Prevalence and clinical predictors of pulmonary tuberculosis among isolated inpatients: a prospective study.

Guidelines help to prevent the transmission of Mycobacterium tuberculosis in healthcare settings, but may also result in the unnecessary isolation of many patients. We performed a prospective study to assess the prevalence and identify clinical predictors of culture-proven tuberculosis among inpatients isolated for suspected pulmonary tuberculosis (PTB) at our hospital. We also wished to validate a pre-existing clinical decision rule to improve our isolation policy. From August 2005 to January 2007, 134 patients isolated on admission to the ward for suspicion of PTB were prospectively enrolled. The admitting team made the decision to isolate patients on the basis of clinical and radiological findings, without the use of the clinical decision rule, and graded the overall suspicion of PTB. Twenty-six of the 134 isolated patients had PTB (prevalence: 19.4%), as well as one patient not isolated at admission. Univariate analysis revealed that PTB was significantly associated with young age, lack of human immunodeficiency virus (HIV) infection, weight loss, night sweats, fever, upper lobe disease and, especially, cavitary lesions on chest X-ray (adjusted OR 25.4, p <0.0001). Low suspicion of PTB by the admitting team and low clinical decision rule score had negative predictive values of 98.5% and 95.8% for PTB, respectively. Use of the clinical decision rule in addition to the team assessment would have led to the isolation of the patient with PTB not isolated on admission, and avoided 16 (14.8%) unnecessary isolations. In conclusion, the prevalence of PTB among isolated inpatients was high, and the use of a clinical decision rule in addition to clinical impression might improve isolation decisions.

Regulation of prohepcidin processing and activity by the subtilisin-like proprotein convertases Furin, PC5, PACE4 and PC7.

BACKGROUND AND AIMS: Hepcidin is an iron homoeostasis regulator peptide. Loss-of-function mutations cause juvenile haemochromatosis while its over-expression results in anaemia. However, the mechanism and function of preprohepcidin conversion to mature hepcidins (25, 22 and 20 amino acid C-terminal peptides) are not well known. After removal of the signal peptide, the first proteolytic cleavage occurs within the basic motif RRRRR(59)DT, suggesting the involvement of proprotein convertase (PC) family members in this process. METHODS AND RESULTS: Using cell transfection experiments, the processing of preprohepcidin in the human hepatocyte line Huh-7 was found to be inhibited by the Furin inhibitors serpin alpha1-antitrypsin (alpha1-PDX) and prosegment preproFurin (ppFurin). Site-directed mutagenesis analysis confirmed the RRRRR(59)DT preprohepcidin cleavage site. In parallel, the lack of preprohepcidin processing found in the PC activity-deficient cell line LoVo was restored by the expression of Furin, paired basic amino acid cleaving enzyme 4 (PACE4), PC5 or PC7. This finding is consistent with the in vitro digestions of a synthetic peptide mimicking the cleavage site of preprohepcidin. In addition, during mouse embryonic development the major expression of hepcidin found in the liver coincided with that of Furin. While hepcidin induces the degradation of the iron transporter ferroportin, its RRRRR(59) to SSSSS(59) mutant is not active. CONCLUSIONS: These results demonstrate the key role of the convertases Furin, PACE4, PC5 and/or PC7 in the generation and secretion of active hepcidin and suggest that the control of hepcidin processing as a potential therapeutic/diagnostic strategy in hepcidin-related disorders such as haemochromatosis, inflammatory diseases, anaemia and cancer.

Microbial contamination of BM products before and after processing: a report of incidence and immediate adverse events in 257 grafts.

BACKGROUND: The incidence and potential clinical consequences of bacterial contamination of autologous and allogeneic BM products remains open to question. We report our experience of bacterial contamination of BM grafts and adverse events that occurred after transplantation. METHODS: From January 2003 to February 2006, 257 BM harvests were processed and infused at our institution. Analysis of microbial contamination incidence before and after processing, sensitivity spectra of isolated bacteria and adverse events after graft infusion were analyzed. RESULTS: Nineteen of 257 BM (7.4%) were contaminated. Coagulase-negative Staphylococcus (n=9) and Propionibacterium acnes (n=6) were the most frequently isolated microorganisms. Two of nine coagulase-negative staphylococci were found to be resistant to erythromycin and two of six P. acnes to fosfomycin and gentamycin. The frequency and severity of immediate adverse events reported in patients receiving a contaminated graft were similar to those observed in patients receiving a non-contaminated product. No major adverse sequelae occurred after infusion of contaminated grafts. Finally, none of the patients transplanted with a contaminated graft developed bacteriemia that could have been related to the isolated microorganism. DISCUSSION: Microbial contamination of BM progenitor cell grafts does not induce severe clinical complications or infectious diseases after infusion. The vast majority of isolated pathogens were skin contaminants.

[Granulysin: antimicrobial molecule of innate and acquired immunity in human tuberculosis]. / Granulysine: médiateur de l'immunité innée et de l'immunité acquise dans la tuberculose chez l'homme.

The recent global increase in cases of tuberculosis and the emergence of multidrug-resistant strains of tuberculosis have focused attention on the molecular mechanisms of human antimycobacterial immunity. The macrophage is not only the primary site for Mycobacterium tuberculosis growth but also ordinarily provides the primary lines of host defense against invading pathogens in its role as an effector of innate immunity. The ability of M. tuberculosis to survive and replicate in the host macrophage is critical to its pathogenesis, emphasizing a need for a clearer understanding of its interactions with the host macrophage. Macrophages use varied strategies to kill and destroy invading organisms, including production of reactive nitrogen and oxygen intermediates, phagosome maturation and acidification, fusion with lysosomes, exposure to defensins and host cell apoptosis. In human, granulysin is a recently identified antimicrobial protein expressed on cytotoxic T cells, natural killer (NK) cells and NKT cells. It has been shown that granulysin contributes to the defense mechanisms against mycobacterial infection. We hypothesized that human macrophages may possess antimicrobial substances, such as granulysin, and play a role in the defense mechanism.

Evaluation of the automated phoenix system for potential routine use in the clinical microbiology laboratory.

A comparative study was designed to evaluate the identification (ID) and antimicrobial susceptibility testing (AST) performances of the BD Phoenix Automated Microbiology System (Becton Dickinson Diagnostic Systems [BD], Pont de Claix, France). A total of 305 single clinical isolates were collected, and comparisons were made with routine manual methods in use in our microbiology laboratories. The percentages of correct IDs were 93.3, 89.4, 91.8, and 85.7% for enterobacteria, nonfermenting gram-negative bacilli, staphylococci, and streptococci-enterococci, respectively. The median ID time was 3 h, and the median time for AST was 10 h 30 min. AST results showed variable percentages of errors for the different antibiotics. None of the enterobacteria and 0.3% of Pseudomonas aeruginosa isolates showed a very major error (VME). Only one strain of Staphylococcus aureus showed a VME with oxacillin. We demonstrate here the efficiency of the Phoenix system, which can be used for the majority of strains encountered in a university-based laboratory, for ID and AST.

Kinetics of circulating antibodies, immune complex and specific antibody-secreting cells in tuberculosis patients during 6 months of antimicrobial therapy.

OBJECTIVE: During the follow-up of a group of patients with active tuberculosis, the predictive potential of several antibody-based assays was evaluated in monitoring treatment efficacy. DESIGN: Eleven patients with bacteriologically documented pulmonary tuberculosis and two patients with tuberculosis pleurisy were studied over a period of 6 months, from the day before treatment to its completion. The kinetics of the humoral response to Mycobacterium tuberculosis was determined by the number of specific circulating antibody secreting cells (ASC) (ELISPOT assay), as well as the titres of specific circulating antibody and specific antibody present in circulating immune complexes (quantitative ELISA). RESULTS: Follow-up ELISPOT assays, performed after initiation of tuberculosis therapy showed a rapid increase of ASC, during the first week, followed by rapid 3-10 fold decline of ASC in 12 of 13 patients tested. This decline occurred more rapidly than the mycobacterial culture conversion. In contrast, follow-up of ELISA assays did not give relevant information in assessing the outcome of treatment. CONCLUSION: In comparison with direct detection of tubercle bacilli in sputum samples, the rapid clearance of specific circulating ASC occurring early on after the onset of therapy could suggest a potential usefulness of ELISPOT in monitoring therapeutic response.

Mycobacteriosis in the compromised host.

The studies of rare genetic defects, the preliminary results of population-based studies, being validated by the experimental immunocompromised animal models and the current observations accumulated in immunocompromised patients with mycobacterial diseases provide us with insights into the importance of the macrophage activation pathway in controlling human infection with pathogenic and non pathogenic intracellular multiplying mycobacteria. Initial cytokine production by infected macrophages and/or dendritic cells could be crucial in the overall regulation of self cure, acquired protection or immunopathological sequelae expressing the disease. Knowledge of molecular and genetic cross-talks between phagocytic and specialized antigen presenting cells and different mycobacterial products associated with persistence or replication of the intracellular bacteria, could provide further informations on the global immune regulation of the early host responses to infection and the following events. It seems likely that the development of mycobacterial infections in humans will turn out to be as much dependent on the genetic make up of the host as or the virulence of the bacteria.

Down-regulation of human granzyme B expression by glucocorticoids. Dexamethasone inhibits binding to the Ikaros and AP-1 regulatory elements of the granzyme B promoter.

The serine protease granzyme B is an essential component of the granule exocytosis pathway, a major apoptotic mechanism used by cytotoxic T lymphocytes and natural killer cells to induce target cell apoptosis. Granzyme B gene transcription is induced in activated lymphocytes upon antigenic stimulation, and several regulatory regions including CBF, AP-1, and Ikaros binding sites have been shown to be essential in the control of granzyme B promoter activation. Dexamethasone, a glucocorticoid that is widely used as an immunomodulatory and anti-inflammatory agent, inhibits granzyme B mRNA transcript in phytohemagglutinin-activated peripheral blood mononuclear cells. Transfection of a reporter construct containing the -148 to +60 region of the human granzyme B promoter demonstrated that this region was the target for dexamethasone repression. Mutation of Ikaros or AP-1 binding sites in the context of the granzyme B promoter demonstrated that both sites participate in dexamethasone-mediated inhibition of the granzyme B promoter activity. Electromobility shift assay revealed that dexamethasone abolished the binding of nuclear transcription factors to the Ikaros binding site and reduced AP-1 binding activity. These results indicate that dexamethasone is able to abrogate the transcriptional activity of the human granzyme B gene promoter by inhibiting the binding of nuclear factors at the AP-1 and Ikaros sites.

Identification of human granzyme B promoter regulatory elements interacting with activated T-cell-specific proteins: implication of Ikaros and CBF binding sites in promoter activation.

Granzyme B serine protease is found in the granules of activated cytotoxic T cells and in natural and lymphokine-activated killer cells. This protease plays a critical role in the rapid induction of target cell DNA fragmentation. The DNA regulatory elements that are responsible for the specificity of granzyme B gene transcription in activated T-cells reside between nt -148 and +60 (relative to the transcription start point at +1) of the human granzyme B gene promoter. This region contains binding sites for the transcription factors Ikaros, CBF, Ets, and AP-1. Mutational analysis of the human granzyme B promoter reveals that the Ikaros binding site (-143 to -114) and the AP-1/CBF binding site (-103 to -77) are essential for the activation of transcription in phytohemagglutinin-activated peripheral blood lymphocytes, whereas mutation of the Ets binding site does not affect promoter activity in these cells.

Cord blood T lymphocytes lack constitutive perforin expression in contrast to adult peripheral blood T lymphocytes.

Perforin is the cytolytic pore-forming protein, which alone can be responsible for the lethal hit in one of the killing mechanisms used by natural killer (NK) cells or cytotoxic T lymphocytes. In this study, perforin expression was investigated in cord blood (CB) lymphocytes to determine their killing potential in vivo. The majority of CB CD3- NK cells had the protein. Compared with adult perforin-positive NK cells, a significantly lower percentage of cells expressing CD56 and CD57, the related neural cell adhesion molecules, was found (P = .0001). Perforin was also present in a unique immature CB NK-cell subset, characterized by cytoplasmic CD3 antigen (Ag) expression. In CB, very few CD8 perforin-positive T lymphocytes could be detected, but they were in significant numbers in adult peripheral blood (P = .02). A substantial proportion of these cells (70% +/- 23%) lacked the CD28 T-cell coactivation Ag, and they were able to exert NK-like, major histocompatibility complex nonrestricted cytolytic activity. CD4+ and gamma delta-T cells expressing perforin were absent from CB, but low numbers of such cells were detected in adult peripheral blood (P = .0001). Therefore, the spontaneous cytolytic activity of CB lymphocytes appeared to be dependent on well-represented perforin-positive NK cells, which were shown to efficiently lyse NK-sensitive target cells.

Immunological aspects of pulmonary tuberculosis in children.

In recent years new information has been collected about the immunological responses to pathogenic mycobacteria. More information on cellular and molecular responses of cells in murine and human tuberculosis has been produced and T cells' role in the production of selected cytokines has been clarified. Studies in mice have provided insight into the phases of the T cell response to virulent M. tuberculosis, the role of various T cell subsets, and the repertoire of antigens recognized by these cells. However, despite this new information, some of which has been confirmed in humans, large gaps remain in our knowledge about the immune response to this infection, particularly concerning cellular or molecular mechanisms involved in acquired protection. Even if some extrapolations from adult data can be made, large gaps in our knowledge exist on the potential immune defects in young infants who are prone to develop tuberculosis soon after infection.

Granzyme B and perforin can be used as predictive markers of acute rejection in heart transplantation.

We have investigated perforin and granzyme B expression in graft-infiltrating lymphocytes of patients who underwent heart transplantation. Those proteins are commonly present in the cytoplasmic granules of cytotoxic T lymphocytes and are released upon effector-target cell interaction. From 28 patients 103 endomyocardial biopsies were obtained and examined by histology and immunocytochemical analysis using relevant monoclonal antibodies. We found that "high" biopsy histological grades were associated with perforin and granzyme B expression in graft-infiltrating lymphocytes of patients with acute severe rejection crisis. In contrast, these markers were not detected in patients without rejection or during graft stabilization. Interestingly, in patients with mild rejection and "low" histological grades, two groups could be distinguished with a differential expression of the two intracytoplasmic proteins. The presence of perforin and granzyme B-expressing cells was found to be predictive of rapid progression to severe rejection, so that this situation required additional treatment; in contrast, their absence seemed to correlate with a good graft outcome without additional treatment. Moreover, perforin and granzyme B expression seemed to be down-regulated by immunosuppressive drugs, which coincided with graft stabilization. In conclusion, our data suggest that detection of granzyme B and perforin in graft-infiltrating lymphocytes might be helpful for routinely monitoring heart transplant patients.

[Bactericidal activity of cells of the immune system]. / Pouvoir bactéricide des cellules du système immunitaire.

The acquisition of a bactericidal activity by the cellular immune system is one of the mechanisms of prime importance for the development of the cellular resistance to infection. It involves the specific recognition of the pathogenic microorganism associated with the direct or indirect activation of effector cells. The in vivo type of replication of the infectious microorganism provides the adaptive response of the host. This response differs according to extracellular or intracellular multiplying microorganism. The acquired resistance to extracellular multiplying bacteria (ie, capsulated bacteria) is linked to the B cell production of specific antibodies that are associated with the activated complement fragments, that enable bacterial opsonization, activation and recruiting of polymorphonuclear phagocytes. These cells did acquired a greater capacity to ingest and to kill bacteria at sites of infection. Such phenomenon could be also associated with acute purulent necrotizing lesions. On the other hand, intracellular multiplying bacteria are mostly associated with chronic granulomatous non pyogenic lesions, and such bacteria are also able to survive and to replicate into the non activated professional mononuclear phagocytes. The acquired resistance against such bacteria is linked to the occurrence of a non specific bactericidal capacity of macrophages, that inhibit and/or kill replicative intracellular microorganisms. Such macrophage bactericidal capacity is dependent upon the gradual and stepwise cellular activation provided by several extracellular and membrane associated signals. Sequential signals are produced by the specific recognition of committed T CD4+ lymphocytes mediated by the infected macrophages through the MHC class 2 restriction of the peptide presentation. One of the major signal of cellular communication to induce macrophage priming involves the production of Interferon gamma (IFN gamma). Secondary signals are needed to induced full macrophage activation defined by phenotypic, metabolic and functional alterations which include their acquired increase capacity to kill intracellular bacteria and tumor cells. Secondary signals involve the production of cytokines such as TNF alpha, IL2 and GM-CSF being transduced by bacterial cell wall products (such as lipopolysaccharide, teichoic acid, or lipoarabinomanan) or by the calcitriol pathway recently described during mycobacterial infection.(ABSTRACT TRUNCATED AT 250 WORDS)

A promoter element of the human serine esterase granzyme B gene controls specific transcription in activated T cells.

The human granzyme B gene encodes a serine protease expressed specifically in cytoplasmic granules of cytotoxic T lymphocytes, released upon effector-target cell interaction. Previous studies have shown that granzyme B mRNA was induced in T lymphocytes after antigenic or mitogenic stimulation. To study the regulation of human granzyme B gene expression during lymphocyte activation we analyzed its 5' flanking region using chloramphenicol acetyl transferase (CAT) reporter gene constructs. We show that a 208-bp fragment (-148 to +60) containing an NF-AT (nuclear factor of activated T cells)-binding site promotes CAT expression in phytohemagglutinin-activated T lymphocytes, in immobilized monoclonal anti-CD3 antibody-activated Jurkat T cell line while it is inactive in unstimulated PEER and Jurkat T cells lines or B Epstein-Barr virus-transformed cell lines.

[Value of agglutination tests for the rapid identification of isolated streptococci in medical microbiology]. / Intérêt des tests d'agglutination pour l'identification rapide des streptocoques isolés en microbiologie médicale.

The clinical need for rapidly and correctly differentiating pathogenic streptococci into Lancefield groups prompted the development of rapid agglutination techniques by the direct colony method. Pastorex Streptogroupe (Diagnostics Pasteur, France) and Streptex (Wellcome Diagnostics, USA) were tested on 90 streptococcal isolates and compared with the identification obtained by conventional procedures. No cross-reactions were observed with any of the 33 strains of enterococci and of the seven strains representative of other genus among Gram-positive bacteria. When combined with colonial morphology and hemolytic reaction, both Pastorex and Streptex were specific tests for identifying beta-hemolytic streptococci, with Pastorex being slightly faster.