 8-mer peptides corresponding to these two regions coupled to keyhole limpet hemocyanin. Prolonged immunization with a 23-mer peptide (positions 40 to 62) resulted in the formation of antibodies reacting with the peptide as well as native ESAT-6. A double-antibody ELISA was then developed with monoclonal antibody HYB76-8 as a capture antibody, antigen for testing in the second layer, and antipeptide antibody in the third layer. The assay was suitable for quantification of ESAT-6 in M. tuberculosis antigen preparations, showing no reactivity with M. bovis BCG Tokyo culture fluid, used as a negative control, or with MPT64 or antigen 85B, previously shown to cross-react with HYB76-8. This capture ELISA permitted the identification of ESAT-6 expression from vaccinia virus constructs containing the esat-6 gene; this expression could not be identified by standard immunoblotting.

Human cytolytic and interferon gamma-secreting CD8+ T lymphocytes specific for Mycobacterium tuberculosis.

Protective immunity to Mycobacterium tuberculosis is poorly understood, but mounting evidence, at least in animal models, implicates major histocompatibility complex class I-restricted CD8+ T cells as an essential component. By using a highly sensitive assay for single cell interferon gamma release, we screened an array of M. tuberculosis antigen-derived peptides congruent with HLA class I allele-specific motifs. We identified CD8+ T cells specific for epitopes in the early secretory antigenic target 6 during active tuberculosis, after clinical recovery and in healthy contacts. Unrestimulated cells exhibited peptide-specific interferon gamma secretion, whereas lines or clones recognized endogenously processed antigen and showed cytolytic activity. These results provide direct evidence for the involvement of CD8+ cytotoxic T lymphocytes in host defense against M. tuberculosis in humans and support current attempts to generate protective cytotoxic T lymphocyte responses against M. tuberculosis by vaccination.

Pneumocystis carinii pneumonia in Zimbabwe.

Pneumocystis carinii pneumonia (PCP) is said to be rare in Africa, with reported rates of 0-22% in human-immunodeficiency-virus (HIV) infected individuals with respiratory symptoms. Over one year in a central hospital in southern Africa, 64 HIV-infected patients with acute diffuse pneumonia unresponsive to penicillin and sputum smear-negative for acid-fast bacilli underwent fibreoptic bronchoscopy. Bronchoalveolar lavage fluid was assessed for bacteria, fungi, Pneumocystis carinii, and mycobacteria. 21 patients (33%) had PCP and 24 (39%) had tuberculosis; 6 of these had both infections. 5 patients had Kaposi's sarcoma (KS) associated with PCP, tuberculosis, or another infection, in 1 patient KS was the only finding, and in 21 no pathogen was identified. A logistic regression model was used to assess clinical, radiographic, and arterial blood gas predictors of PCP and tuberculosis. Fine reticulonodular shadowing on the chest radiograph (nodular component < 1 mm) was the strongest independent predictor of PCP (odds ratio 8.5 [95% CI 6.1-10.9]). A respiratory rate of more than 40/min was the best clinical predictor of PCP (odds ratio 11.2 [95% CI 8.8-13.6]). Median CD4+ T cell count for all cases of PCP was 134/microL (range 5-355) and for tuberculosis without PCP 206/microL (range 61-787). In resource-limited countries, a regionally appropriate management algorithm is required.

PIP: The authors enrolled 64 patients in a large central hospital in Harare, Zimbabwe, over a 12-month period from May 1992 in their study of the prevalence of Pneumocystis carinii pneumonia (PCP) among HIV-infected individuals with acute diffuse pneumonia unresponsive to penicillin and sputum smear-negative for acid-fast bacilli. Subjects underwent fiberoptic bronchoscopy, while bronchoalveolar lavage fluid was assessed for bacteria, fungi, Pneumocystis carinii, and mycobacteria. 21 patients had PCP and 24 had tuberculosis (TB); 6 of these had both infections. 5 patients had Kaposi's sarcoma (KS) associated with PCP, TB, or another infection. KS was the only finding in 1 patient, and no pathogen was identified in 21 patients. Fine reticulonodular shadowing on the chest radiograph and a respiratory rate of more than 40 per minute were the strongest independent predictor of PCP and the best clinical predictor of PCP, respectively. Median CD4+ T cell count for all cases of PCP was 134/mcl (range, 5-355) and for TB without PCP 206/mcl (range, 61-787).

Escalating threat from tuberculosis: the third epidemic.

PIP: A 1994 study reported on cases of drug-resistant tuberculosis (TB) at the Chest Service at Bellevue Hospital, in New York City. 20 years of TB laboratory susceptibility tests were reviewed in 4681 cases. Combined resistance to isoniazid and rifampicin rose from 2.5% in 1971 to 16% in 1991. Over 75% of these cases in 1991 were resistant to rifampicin, isoniazid, streptomycin, and ethambutol. Most of the patients belonged to one or more of the following groups: young, Black or Hispanic, unemployed, homeless, male, HIV-infected, and drug abuser. Clinical characteristics were: anergy, fever, cough, night sweats, weight loss, radiograph bilateral infiltrates, adenopathy, cavities, miliary shadowing, and normal chest radiograph. Overall, in 1993 in the US, 3% of all new cases and 6.9% of recurrent cases were resistant to both rifampicin and isoniazid. This resurgence of TB in industrialized countries has been ascribed to: 1) immigration of foreign populations at high risk of developing TB, 2) coinfection with HIV, and 3) an increase in high risk groups. The WHO stresses the importance of identifying meaningful denominators when discussing both primary and acquired drug resistance rates. Restriction fragment length polymorphism (RFLP) is the molecular technique that differentiates individual strains of Mycobacterium tuberculosis. Three recent studies from New York used RFLP to demonstrate the clustering of multidrug-resistant TB. Solutions to the problem consist of adequate infrastructure, i.e., a national TB program; prescription of combined preparations; inducement or enforcement of compliance using directly observed therapy (DOT) (a DOT protocol employed in Denver, Colorado, used 2 weeks of therapy followed by 24 weeks of twice weekly intermittent therapy); prevention of nosocomial spread by isolation of smear-positive cases during the first 2 weeks of treatment; rapid diagnosis of TB and drug susceptibility (within 10-21 days using radiometric culture, nucleic acid probes, and high performance liquid chromatography of mycolic acids); and treatment by five or six drugs.^ieng

Genetic diversity in human-derived Pneumocystis carinii isolates from four geographical locations shown by analysis of mitochondrial rRNA gene sequences.

The opportunistic fungal pathogen Pneumocystis carinii is a frequent cause of pneumonia in immunocompromised hosts. In this study, we have compared the DNA sequences of a portion of the mitochondrial large-subunit rRNA gene of P. carinii (an informative locus showing up to 27% differences among isolates of P. carinii from human-, rat-, mouse-, ferret-, rabbit-, and horse-infected lungs) obtained from human-derived isolates from widely disparate geographical areas, including Britain, the United States, Brazil, and Zimbabwe. A single-base polymorphism which varied among samples was identified. Apart from this nucleotide, the DNA sequences of all samples were identical. The sequences of the British samples were shown to be stable over a period of 4 years. These data suggest that there is relatively low genetic diversity among isolates of human-derived P. carinii from different global regions.

Salmonella and shigella bacteraemia in Zimbabwe.

In patients with HIV infection, non-typhoidal salmonellae are a recognised cause of bacteraemia. This association was initially demonstrated in the United States, but has more recently been found in Kenyan patients. This prompted us to review the cases of patients with enterobacteriaceae bacteraemia admitted to Parirenyatwa Hospital, Harare. Non-typhoidal salmonella bacteraemia as compared with typhoid fever was significantly more common in HIV infected patients than in non-HIV infected patients (p < 0.01). It was also a cause of bacteraemia in patients with other immuno-suppressive conditions and in some patients without identifiable risk factors.

PIP: The case notes of patients with blood cultures positive for enterobacteriaceae were examined retrospectively over a 6-month period in Parirenyatwa Hospital, Harare, Zimbabwe. Speciation was possible for Salmonella typhi and shigellae only. Nontyphoidal salmonellae were serotyped. Salmonella or shigella bacteremia was identified in 51 patients. There were 14 isolates of S. typhi, 32 isolates of nontyphoidal salmonellae, and 5 isolates of shigellae species. The case notes of 38 patients could be identified for review, and of these HIV serology was available for 15 seropositive and 15 seronegative patients. The male to female ratio was approximately 3:1 for both groups and the mean age was 29.7 +or- 21. Nontyphoidal bacteremias as compared with typhoid fever were strongly associated with HIV seropositivity [p 0.01]. 3 out of 8 HIV-negative patients with nontyphoidal bacteremia had another underlying immunosuppressive disease [2 had myeloma and 1 patient had cirrhosis with complicating hepatoma]. 2 patients with nontyphoidal bacteremia whose HIV status was unknown also had another immunosuppressing disease [acute myeloid leukemia and idiopathic pancytopenia]. 13 out of 15 HIV-positive patients showed other signs of HIV infection [oral candida, herpes zoster, persistent generalized lymphadenopathy]. 3 out of 11 patients [27%] with typhoid died, while 11 out of 27 patients [40.7%] with nontyphi bacteremia died. Most strains of S. typhimurium were included in serogroup B, which accounted for 37% of nontyphoidal isolates. Earlier studies identified invasive salmonellosis in patients with other AIDS defining diseases. In Nairobi clinical features of HIV infection were found in 64% of bacteremic HIV-positive patients, but only 28% of patients fulfilled the CDC clinical case definition for AIDS. A more recent study from Nairobi demonstrated that S. typhimurium bacteremia is a common cause of intercurrent infection in HIV-positive tuberculous patients.