Critical research concepts in tuberculosis vaccine development.

A new and improved vaccine against tuberculosis (TB) would provide a powerful tool to conquer one of the most insidious infectious diseases of mankind. Protection afforded by bacillus Calmette-Guérin (BCG) has been shown to be limited and inconsistent, especially in adults that are known to transmit TB disease. In the last two decades, several new vaccines have been developed and tested with the aim to elicit robust and long-lived T-cell responses against Mycobacterium tuberculosis antigens. Although much progress has been made in the TB vaccine field, there is an urgent need to address critical research questions about TB immunity with a special focus on designing vaccines aimed at preventing infection and transmission of TB. Here, we discuss the rationale behind the current immunization strategies being implemented for TB vaccines and provide some suggestions for hypothesis driven research to encourage the development of novel TB vaccines.

Molecular epidemiology of Enterococcus faecium isolates from an Italian hospital.

BACKGROUND: Outbreaks of vancomycin-resistant Enterococcus faecium (VRE) strains is an emerging problem worldwide. Even if still relatively uncommon in European hospitals, infections caused by VRE have also been increasing recently in this continent. METHODS: In this study, we characterized 50 consecutive VRE and 23 vancomycin-sensitive E. faecium (VSE) isolates collected in an Italian hospital. The presence of the esp gene and that of genes encoding resistance to glycopeptides was investigated by polymerase chain reaction (PCR). All of the isolates were typed by multi-locus sequence typing (MLST), and a selection of them also by pulsed-field gel electrophoresis (PFGE). RESULTS: We found that all of the VRE and 18 (78%) of the VSE strains belonged to the single clonal complex-17 (CC17). The most represented sequence type (ST) was ST78 (34% of the isolates). When further analyzed by PFGE, ST78 isolates were subdivided into five pulsotypes, four of them closely related. The strong association between the esp gene and CC17 was confirmed. Interestingly, such an association was higher among vancomycin-resistant isolates. Most of the esp-positive isolates (34/46, 74%) encoded Esp4, a rare variant of this protein characterized by the absence of A repeats. CONCLUSIONS: Our findings underscore the role of the CC17 lineage in the nosocomial spread of VRE and VSE, and its rapid local evolution, underscoring the need for programs designed to provide early detection in order to prevent its spreading among the nosocomial population.

The Mycobacterium tuberculosis sigma factor sigmaB is required for full response to cell envelope stress and hypoxia in vitro, but it is dispensable for in vivo growth.

The numerous sigma (sigma) factors present in Mycobacterium tuberculosis are indicative of the adaptability of this pathogen to different environmental conditions. In this report, we describe the M. tuberculosis sigma(B) regulon and the phenotypes of an M. tuberculosis sigB mutant strain exposed to cell envelope stress, oxidative stress, and hypoxia. The sigB mutant was especially defective in survival under hypoxic conditions in vitro, but it was not attenuated for growth in THP-1 cells or during mouse and guinea pig infection.

Assessment of the lightcycler PCR assay for diagnosis of invasive aspergillosis in paediatric patients with onco-haematological diseases.

A reliable diagnosis of invasive aspergillosis (IA) is hampered by the difficulty in obtaining suitable tissue samples. To evaluate the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the LightCycler PCR for the diagnosis of IA, 536 blood samples were collected over a 22-month period from 62 paediatric patients (median age 10 years, range 1-18) considered at risk of IA. The galactomannan antigen (GM) and fungal DNA were assessed on serial blood samples. IA was diagnosed in eight of 62 patients (13%): proven, five, probable, three. Sensitivity, specificity, PPV and NPV of LightCycler PCR varied according to the number of positive samples used to define positivity: 88%; 37%; 17% and 95% for single sample positivity; and 63%, 81%, 33% and 94% for serial sample positivity respectively. The concordance between positivity of LightCycler PCR assay and the diagnosis of IA was 79%. The single positivity of LightCycler PCR assay showed a good sensitivity for the diagnosis of IA in paediatric patients. The high NPV makes LightCycler PCR a promising tool in addition to GM testing to design a strategy of pre-emptive antifungal therapy, although further validation studies are needed.

[Diagnostic pathway in a case with severe degree of hypertension]. / Iter diagnostico di un caso di ipertensione arteriosa di grado severo.

A 29- year-old male was admitted because of exertion dyspnea and intense headache. These symptoms were associated with severe hypertension, small multiple areas of cerebral ischemia, thrombocytopenia, prolonged aPTT and renal failure. The diagnostic tests performed during hospitalization resulted in a diagnosis of Primary Antiphospholipids Syndrome. The renal biopsy sample suggested histopathological features of uncommon simultaneous occurrence of antiphospholipids nephropathy and a "collapsing variant" of segmental focal glomerulosclerosis. It is fundamental to be aware that this syndrome is very likely to occur, and therefore to perform antiphospholipids antibodies assessment, since only an anticoagulant therapy proves effective; nevertheless, in view of the pathological renal findings, other therapies such as steroids might be added.

Antiviral activity in vitro of Urtica dioica L., Parietaria diffusa M. et K. and Sambucus nigra L.

Parietaria diffusa M. et K., Urtica dioica L. (Urticaceae) and Sambucus nigra L. (Caprifoliaceae) are plants usually used in popular medicine of central Italy for treating numerous diseases, first of all Herpes zoster. Several plant products have been described as potential antiviral agents, with special attention being devoted to those having retroviruses as etiological agents, including acquired immunodeficiency syndrome (AIDS), in which a retrovirus, the designated human immunodeficiency virus HIV, has been clearly identified as the primary cause of this disease. The present study proposes a preliminary screening of the antiviral activity of Parietaria diffusa, Sambucus nigra and Urtica dioica preparation against the feline immunodeficiency virus (FIV) infection. The feline immunodeficiency virus is a widespread lentivirus of domestic cats sharing numerous biological and pathogenic features with the human immunodeficiency virus (HIV). FIV infection in cats has therefore been proposed as an animal model for AIDS studies with respect to pathogenesis, chemotherapy, and vaccine development [Pedersen, N.C., 1993. Feline immunodeficiency virus infection. In: Levy, J.A. (Ed.), The Retroviridae. Plenum Press, New York; Bendinelli, M., Pistello, M., Lombardi, S., Poli, A., Garzelli, C., Matteucci, D., Ceccherini-Nelli, L., Malvaldi, G., Tozzini, F., 1995. Feline immunodeficiency virus: an interesting model for AIDS studies and an important cat pathogen. Clinical Microbiology Revue 8, 87-112; North, T.W., LaCasse, R.A., 1995. Testing anti-HIV drugs in the FIV model. Nature Medicine 1, 410-411; Matteucci, D., Pistello, M., Mazzetti, P., Giannechini, S., Isola, P., Merico, A., Zaccaro, L., Rizzati, A., Bendinelli, M., 2000. AIDS vaccination studies using feline immunodeficiency virus as a model: immunisation with inactivated whole virus suppresses viraemia levels following intravaginal challenge with infected cells but non-following intravenous challenge with cell-free virus. Vaccine 18, 119-130]. Early studies showed that some of them presented antiviral activity against infection of FIV as assayed by syncytia formation using feline kidney Crandell cells (CrFK).

Engineered E. coli delivers therapeutic genes to the colonic mucosa.

Taking advantage of the proximity of bowel mucosa to luminal bacteria, we have attempted to deliver a therapeutic gene to the colonic mucosa by oral administration of an invasive and non-pathogenic Escherichia coli. E. coli diamenopimelate (dap) auxotroph, harboring plasmid pGB2Omegainv-hly, express the inv gene from Yersinia pseudotubercolosis that confers the ability to invade nonprofessional phagocytic cells and the hly gene from Listeria monocytogenes that allows expression of lystreriolysin O, a perforin cytolysin able to perfore phagosomal membranes. This bacterial vector invades and transfers functional DNA to epithelial cells in vitro. We have shown that this strain carrying a therapeutic gene (pC1OmegaTGF-beta1) can significantly reduce the severity of experimental colitis in mice. However, as a consequence of mucosal barrier disruption during colitis, vector-specific mRNA transcripts could be recovered from the colon and also from extra-colonic tissues. We therefore replaced the constitutive CMV promoter in pC1OmegaTGF-beta1 by the inflammation-inducible interleukin-8 promoter generating plasmid pC1OmegaTGF-beta1IND. Plasmid-specific TGF-beta1 mRNA transcripts were detectable in mouse CMT-93 epithelial cells incubated with E. coli BM2710/pGB2Omegainv-hly carrying pC1OmegaTGF-beta1IND following exposure to inflammatory cytokines. Furthermore, the transcripts were detectable only within inflamed tissues and the therapeutic effects were comparable to those in animals treated with E. coli BM2710/pGB2Omegainv-hly+pC1OmegaTGF-beta1. In summary, engineered enteric bacteria can efficiently deliver in vivo therapeutic genes to the intact intestinal mucosa and regulation expression of the therapeutic gene by an inflammation-inducible promoter prevents its dissemination during colitis.

[Aspects of the on-line hemodiafiltration with regeneration and reinfusion of the ultrafiltrate (HFR): multicenter study]. / Aspetti dell'emodiafiltrazione on-line con rigenerazione e reinfusione dell'ultrafiltrato (HFR): uno studio multicentrico.

Despite technological advances in dialysis treatment, survival, morbidity and the quality of life in hemodialysis (HD) patients are affected by long-term complications, often related to the treatment itself. Among these complications, moderate protein and caloric malnutrition are present in approximately 30% of dialysis patients and are viewed as major contributors to increased mortality. In malnutrition pathogenesis, great importance is given to protein catabolism and to the loss of somatic protein and amino acids during dialysis. On the contrary, toxin clearance is believed to influence, positively, both protein anabolism and dietary protein intake. In hemodiafiltration (HDF), the clearance process is potentiated by three mechanisms (diffusion, convection and adsorption) and this could have a favorable effect on malnutrition. In addition, the reinfusion of regenerated ultrafiltrate (UF) would avoid the loss of large amounts of useful solutes as occurs with standard HD. In fact, all amino acids are present in the UF, which is not important in standard HD, but could be a problem in hemodiafiltration reinfusion (HFR). We treated 16 patients with HFR during the previous 3 months (the study will last for 12 months). Patients had been previously treated with bicarbonate dialysis for at least 6 months. The clinical tolerance of HFR was excellent and the technique appeared to be quite simple. The preliminary biochemical results demonstrated the stabilization of some parameters (such as urea and uric acid) with an adequate clearance of small molecules, while variables related to nutritional status (body weight, serum albumin and serum transferrin) did not change substantially. Surprisingly, the loss of both branched chain amino acids (BCAA) and essential amino acids (EAA) seemed slightly lower in HFR compared with standard HD. However, the reduced loss of amino acids (AA) observed with HFR should take into account other factors, such as absorption on adsorbent material and the basal plasma AA concentrations. Therefore, although each patient is in control of himself, it is difficult to draw any definite conclusions after only 3 months. However, it is evident that the loss of AA in HFR is quite modest and is not increased by the fact that it is a hemofiltration technique with all the consequent positive effects.

[Renal biopsy: outpatient procedure?]. / Biopsia renale: possibile procedura ambulatoriale?

BACKGROUND: The renal biopsy is usually performed as an in-patient procedure, with patients admitted to hospital for at least 24 hours. We have carried out renal biopsies on two groups of patients. In the first group, patients rest in the hospital for 8 hours following the procedure. They are discharged after undergoing ultrasonography and a TC scan. These patients return to the hospital after 24 hours to verify possible post-biopsy complications. In the comparison group, patients remain in hospital for 24 hours. RESULTS: In both groups, the only observed complication was asymptomatic postbiopsy hematoma. No major complications were present in either group. CONCLUSIONS: In selected cases, renal biopsy performed by an expert practitioner as an outpatient procedure is safe and does not require 24-hour observation.

Curing animals with plants: traditional usage in Tuscany (Italy).

Tuscany is an area rich in traditions, many of an ethnobotanical nature, and those of veterinary practice are of special interest. Almost a 100 different plant species are used to treat animals; sometimes old remedies are used to cure similar human ailments, other times the cure is used exclusively for veterinary treatment.

An ethno-pharmacobotanical survey in the Sarrabus district (south-east Sardinia).

The therapeutic uses and methods of administration of 70 plants in the traditional medicine of Sarrabus (south-east Sardinia, Italy) are documented. Among these species, some were not reported previously for Sardinia, while others turn out to have an original therapeutic use.

The Mycobacterium tuberculosis ECF sigma factor sigmaE: role in global gene expression and survival in macrophages.

In previously published work, we identified three Mycobacterium tuberculosis sigma (sigma) factor genes responding to heat shock (sigB, sigE and sigH). Two of them (sigB and sigE) also responded to SDS exposure. As these responses to stress suggested that the sigma factors encoded by these genes could be involved in pathogenicity, we are studying their role in physiology and virulence. In this work, we characterize a sigE mutant of M. tuberculosis H37Rv. The sigE mutant strain was more sensitive than the wild-type strain to heat shock, SDS and various oxidative stresses. It was also defective in the ability to grow inside both human and murine unactivated macrophages and was more sensitive than the wild-type strain to the killing activity of activated murine macrophages. Using microarray technology and quantitative reverse transcription-polymerase chain reaction (RT-PCR), we started to define the sigmaE regulon of M. tuberculosis and its involvement in the global regulation of the stress induced by SDS. We showed the requirement for a functional sigE gene for full expression of sigB and for its induction after SDS exposure but not after heat shock. We also identified several genes that are no longer induced when sigmaE is absent. These genes encode proteins belonging to different classes including transcriptional regulators, enzymes involved in fatty acid degradation and classical heat shock proteins.

Real Time PCR Using Molecular Beacons : A New Tool to Identify Point Mutations and to Analyze Gene Expression in Mycobacterium tuberculosis.

Molecular beacons are a novel family of hybridization probes, which emit fluorescence upon interaction with their target. They are hairpin-shaped oligonucleotides with a central part complementary to the target, flanked by two 5 6 base pair (bp) inverted repeats, which can form a stable stem. A fluorescent moiety is covalently linked to the 5' end of the molecule, whereas the quenching moiety, 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), is covalently linked to the 3' end. The stem keeps the two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. When molecular beacons bind to their target, they undergo a conformational change that results in the restoration of fluorescence of the internally quenched fluorophore (1) (Fig. 1). Molecular beacons are extremely specific, and can clearly discriminate between targets differing only by a single nucleotide (2,3). When present in a PCR reaction where their target is the amplification product, molecular beacons can form a stable hybrid with the amplicon during the annealing step. The intensity of fluorescence at the annealing step in each amplification cycle is a direct measure of amplicon concentration (2,4) (Fig. 2). Another interesting feature of molecular beacons is that they can be coupled to a variety of differently colored fluorophores. This allows multiplex PCR reactions where different DNA fragments can be amplified and detected simultaneously in the same tube (2,3). Fig. 1. Operation of molecular beacons. On their own, these molecules are nonfluorescent, because the stem hybrid keeps the fluorophore (◯) close to the quencher (•). When the probe sequence in the loop hybridizes to its target, forming a rigid double helix, a conformational reorganization occurs that separates the quencher from the fluorophore, restoring fluorescence (1). Fig. 2. Real time measurement of amplicon synthesis during PCR using molecular beacons. (A) Four PCR reactions were initiated with a different number of template molecules (indicated). The concentration of amplicons present after each cycle of amplification was determined by measuring fluorescence during the last few seconds of the annealing step. (B) Inverted relationship between the threshold cycle (the cycle at which the fluorescent signal becomes detectable above the background) and the logarithm of the initial number of template molecules. In this example, the target is M. tuberculosis H37Rv chromosomal DNA. The primers-molecular beacon set used in the reaction was specific for sigA (reprinted from ref. 4).

Ethnopharmacobotany in Tuscany: plants used as antihypertensives.

Among the more than 400 plants used in popular medicine in Tuscany, over 30 are used in the therapy of hypertension. For some of them their use is already known while for others there is no documentation. In this work we present the first results obtained from research carried out on antihypertensive plants belonging to Gentianaceae and in particular Gentiana kokiana.

Immunogenicity of the B monomer of Escherichia coli heat-labile toxin expressed on the surface of Streptococcus gordonii.

The B monomer of the Escherichia coli heat-labile toxin (LTB) was expressed on the surface of the human oral commensal bacterium Streptococcus gordonii. Recombinant bacteria expressing LTB were used to immunize BALB/c mice subcutaneously and intragastrically. The LTB monomer expressed on the streptococcal surface proved to be highly immunogenic, as LTB-specific immunoglobulin G (IgG) serum titers of 140,000 were induced after systemic immunization. Most significantly, these antibodies were capable of neutralizing the enterotoxin in a cell neutralization assay. Following mucosal delivery, antigen-specific IgA antibodies were found in feces and antigen-specific IgG antibodies were found in sera. Analysis of serum IgG subclasses showed a clear predominance of IgG1 when recombinant bacteria were inoculated subcutaneously, while a prevalence of IgG2a was observed upon intragastric delivery, suggesting, in this case, the recruitment of a Th1 type of immune response.

Ethnopharmacobotanical studies of the Tuscan Archipelago.

From an ethno-pharmacobotanical point of view, Tuscany is a region with very rich and interesting traditions. The Tuscan Archipelago, particularly due to its geographical position and its history, presents a large variety of plant species used in popular medicine in numerous pathologies, including several viral infections. Over 100 species of plants are used in popular medicine in this region.

Differential expression of 10 sigma factor genes in Mycobacterium tuberculosis.

The ability of Mycobacterium tuberculosis to adapt to different environments in the infected host is essential for its pathogenicity. Consequently, this organism must be able to modulate gene expression to respond to the changing conditions it encounters during infection. In this paper we begin a comprehensive study of M. tuberculosis gene regulation, characterizing the transcript levels of 10 of its 13 putative sigma factor genes. We developed a real-time RT-PCR assay using a family of novel fluorescent probes called molecular beacons to quantitatively measure the different mRNAs. Three sigma factor genes were identified that have increased mRNA levels after heat shock, two of which also responded to detergent stress. In addition, we also identified a sigma factor gene whose mRNA increased after mild cold shock and a second that responded to conditions of low aeration.

Characterization of emb, a gene encoding the major adhesin of Streptococcus defectivus.

Streptococcus defectivus is one of the nutritionally variant streptococci, a class of viridans group streptococci first isolated from patients with endocarditis and otitis media. In previous studies, NVS-47, a clinical isolate of S. defectivus, was shown to bind to the extracellular matrix. A high-molecular-weight surface protein was identified and proposed to be responsible for mediating this binding. In the present study, the gene encoding this protein was identified by transposon mutagenesis and characterized. The gene (emb) was found to be larger than 14 kb and was partially sequenced. It encodes a protein containing at least 50 repeats of 77 amino acids predicted to assume an alternating coiled-coil conformation. The domain responsible for extracellular matrix binding was mapped to the N terminus of the protein. From sequence analysis, Emb is proposed to be the prototype of a new family of streptococcal fibrillar proteins.

Insertion vectors for construction of recombinant conjugative transposons in Bacillus subtilis and Enterococcus faecalis.

The broad-host range of conjugal transfer and the chromosomal location make conjugative transposons (CT) attractive candidates as tools for genetic manipulation of a large variety of bacteria. In this paper we describe insertion vectors capable of integrating into Tn916, the prototype of CT in Gram-positive bacteria. The integration of vectors into a single chromosomal copy of Tn916 was studied both after natural transformation of Bacillus subtilis, and after electroporation in Enterococcus faecalis. Integration occurred either by double or by single crossover, and the integrated DNA segment was shown to be highly stable. All recombinant CT (rCT) were still able to excise from the chromosome to form circular intermediates, the first step of both transposition and conjugal transfer. All classes of rCT generated by insertion vector pSMB47 were capable of conjugal transfer, while using pVMB11 it was possible to generate non-conjugative rCT.

Extra and intracellular expression of Mycobacterium tuberculosis genes.

To understand how Mycobacterium tuberculosis survives and grows in an infected host, we are studying the mycobacterial transcriptional machinery and its response to stresses encountered in vitro and in vivo. Much has been learned about sigma factors and other transcriptional regulators concerning their roles in controlling mycobacterial gene expression. It has recently been shown that sigma A is the essential housekeeping sigma factor and the alternative sigma factor sigma B, not essential for growth in a laboratory setting, is required for a robust protective response to various environmental stresses. We are also studying the mechanism by which the R522H mutation in sigma A prevents the transcription of certain genes, including some that are believed necessary for virulence. Also under investigation is the mycobacterial iron acquisition apparatus and its regulation, as metabolism of this essential element plays a key role in microbial pathogenesis. We have identified and characterized the major mycobacterial iron regulator IdeR that blocks the synthesis of the iron uptake machinery and have identified target genes in M. smegmatis and M. tuberculosis that are directly repressed by IdeR. Recent studies have examined the control of M. tuberculosis gene expression in vivo. Among these new approaches are an in vivo expression technology system to identify M. tuberculosis genes that are induced in macrophages and mice and a novel RT-PCR method that allows an accurate comparison between the levels of specific mRNAs in M. tuberculosis grown in vitro with those found in bacteria growing in human macrophages.