Biomedical subjects
Publications and source records attributed to G Delogu.
Mycobacterium neoaurum is a novel species of Mycobacteria, until now only isolated from catheters in immunosuppressed patients. This report describes the isolation and identification of M. neoaurum from urine obtained from a hospitalized patient.
BACKGROUND: Surgery and general anesthesia induce excessive apoptosis on peripheral lymphocytes and this phenomenon significantly contributes to the postoperative lymphocytopenia. However, the role played by anesthetic agents on this event remains to be elucidated. In this study we examined whether an anesthetic compound such as propofol is able to exert, in vitro, a proapoptotic effect on T cells. METHODS: Lymphocytes were isolated from heparinized venous blood in healthy volunteers and were incubated with propofol at clinically relevant concentration (5 mg/ml) and at 10 times this concentration (50 mg/ml). The counts of cells either bearing the Fas/FasL phenotype or expressing the intracellular Bcl2 protein were determined by means of flow cytometry. Assessment of lymphocytes undergoing apoptosis was made both by staining of apoptotic nuclei with propidium iodide (PI) and by phenotypic analysis of apoptotic cells with 7-amino-actinomycin D (7-AAD). RESULTS: Treated lymphocytes exhibit a significantly enhanced expression of Fas/FssL system associated with a decrease of Bcl2 expression at both 5 and 50 mg/ml propofol concentrations (p<0.05). On the other hand, the rate of apoptotic cells was not significantly different as compared to control in the absence of propofol. CONCLUSIONS: Propofol impairs, in vitro, both Fas/FasL and Bcl2 expressions on lymphocytes at clinically relevant concentrations but fails to induce apoptotic cell death. It is suggested that the antioxidative properties of the drug could inhibit some way the mechanisms by which mitochondrial free-radicals mediating apoptosis ultimately lead to cell death execution.
BACKGROUND: Surgery and anesthesia cause depression of cell-mediated immunity in the postoperative period, including a reduction in the numbers of circulating lymphocytes. It has been claimed that this immunosuppression is associated with an increased incidence of postoperative infections. HYPOTHESIS: Lymphocytopenia following surgical trauma depends on a dysregulated expression of death/and survival factors associated with apoptosis that, in turn, interferes with the occurrence of postsurgical infections. DESIGN: Fifteen subjects undergoing elective surgery under general anesthesia entered the study. The data of the patients who had infections during the postoperative outcome were compared with the data of those who did not. The data were collected prospectively. MAIN OUTCOME MEASURES: Peripheral blood samples were drawn before the operation, and 24 hours and 96 hours after the operation. Lymphocytes were isolated and examined for quantification and phenotypic analysis of apoptosis using the 7-amino-actinomycin D method, as well as for Fas and Fas ligand, interleukin 1-converting enzyme p20/caspase-1, Bcl-2, and p35 expression. The rate of apoptotic cells was correlated with the incidence of postoperative infections. SETTING: University hospital. RESULTS: Twenty-four hours after surgery, CD4(+) and CD8(+) cells exhibited a significantly higher frequency of apoptosis as well as of Fas and Fas ligand and interleukin 1-converting enzyme p20/caspase-1 expressions than preoperatively. This increase was paralleled by a significant down-regulation of antiapoptotic factors such as Bcl-2. However, the expression of the proapoptotic factor p35 was reduced. In addition, we found a relationship between the rate of the apoptotic CD8(+) subset and the occurrence of infectious complications during the postoperative course. At 96 hours after surgery, the variables studied returned to the baseline levels. CONCLUSIONS: In the early postoperative period, surgical trauma under general anesthesia induces an intracellular perturbation on peripheral lymphocytes, resulting in both up-regulation of death-signaling factors and down-regulation of survival-signaling factors. The increased apoptosis of CD8(+) lymphocytes, but not of CD4(+) cells, seemed to be associated with a greater risk of postsurgical infections.
Mycobacterium avium is a facultative intracellular microorganism, able to survive and multiply within mammalian macrophages by circumventing antimicrobial mechanisms. In this study we hypothesize that pre-existing M. avium infection could result in macrophage superinfections by other microorganisms. We found that 24 h after ingestion of M. avium at a low multiplicity of infection, macrophages are unable to efficiently produce superoxide anions when over-stimulated with phorbol esters, and that the generation of oxidative burst is only partially restored 72 h after bacteria ingestion. We also demonstrate that intracellular killing of Cryptococcus neoformans is markedly impaired in human macrophages that have previously ingested M. avium (but not other bacteria such as Escherichia coli). This inhibitory effect is observed with live mycobacteria, but not when heat-inactivated bacteria are ingested. In contrast, when Candida albicans is given to macrophages instead of C. neoformans, an enhancement of intracellular killing is observed, suggesting that cytocidal mechanisms other than respiratory burst are involved in the anti- Candidacidal activity of macrophages.
Genetic immunization is a promising new technology for developing vaccines against tuberculosis that are more effective. In the present study, we evaluated the effects of intracellular turnover of antigens expressed by DNA vaccines on the immune response induced by these vaccines in a mouse model of pulmonary tuberculosis. The mycobacterial culture filtrate protein MPT64 was expressed as a chimeric protein fused to one of three variants of the ubiquitin protein (UbG, UbA, and UbGR) known to differentially affect the intracellular processing of the coexpressed antigens. Immunoblot analysis of cell lysates of in vitro-transfected cells showed substantial differences in the degradation rate of ubiquinated MPT64 (i.e., UbG64 < UbA64 < UbGR64). The specific immune response generated in mice correlated with the stability of the ubiquitin-conjugated antigen. The UbA64 DNA vaccine induced a weak humoral response compared to UbG64, and a mixed population of interleukin-4 (IL-4)- and gamma interferon (IFN-gamma)-secreting cells. Vaccination with the UbGR64 plasmid generated a strong Th1 cell response (high IFN-gamma, low IL-4) in the absence of a detectable humoral response. Aerogenic challenge of vaccinated mice with Mycobacterium tuberculosis indicated that immunization with both the UbA64- and UbGR64-expressing plasmids evoked an enhanced protective response compared to the vector control. The expression of mycobacterial antigens from DNA vaccines as fusion proteins with a destabilizing ubiquitin molecule (UbA or UbGR) shifted the host response toward a stronger Th1-type immunity which was characterized by low specific antibody levels, high numbers of IFN-gamma-secreting cells, and significant resistance to a tuberculous challenge.
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Hexakis[butoxytris(ethoxy)]cyclophosphazene (3a), hexakis[dodecyloxytetrakis (ethoxy)]cyclophosphazene (3b) and hexakis[hexadecyloxyeicosanekis(ethoxy)]cyclophosphazene+ ++ (3c) were synthesised and their ability to form niosomes was studied. All synthesised compounds in the presence of cholesterol were shown to form vesicles, which aggregated strongly. To prevent aggregation, dicetylphosphate was used. The capacity of the sonicated and unsonicated niosomes to encapsulate hydrophile and lipophile molecules was also studied using carboxyfluorescein and diphenylhexatriene.
OBJECTIVES: To investigate the concentrations of mononuclear cell-associated ceramide and serum tumor necrosis factor-alpha (TNF-alpha) in patients with sepsis and to assess their predictive value for the development of multiple organ dysfunction syndrome (MODS). DESIGN: Prospective, cohort study. SETTING: Intensive care unit and two research laboratories at a university hospital. PATIENTS: Twenty-three adult patients admitted to an intensive care unit meeting the criteria for diagnosis of sepsis. INTERVENTIONS: Blood samples were collected at the time when diagnosis of sepsis was made. MEASUREMENTS AND MAIN RESULTS: Mononuclear cell-associated ceramide and serum TNF-alpha were significantly elevated in the samples from the septic patients compared with the control individuals (318.01+/-270.15 pmol/10(6) cells vs. 99.90+/-52.75 pmol/10(6) cells; p<.001, and 28.52+/-18.77 pg/mL vs. 10.43+/-3.37 pg/mL; p<.0001, respectively), and a direct correlation linked ceramide and TNF-alpha concentrations (r2 = .90, p<.00001). In the septic patients who went on to develop MODS, ceramide and TNF-alpha were significantly higher compared with the no MODS patients (489.22+/-264.93 pmol/10(6) cells vs. 131.23+/-99.02 pmol/10(6) cells; p<.0001, and 40.96+/-18 pg/mL vs. 14.95+/-5.60 pg/mL; p<.001, respectively). The receiver operating characteristic curves demonstrated that both TNF-alpha and ceramide were prognostic of MODS, but ceramide concentrations were more efficient predictors. CONCLUSIONS: These observations suggest that mononuclear cells of peripheral blood from patients with sepsis are committed to undergo apoptosis, because there is evidence that ceramide acts as an endogenous mediator of apoptosis. The strong correlation we found between cell-associated ceramide and serum TNF-alpha supports the hypothesis that this cytokine plays an important role in activating the sphingomyelin pathway and ceramide generation in patients with sepsis. In addition, this study provides evidence that consistent concentrations of mononuclear cell-associated ceramide may predict progression toward MODS in septic patients.
Novel tuberculosis DNA vaccines encoding native ESAT-6, MPT-64, KatG, or HBHA mycobacterial proteins or the same proteins fused to tissue plasminogen activator (TPA) signal sequences were evaluated for their capacity to elicit humoral, cell-mediated, and protective immune responses in vaccinated mice. While all eight plasmids induced specific humoral responses, the constructs expressing the TPA fusions generally evoked higher antibody responses in vaccinated hosts. Although most of the DNA vaccines tested induced a substantial gamma interferon response in the spleen, the antigen-specific lung responses were 2- to 10-fold lower than the splenic responses at the time of challenge. DNA vaccines encoding the ESAT-6, MPT-64, and KatG antigens fused to TPA signal sequences evoked significant protective responses in mice aerogenically challenged with low doses of Mycobacterium tuberculosis Erdman 17 to 21 days after the final immunization. However, the protective response induced by live Mycobacterium bovis BCG vaccine was greater than the response induced by any of the DNA vaccines tested. These results suggest that the tuberculosis DNA vaccines were able to elicit substantial immune responses in suitably vaccinated mice, but further refinements to the constructs or the use of alternative immunization strategies will be needed to improve the efficacy of these vaccine candidates.
Identification and characterization of mycobacterial adhesins and complementary host receptors required for colonization and dissemination of mycobacteria in host tissues are needed for a more complete understanding of the pathogenesis of diseases caused by these bacteria and for the development of effective vaccines. Previous investigations have demonstrated that a 28-kDa heparin-binding mycobacterial surface protein, HBHA, can agglutinate erythrocytes and promote mycobacterial aggregation in vitro. In this study, further molecular and biochemical analysis of HBHA demonstrates that it has three functional domains: a transmembrane domain of 18 amino acids residing near the N terminus, a large domain of 81 amino acids consistent with an alpha-helical coiled-coil region, and a Lys-Pro-Ala-rich C-terminal domain that mediates binding to proteoglycans. Using His-tagged recombinant HBHA proteins and nickel chromatography we demonstrate that HBHA polypeptides which contain the coiled-coil region form multimers. This tendency to oligomerize may be responsible for the induction of mycobacterial aggregation since a truncated N-terminal HBHA fragment containing the coiled-coil domain promotes mycobacterial aggregation. Conversely, a truncated C-terminal HBHA fragment which contains Lys-Pro-Ala-rich repeats binds to the proteoglycan decorin. These results indicate that HBHA contains at least three distinct domains which facilitate intercalation into surface membranes, promote bacterium-bacterium interactions, and mediate the attachment to sulfated glycoconjugates found in host tissues.
To verify the impact on stress response and the influence of anesthesia on endocrine/immunologic changes, we have investigated the plasma level of norepinephrine, cortisol, TNFalpha, and IL-6 in 46 patients scheduled for laparotomy and laparoscopic cholecystectomy at 2, 6, 12, and 24 h after the operation. Among subjects who underwent open approach, 9 received fentanyl anesthesia and 13 received isoflurane anesthesia. In the laparoscopy group, 14 patients were given fentanyl anesthesia and 10 were given isoflurane anesthesia. The results obtained confirmed that laparoscopic cholecystectomy is associated with a lesser immunoendocrine response, and the two anesthesia models do not interfere with plasma changes of the assessed hormones and cytokines.
Ewe milk and ewe cheese samples were analysed by matrix-assisted laser desorption/ionization mass spectrometry and their protein profiles were compared with those obtained from bovine milk and bovine cheeses. Various mixtures of bovine and ewe cheeses in different weight ratios were analysed, leading to a reproducible calibration curve, which has been successfully employed in determining the percentage of bovine milk fraudulently added to ewe milk in the production of marketed ewe cheese.
We have identified and sequenced a cDNA clone coding for Trichomonas vaginalis alpha-actinin. Analysis of the obtained sequence revealed that the 2,857-nucleotide-long cDNA contained an open reading frame encoding 849 amino acids which showed consistent homology with alpha-actinins of different species. Such homology was particularly significant in regions which have been reported to represent the actin-binding and Ca2+-binding domains in other alpha-actinins. The deduced protein was also characterized by the presence of a divergent central region thought to play a role in its high immunogenicity. A study of protein localization performed by immunofluorescence revealed that the protein is diffusely distributed throughout the T. vaginalis cytoplasm when the cell is pear shaped. When parasites adhere and transform into the amoeboid morphology, the protein is located only in areas close to the cytoplasmic membrane and colocalizes with actin. Concomitantly with transformation into the amoeboid morphology, alpha-actinin mRNA expression is upregulated.
The presence of enterobacterial repetitive intergenic consensus (ERIC) sequences was demonstrated for the first time in the genome of Mycobacterium tuberculosis; these sequences have been found in transcribed regions of the chromosomes of gram-negative bacteria. In this study genetic diversity among clinical isolates of M. tuberculosis was determined by PCR with ERIC primers (ERIC-PCR). The study isolates comprised 71 clinical isolates collected from Sardinia, Italy. ERIC-PCR was able to identify 59 distinct profiles. The results obtained were compared with IS6110 and PCR-GTG fingerprinting. We found that the level of differentiation obtained by ERIC-PCR is greater than that obtained by IS6110 fingerprinting and comparable to that obtained by PCR-GTG. This method of fingerprinting is rapid and sensitive and can be applied to the study of the epidemiology of M. tuberculosis infections, especially when IS6110 fingerprinting is not of any help.
In this study classical ribotyping based on hybridization of an enteroccocal ribosomal operon previously cloned from Enterococcus hirae (Sechi and Daneo-Moore, 1993) with XbaI cut chromosomal DNA and PCR-ribotyping were used to characterize the molecular epidemiology of 131 Enterococcus faecium, with high-level resistance to gentamicin, isolated from different hospitals in Italy and the United States. The ribotyping was able to differentiate all 131 clinical isolates into 96 family patterns. These family patterns appeared to be useful in establishing epidemiological spread. The results obtained were in agreement with those previously published, suggesting the presence of five to six operons in the Enterococcus genus (Sechi et al., 1994). We performed PCR-ribotyping, based on conserved sequences at the 3' end of the enterococcal 16S rrn and the 5' end of the 23S rrn, on 131 clinical isolates as well as on several enterococcal ATCC strains tested. The results were then compared with those obtained with the classical ribotyping method. The results suggest the presence of at least four classes of intergenic spacers among enterococci, but these classes are not helpful in differentiating between Enterococci or among Enterococcal isolates.
PURPOSE: This study investigates heat shock protein 70 (HSP70) expression by peripheral blood mononuclear cells (PBMCs) of septic patients admitted to an intensive care unit and examines the possibility of a correlation between HSP70 levels and plasma tumor necrosis factor alpha (TNF-alpha) concentrations. Additionally, we evaluated whether the HSP70 production could be regarded as a prognostic factor for the development of septic shock as well as for patient survival. MATERIALS AND METHODS: Blood samples of 29 patients were taken 24 hours after the diagnosis of sepsis. HSP70 expression and TNF-alpha level were measured using indirect immunofluorescent analysis and a commercially available enzyme-linked immunosorbent assay method, respectively. RESULTS: PBMCs expressed significantly high levels of HSP70 (11.9 +/- 5.6 [sd]) compared with those of the healthy control group (3.2 +/- 2.1% positive cells). Such enhanced levels were correlated to plasma TNF-alpha concentrations (r = .99, P < .01). This study failed to demonstrate a relationship between HSP70 production and clinical outcome. CONCLUSION: These findings give further evidence that also in humans, heat shock response is activated during sepsis. The correlation observed between HSP70 overproduction and TNF-alpha plasma concentrations suggests that HSP70 exerts a possible protective effect against TNF-alpha cytotoxicity. Such hypothesis has not been confirmed by our clinical data.
This study was carried out to evaluate the rate of recovery and time required for detection of mycobacteria from pulmonary and extrapulmonary human clinical samples, by using a fluorescence-quenching-based oxygen sensor (BACTEC 9000 MB; Becton Dickinson Microbiology Systems, Sparks, Md.). The results were compared with those obtained by microscopy, conventional culture in Lowenstein-Jensen (LJ) medium, and a BACTEC radiometric system (BACTEC 460 TB; Becton Dickinson). Of the 779 clinical samples processed, 364 from pulmonary sites and 415 from extrapulmonary sites, 62 (7.9%) were positive for mycobacterial isolates; of the positive samples, 59 (95.1%) were detected with the fluorescent BACTEC 9000 MB system, 57 (91.9%) were detected with the radiometric system (BACTEC 460 TB), and 43 (69.3%) were detected with LJ conventional culture. The mean times to detection of all mycobacteria with BACTEC 9000 MB and BACTEC 460 TB were similar (10.3 and 10.0 days, respectively). The results obtained indicate that the nonradiometric BACTEC (BACTEC 9000 MB) system is as efficient as Bactec 460 TB and significantly more efficient than LJ for the rapid recovery of mycobacteria from both pulmonary and extrapulmonary clinical specimens. Though the BACTEC 9000 MB system is recommended for respiratory specimens, we demonstrated that it can be successfully used also for recovery of mycobacteria from clinical specimens from various extrapulmonary sites.