PE_PGRS30 is required for the full virulence of Mycobacterium tuberculosis.

The role and function of PE_PGRS proteins of Mycobacterium tuberculosis (Mtb) remains elusive. In this study for the first time, Mtb isogenic mutants missing selected PE_PGRSs were used to investigate their role in the pathogenesis of tuberculosis (TB). We demonstrate that the MtbΔPE_PGRS30 mutant was impaired in its ability to colonize lung tissue and to cause tissue damage, specifically during the chronic steps of infection. Inactivation of PE_PGRS30 resulted in an attenuated phenotype in murine and human macrophages due to the inability of the Mtb mutant to inhibit phagosome-lysosome fusion. Using a series of functional deletion mutants of PE_PGRS30 to complement MtbΔPE_PGRS30, we show that the unique C-terminal domain of the protein is not required for the full virulence. Interestingly, when Mycobacterium smegmatis recombinant strain expressing PE_PGRS30 was used to infect macrophages or mice in vivo, we observed enhanced cytotoxicity and cell death, and this effect was dependent upon the PGRS domain of the protein.Taken together these results indicate that PE_PGRS30 is necessary for the full virulence of Mtb and sufficient to induce cell death in host cells by the otherwise non-pathogenic species M. smegmatis, clearly demonstrating that PE_PGRS30 is an Mtb virulence factor.

Surface expression of MPT64 as a fusion with the PE domain of PE_PGRS33 enhances Mycobacterium bovis BCG protective activity against Mycobacterium tuberculosis in mice.

To improve the current vaccine against tuberculosis, a recombinant strain of Mycobacterium bovis bacillus Calmette-Guérin (rBCG) expressing a Mycobacterium tuberculosis vaccine candidate antigen (MPT64) in strong association with the mycobacterial cell wall was developed. To deliver the candidate antigen on the surface, we fused the mpt64 gene to the sequence encoding the PE domain of the PE_PGRS33 protein of M. tuberculosis (to create strain (H)PE-ΔMPT64-BCG), which we have previously shown to transport proteins to the bacterial surface. In a series of protection experiments in the mouse model of tuberculosis, we showed that (i) immunization of mice with (H)PE-ΔMPT64-BCG provides levels of protection significantly higher than those afforded by the parental BCG strain, as assessed by bacterial colonization in lungs and spleens and by lung involvement (at both 28 and 70 days postchallenge), (ii) rBCG strains expressing MPT64 provide better protection than the parental BCG strain only when this antigen is surface expressed, and (iii) the (H)PE-ΔMPT64-BCG-induced MPT64-specific T cell repertoire when characterized by ß chain variable region-ß chain joining region (BV-BJ) spectratyping indicates that protection is correlated with the ability to recruit gamma interferon (IFN-γ)-secreting T cells carrying the BV8.3-BJ1.5 (172 bp) shared rearrangement. These results demonstrate that (H)PE-ΔMPT64-BCG is one of the most effective new vaccines tested so far in the mouse model of tuberculosis and underscore the impact of antigen cellular localization on the induction of the specific immune response induced by rBCG.

Molecular characterization of Sardinian Mycobacterium tuberculosis isolates by IS6110 restriction fragment length polymorphism, MIRU-VNTR and rep-PCR.

An evaluation of the utility of rep PCR typing compared to the 15 loci discriminatory set of MIRU-VNTR was undertaken. Twenty-nine isolates of Mycobacterium tuberculosis from patients were examined. Genomic DNA was extracted from the isolates by standard method. The number of copies of tandem repeats of the 15 MIRU-VNTR loci was determined by PCR amplification and agarose gel electrophoresis of the amplicons. M. tuberculosis outbreak-related strains were distinguished from other isolates. MIRU-VNTR typing identified 4 major clusters of strains. The same isolates clustered together after RFLP typing, but rep-PCR identified only 3 of them. The concordance between RFLP and MIRU-VNTR typing was complete, with the exception of two isolates with identical RFLP patterns that differed in the number of tandem repeat copies at two MIRU-VNTR alleles. A further isolate, even sharing the same RFLP pattern, differed by one repeat from the rest of its cluster. We also tested the use of an automated rep-PCR for clinical laboratory applications but it failed to identify the link between two pairs of epidemiologically related strains clustered by the other 2 techniques. For superior discrimination, ease of comparison of results and lower cost, MIRU-VNTR typing should be the favored PCR-based typing tool.

Evaluation of the anti-tuberculosis activity generated by different multigene DNA vaccine constructs.

Development of multigenic constructs expressing Mycobacterium tuberculosis (Mtb) antigens may be a strategy to obtain improved DNA vaccines against tuberculosis (TB). Several multigenic constructs expressing two or three Mtb antigens as fusion proteins were developed, both as tPA- and ubiquitin-fusion proteins. To demonstrate proper protein expression and intracellular turnover all multiantigens were tagged with the HA epitope and constructs were used to transfect rhabdomyosarcoma (RD) cells. Antigen expression was demonstrated by immunofluorescence using anti-HA antibodies. C57Bl/6 mice were immunized with selected constructs and protective activity was assessed following aerogenic challenge with Mtb. Several of these constructs induced a significant level of protection in the lung and in the spleen. Immunization with the construct expressing tPA85B-ES6 induced a level of protection that approached that provided by BCG. Immunization with a combination of these constructs induced levels of protection that were not superior to those elicited by a single combination, and immunization with a construct expressing five Mtb antigens could not provide an improved level of protection compared to tPA85B-ES6. We conclude that the activity of a DNA vaccine based on tPA85B-ES6 cannot be enhanced by broadening the antigen repertoire with other highly immunogenic secreted Mtb proteins.

Laparoscopic harvest of an omental flap to reconstruct an infected sternotomy wound.

Sternotomy dehiscence is associated with a high mortality rate. In most cases this complication may be treated by simple debridement and antibiotic therapy, but sometimes it is necessary to fill the sternal defect with viable tissue. The greater omentum seems to be the ideal tissue to be transposed because of its malleability, good vascularization, and high lymphatic tissue content. The transposition of the greater omentum usually requires a midline laparotomy for the preparation of the flap, with significant laparotomy-related morbidity. Laparoscopic access may represent an effective alternative for preparing and transposing the omental flap. The key points of the laparoscopic technique are (1) the coloepiploic detachment, (2) the section of the anastomotic arterial branches between the Barkow's arcade and the gastroepiploic arcade, (3) the mobilization of the greater omentum pedicled on the right gastroepiploic artery, and (4) its transposition into the mediastinum, taking care to avoid twisting the gastric greater curvature and the flap itself.

PE_PGRS33 Contributes to Mycobacterium tuberculosis Entry in Macrophages through Interaction with TLR2.

PE_PGRS represent a large family of proteins typical of pathogenic mycobacteria whose members are characterized by an N-terminal PE domain followed by a large Gly-Ala repeat-rich C-terminal domain. Despite the abundance of PE_PGRS-coding genes in the Mycobacterium tuberculosis (Mtb) genome their role and function in the biology and pathogenesis still remains elusive. In this study, we generated and characterized an Mtb H37Rv mutant (MtbΔ33) in which the structural gene of PE_PGRS33, a prototypical member of the protein family, was inactivated. We showed that this mutant entered macrophages with an efficiency up to ten times lower than parental or complemented strains, while its efficiency in infecting pneumocytes remained unaffected. Interestingly, the lack of PE_PGRS33 did not affect the intracellular growth of this mutant in macrophages. Using a series of functional deletion mutants of the PE_PGRS33 gene to complement the MtbΔ33 strain, we demonstrated that the PGRS domain is required to mediate cell entry into macrophages, with the key domain encompassing position 140-260 amino acids of PE_PGRS33. PE_PGRS33-mediated entry into macrophages was abolished in TLR2-deficient mice, as well as following treatment with wortmannin or an antibody against the complement receptor 3 (CR3), indicating that PE_PGRS33-mediated entry of Mtb in macrophages occurs through interaction with TLR2.

Accuracy of QuantiFERON-TB Gold Test for Tuberculosis Diagnosis in Children.

OBJECTIVES: To evaluate the accuracy of the QuantiFERON-TB Gold assay (QFT-IT) in children with suspected active or latent TB infection (LTBI). METHODS: A retrospective study was conducted on 621 children (0-14 years old) evaluated for TB infection or disease. Following clinical assessment, children were tested with the QFT-IT assay. RESULTS: Among the 140 active TB suspects, we identified 19 cases of active disease. The overall sensitivity for active TB was 87.5%, ranging from 62.5% in children 25-36 months old to 100% in children older than 49 months. The overall specificity for active TB was 93.6%. Among the 481 children tested for LTBI screening, 38 scored positive and all but 2 had at least one risk factor for TB infection. Among the 26 children with indeterminate results, bacterial, viral or fungal pneumonia were later diagnosed in 11 (42.3%) cases and non-TB related extra-pulmonary infections in 12 (46.1%). CONCLUSIONS: Our results indicate that the children's response to QFT-IT associates to active TB and risk factors for LTBI. Moreover, we show that mitogen response is also found in children of 1 year of age, providing support for QFT-IT use also in young children.

Organization of cortico-cortical associative projections in rats exposed to ethanol during early postnatal life.

The fine organization of cortico-cortical associative projections was investigated in adult rats exposed to inhalation of ethanol during the first postnatal week. Ethanol-treated and control animals received cortical injections of biotinylated dextran amine combined with N-methyl-D-aspartic acid, in order to obtain a Golgi-like retrograde labeling of associative pyramidal neurons. The results obtained from the analysis of labeling can be summarized as follows: (a) there are fewer associative projection neurons in ethanol-treated than in normal animals; (b) the ratio between the number of supragranular and infragranular associative neurons is higher in ethanol-treated animals compared to controls; (c) the basal dendrites of pyramidal associative cells of layer 2/3 display a simplified dendritic branching in ethanol exposed cases as compared to controls; (d) the cluster analysis shows that normal dendrites can be clearly subdivided into different groups according to their geometric properties, whereas dendrites from animals exposed to ethanol follow less robust grouping criteria. These differences are discussed in consideration of the functional alterations that characterize the fetal alcohol syndrome.

A prospective randomized clinical trial on pain control after major abdominal surgery.

This study was conducted in order to investigate the advantages and limitations of four analgesic modalities: a) epidural morphine; b) intravenous morphine; c) patient controlled intravenous morphine (patient-controlled analgesia); and d) non-steroidal anti-inflammatory drugs. Eighty patients undergoing major abdominal surgical procedures were prospectively and randomly treated with one of the above-mentioned analgesic methods. Evaluation of pain perception was done using the visual analogue pain score and the simple descriptive scale 4 hours after the procedure, in the early morning on postoperative day 1 and in the afternoon on postoperative days 1, 2 and 3. The need for supplementary analgesia and the onset of complications, if any, were also evaluated for each patient. Patient-controlled intravenous morphine yielded the best analgesic effect over the entire period. Epidural morphine was more effective in the very early postoperative period compared to modalities (b) and (d). Non-steroidal anti-inflammatory drugs, on the other hand, were more effective on the later postoperative days. None of the patients in group C needed supplementary analgesia, as against 20% in group A, 55% in group B and 40% in group D. Patients with hypochondriasis scores > 70 or depression scores > 70 required supplementation of analgesia more often. Morphine proved to be the drug of choice. Drug titration may be modulated in relation to the psychological characteristics of the patient. The best drug titration modality, in fact, is patient-controlled analgesia.

[Identification of gallbladder pedicle anatomy during laparoscopic cholecystectomy]. / In tema di riconoscimento dell'anatomia dell'ilo colecistico in corso di colecistectomia laparoscopica.

Laparoscopic cholecystectomy is widely accepted nowadays as the gold standard in the treatment of cholelithiasis. This new technique was initially associated with a significant increase in morbidity, and in particular in iatrogenic biliary injuries and arterial haemorrhages, perhaps due to a lack of knowledge of the "laparoscopic anatomy" of the gallbladder pedicle. In this technique the anatomical structures are viewed on a two-dimensional video monitor, and the dissection is performed with long instruments without manual sensitivity. Therefore, the laparoscopic surgeon has to deal with new anatomical views and must be aware of the possible arterial and biliary variants. In this review we describe our technique of laparoscopic cholecystectomy, with particular reference to manoeuvres useful for identifying the various anatomical structures at the gallbladder hilum. In our opinion, it is mandatory to avoid cutting any duct if its identity has yet to be established. For this reason, we pay great attention to the anatomical dissection of Calot's triangle, in order to accurately identify the cystic duct and the cystic artery and any other vascular or biliary structures. Routine intraoperative cholangiography may be useful for identifying the biliary anatomy. When in doubt, the surgeon should not hesitate to convert the procedure to open surgery.

Homologous prime boosting based on intranasal delivery of non-pathogenic invasive Escherichia coli expressing MPT64, decreases Mycobacterium tuberculosis dissemination.

Protein-subunit vaccines as boosting strategies against tuberculosis (TB) infection are currently in the pipeline of TB vaccine research. Their main limitation is represented by their poor immunogenicity, which makes it necessary to couple protein-subunits with adjuvant molecules. In this study, we employed replication-deficient invasive Escherichia coli strains to deliver Mycobacterium tuberculosis proteins to the cytoplasm of non-phagocytic eukaryotic cells using various priming and prime-boosting vaccination protocols. Our results demonstrate that intranasal administration of invasive E. coli expressing the M. tuberculosis protective antigen MPT64 to mice primed with a recombinant BCG strain over-expressing MPT64 on its surface, decrease bacterial burden in mice spleens. Our data suggest that replication-deficient invasive E. coli may represent a suitable platform for BCG/rBCG priming followed by homologous-boosting immunization strategies.

Functional dissection of protein domains involved in the immunomodulatory properties of PE_PGRS33 of Mycobacterium tuberculosis.

PE_PGRSs are a large family of proteins identified in Mycobacterium tuberculosis complex and in few other pathogenic mycobacteria. The PE domain of PE_PGRS33 mediates localization of the protein on the mycobacterial cell surface, where the PGRS domain is available to interact with host components. In this study, PE_PGRS33 and its functional deletion mutants were expressed in M. smegmatis, and in vitro and in vivo assays were used to dissect the protein domains involved in the immunomodulatory properties of the protein. We demonstrate that PE_PGRS33-mediated secretion of TNF-α by macrophages occurs by extracellular interaction with TLR2. Our results also show that while the PGRS domain of the protein is required for triggering TNF-α secretion, mutation in the PE domain affects the pro-inflammatory properties of the protein. These results indicate that PE_PGRS33 is a protein with immunomodulatory activity and that protein stability and localization on the mycobacterial surface can affect these properties.

PPE_MPTR genes are differentially expressed by Mycobacterium tuberculosis in vivo.

The PPE_MPTR protein sub-family is unique to mycobacteria and comprises proteins found only in MTB complex and in few other pathogenic mycobacteria. Very little is known about the precise function of PPE_MPTR, as well as on the expression pattern and the transcriptional regulation of their structural genes. In the present work, real time RT-PCR techniques were used to determine the expression profile of PPE_MPTR genes of Mycobacterium tuberculosis during infection in vivo and in different culture conditions. The PPE_MPTR genes showed a similar expression profile in axenic cultures, with a significant increase of gene expression following exposure to environmental signals such as SDS, isoniazid and ethambutol. The PPE_MPTR genes were expressed in lung and spleen tissues infected by M. tuberculosis, and levels of expression were similar to those of genes encoding M. tuberculosis virulence factors such as hbhA and mpt64. Levels and pattern of gene expression in host tissues were different for each PPE_MPTR gene under study. The results of this study indicate that PPE_MPTR genes are differentially regulated in the lung and spleen tissues during M. tuberculosis infection, suggesting that each gene responds independently to the different and complex environmental signals encountered in host tissues.

Estrogen and progesterone receptors in carpal tunnel syndrome.

Carpal tunnel syndrome (CTS) is a compression median nerve neuropathy common in women at menopausal age. The aim of this work was to study immunohistochemically the expression of estrogen (ER) and progesterone (PR) receptors in CTS and control specimens. Biopsies of transverse carpal ligament (TCL) and flexor tendon synovitis were collected from 23 women and from 7 men undergoing surgery for median nerve decompression at the wrist for CTS. In TCL and synovial tissue, cells expressed ER and PR with statistically significant differences related to the age and sex of patients. Immunoreactivity was observed in fibroblasts of TCL, and in lining cells and fibroblasts of synovial tissue. In women, the number of ER-positive cells in the TCL and synovial tissue increased with the age, peaking at 55-70 years, and then decreasing. PR-immunoreactivity was observed only in fibroblasts of TCL and its expression decreased with age, while no immunolabeling was found in the synovial tissue. In TCL samples, the number of ER- and PR-positive cells in non-CTS patients was significantly lower than in CTS patients. These results demonstrate that ER and PR are present in TCL and flexor tendon synovitis, suggesting a role for sex steroid hormones in the pathogenesis of CTS disease.

Intranasal delivery of DNA encoding antigens of Mycobacterium tuberculosis by non-pathogenic invasive Escherichia coli.

Naturally invasive bacteria have been successfully used for mucosal delivery of DNA vaccines against bacterial, viral and tumour antigens. Recently, an alternative delivery system based on a genetically modified mutant of the non-pathogenic commensal bacterium Escherichia coli, was developed and successfully used to deliver therapeutic genes and immunogenic proteins to epithelial cells in vivo. In this work, we used these recombinant invasive bacteria to deliver DNA vaccines against two Mycobacterium tuberculosis proteins (FbpA, and HtpX) following intranasal administration. Both DNA vaccines were able to induce an antigen-specific T-cell response. Moreover, mice immunized with the recombinant bacteria carrying the DNA vaccine encoding HtpX, were significatively protected from challenge with M. tuberculosis.