Biodegradation ability of two selected microbial autochthonous consortia from a chronically polluted marine coastal area (Priolo Gargallo, Italy).

The aims of this study were: (i) the characterization of the structure of the indigenous microbial community associated with the sediments under study; (ii) the isolation and characterization of microbial consortia able to degrade the aged hydrocarbons contaminating the sediments, and (iii) the assessment of related biodegradation capability of selected consortia. Samples of surface sediments were collected in Priolo Gargallo harbour (Sicily, Italy). The samples were analysed for physical, chemical (GC-FID analysis) and microbiological characteristics (qualitative (16S rDNA clone library) and quantitative (DAPI, CFU and MPN count) analysis). The sediment samples were used for the selection of two microbial consortia (indicated as PSO and PSM) with high biodegradation capacity for crude oil (∼95%) and PAHs (∼63%) respectively. Genetic analysis showed that Alcanivorax and Cycloclasticus were the dominant genera in both the PSO and PSM consortia. Oil-polluted environments naturally develop an elevated biorecovery potential. The presence of a highly specialized microbial flora (adapted to support the contamination) and their stimulation through favourable induced conditions provides a promising recovery strategy. The chance to identify and select indigenous bacteria and/or consortia with a high biodegradation capacity is fundamental for the development and optimization of bioaugmentation strategies especially for those concerning in situ applications.

Bioremediation technologies for polluted seawater sampled after an oil-spill in Taranto Gulf (Italy): A comparison of biostimulation, bioaugmentation and use of a washing agent in microcosm studies.

One of the main challenges of bioremediation is to define efficient protocols having a low environmental impact. We have investigated the effect of three treatments in oily-seawater after a real oil-spill occurred in the Gulf of Taranto (Italy). Biostimulation with inorganic nutrients allowed the biodegradation of the 73±2.4% of hydrocarbons, bioaugmentation with a selected hydrocarbonoclastic consortium consisting of Alcanivorax borkumensis, Alcanivorax dieselolei, Marinobacter hydrocarbonoclasticus, Cycloclasticus sp. 78-ME and Thalassolituus oleivorans degraded 79±3.2%, while the addition of nutrients and a washing agent has allowed the degradation of the 69±2.6%. On the other hand, microbial community was severely affected by the addition of the washing agent and the same product seemed to inhibit the growth of the majority of strains composing the selected consortium at the tested concentration. The use of dispersant should be accurately evaluated also considering its effect on the principal actors of biodegradation.

Biodegradation potentiality of psychrophilic bacterial strain Oleispira antarctica RB-8(T).

The present study is focused on assessing the growth and hydrocarbon-degrading capability of the psychrophilic strain Oleispira antarctica RB-8(T). This study considered six hydrocarbon mixtures that were tested for 22days at two different cultivation temperatures (4 and 15°C). During the incubation period, six sub-aliquots of each culture at different times were processed for total bacterial abundance and GC-FID (gas chromatography-flame ionization detection) hydrocarbon analysis. Results from DNA extraction and DAPI (4',6-diamidino-2-phenylindole) staining showed a linear increase during the first 18days of the experiment in almost all the substrates used; both techniques showed a good match, but the difference in values obtained was approximately one order of magnitude. GC-FID results revealed a substantial hydrocarbon degradation rate in almost all hydrocarbon sources and in particular at 15°C rather than 4°C (for commercial oil engine, oily waste, fuel jet, and crude oil). A more efficient degradation was observed in cultures grown with diesel and bilge water at 4°C.

Tuberculosis specific responses following therapy for TB: Impact of HIV co-infection.

Characterizing perturbations in the immune response to tuberculosis in HIV can develop insights into the pathogenesis of coinfection. HIV+ TB+ and TB monoinfected (TB+) subjects recruited from clinics in Bamako prior to initiation of TB treatment were evaluated at time-points following initiation of therapy. Flow cytometry assessed CD4+/CD8+ T cell subsets and activation markers CD38/HLA-DR. Antigen specific responses to TB proteins were assessed by intracellular cytokine detection and proliferation. HIV+ TB+ subjects had significantly higher markers of immune activation in the CD4+ and CD8+ T cells compared to TB+ subjects. HIV+ TB+ had lower numbers of TB-specific CD4+ T cells at baseline. Plasma IFNγ levels were similar between HIV+ TB+ and TB+ subjects. No differences were observed in in-vitro proliferative capacity to TB antigens between HIV+ TB+ and TB+ subjects. Subjects with HIV+ TB+ coinfection demonstrate in vivo expansion of TB-specific CD4+ T cells. Immunodeficiency associated with CD4+ T cell depletion may be less significant compared to immunosuppression associated with HIV viremia or untreated TB infection.

Analysis of pulegone and its enanthiomeric distribution in mint-flavoured food products.

A procedure for the extraction and determination of pulegone enanthiomers in mint essential oils and mint products (syrups, dried leaves, toothpaste, lozenges, candy and chewing-gum) was developed. The compounds were recovered from the food matrices by employing a simultaneous distillation-extraction (SDE) technique with a Likens-Nickerson apparatus using dichloromethane as an extraction solvent. The analyses were performed by capillary gas chromatography mass spectrometry (GC/MS). Experiments on food products spiked at different pulegone concentrations showed recoveries ranging from 95 to 106%. The detection limit was about 5?mg?l(-1) for both pulegone enanthiomers and good linearity was found in the concentration range 0.5-25?mg?l(-1). In a number of repeated analyses, the pulegone peak height repeatability (RSD) was 0.2%. The pulegone enanthiomers were separated and quantified by enanthioselective multidimensional gas chromatography. The results of analyses conducted on essential mint oils and mint-flavoured food products are reported.

HLA-DM can partially replace the invariant chain for HLA-DR transport and surface expression in transfected endocrine epithelial cells.

The function of HLA class II molecules as peptide presenters to CD4+ T cells depends on the expression of associated molecules such as the invariant chain (Ii) and DM responsible for the correct transport of and high-stability peptide binding to the class II dimers. In organs affected by autoimmune diseases, endocrine epithelial cells express class II molecules, which presumably are involved in the presentation of self-peptides to autoreactive T cells. We have transfected the rat insulinoma cell line RINm5F with different combinations of HLA-DR, Ii and HLA-DM cDNAs and have studied how Ii and DM affect the transport and stability of class II molecules expressed by the different transfectants. Immunofluorescence and biochemical analysis showed that cells transfected with DR and DM in the absence of Ii expressed mostly stable molecules in their surface, and showed a lower accumulation of DR molecules in the endoplasmic reticulum (ER) than cells expressing only DR. This suggests that, in the absence of invariant chain, DM molecules can not only exchange peptides other than class II-associated invariant chain peptide (CLIP) but may also be involved in the transport of class II molecules out of the ER towards the endosomal route. In addition, these data confirm that expression of DR alone or DR+Ii do not allow the formation of sodium dodecyl sulphate (SDS)-stable complexes, that cells expressing DR+Ii have most DR molecules occupied by CLIP and that Ii and DM molecules allow regular routing and peptide loading of class II molecules.

HLA-DM and invariant chain are expressed by thyroid follicular cells, enabling the expression of compact DR molecules.

Thyroid follicular cells (TFC) in Graves' disease (GD) hyperexpress HLA class I and express ectopic HLA class II molecules, probably as a consequence of cytokines produced by infiltrating T cells. This finding led us to postulate that TFC could act as antigen-presenting cells, and in this way be responsible for the induction and/or maintenance of the in situ autoimmune T cell response. Invariant chain (li) and HLA-DM molecules are implicated in the antigen processing and presentation by HLA class II molecules. We have investigated the expression of these molecules by TFC from GD glands. The results demonstrate that class II+ TFC from GD patients also express li and HLA-DM, and this expression is increased after IFN-gamma stimulation. The level of HLA-DM expression by TFC was low but sufficient to catalyze peptide loading into the HLA class II molecules and form stable HLA class II-peptide complexes expressed at the surface of TFC. These results have implications for the understanding of the possible role of HLA class II+ TFC in thyroid autoimmune disease.

Lack of HLA-G soluble isoforms in Graves-Basedow thyrocytes and complete cDNA sequence of the HLA-G*01012 allele.

The presence of HLA-G mRNA has been studied in thyroid follicular cells from autoimmune patients with Graves' disease. Investigating the possible role of the expression of the HLA-G gene in tissue inflammation, we have found four of the six HLA-G mRNA isoforms described: G1, G2, G3 and G4, but not the soluble ones G5 and G6. Soluble G isoforms may be responsible for inducing tolerance and inflammation control and their absence in autoimmune thyroid follicular cells may induce failure of such control. In addition, the complete coding sequence of HLA-G*01012 has been obtained from thyrocytes and it shows only four synonymous changes with respect to the HLA-G*01011 allele; this further supports the existence of an evolutionary pressure for invariance on HLA-G genes.

HLA-DPbeta residue 69 plays a crucial role in allorecognition.

To investigate the contribution to allorecognition of the individual polymorphic positions Glu 69 and Val 36 from the DPB1*02012 allele, DPB1*02012 cDNA was subjected to site-directed mutagenesis and alleles expressing Lys at 69 and Ala at 36 were generated. The lymphoblastoid cell line (LCL) 45.EM1, a previously generated mutant B-LCL which expresses normal levels of DPA mRNA but is not able to transcribe DPB, was transfected with wild-type or mutant DPB1*02012 cDNAs. The ability of two HLA-DPw2 alloreactive CD4+ cytotoxic T-lymphocyte (CTL) clones to lyse the panel of DPB1*02012 wild-type and site-directed mutant B-cell lines was tested. Both CTL clones (8.3 and 8.9) lysed the B-LCL 45.1, which is haploid for HLA and expresses wild-type DPB1*02012, and transfectants expressing Ala at 36 instead of Val, indicating that this polymorphic residue is not critical for T-cell recognition. However, the change of Glu to Lys at 69 prevented recognition by clones 8.3 and 8.9. These data demonstrate that the residue at peptide-binding position 69 is crucial for T-cell receptor recognition and suggest the requirement for a negatively charged residue at this position for allostimulation of these T-cell clones. The side chain of DPbeta-69 is predicted to point into the peptide-binding groove, and the existence of positive(Lys) or negative (Glu) residues probably leads to substantial differences in the allo- or auto-DP-bound peptides or to differences in the conformation of the peptide-MHC complex, which would therefore be responsible for specific DPw2 allorecognition. The binding of a panel of monomorphic and polymorphic anti-HLA-DP monoclonal antibodies (mAbs) to these transfectants was also tested by flow cytometry. The changes at Glu 69 and Val 36 did not affect recognition by any of the monomorphic antibodies tested. However, the binding pattern of some of the polymorphic mAbs was clearly modified. Therefore, even though it is not crucial for T-cell allorecognition, polymorphic residue 36 must be involved in epitopes recognized by some polymorphic anti-DP antibodies, while residue 69 of the DPB molecule is crucial both for T-cell allorecognition and recognition by some mAbs.

Multidimensional Capillary GC-GC for the Analysis of Real Complex Samples. 3. Enantiomeric Distribution of Monoterpene Hydrocarbons and Monoterpene Alcohols of Mandarin Oils.

The enantiomeric distribution of beta-pinene, sabinene, limonene, linalool, terpinen-4-ol, and alpha-terpineol in mandarin oils has been determined using a fully-automated, multidimensional, double-oven GC-GC system. This system allows fractions to be multitransferred during the same GC analysis and the use of the two GCs independently when the multitransfer option is not used. The results obtained allowed the characterization of mandarin essential oil and the determination of extraneous oils added to or contaminating the oil.

Single-cell analysis of intrathyroidal lymphocytes shows differential cytokine expression in Hashimoto's and Graves' disease.

Most human organ-specific autoimmune diseases such as Hashimoto's thyroiditis (HT) are considered to be Th1 mediated, and a quantitative dominance of Th1 cells in thyroid infiltrates from both Graves' disease (GD) and HT affected glands has been reported. However, Th2 dominance would be expected in GD, where thyroid hyperfunction induced by stimulating antibodies predominates over tissue destruction. We have analyzed the interleukin-4 (IL-4), interferon-gamma (IFN-gamma) production by T cells at the single-cell level, both in infiltrating lymphocytes isolated from digested GD and HT thyroid glands and in derived T cell lines, by direct intracellular cytokine detection. Results showed a heterogeneous pattern of cytokine production in bulk GD infiltrates and derived T cell lines, and a similar pattern was observed in the much larger HT infiltrates. Both type 1 and type 2 cytokines were simultaneously produced by the infiltrating populations, and T cells with both patterns as well as intermediate patterns similar to Th0 cells could be detected ex vivo. However, the larger T lymphocytes, presumably activated and responsible for the autoimmune damage, predominantly produced IL-4 in GD and IFN-gamma in HT. The specificity of the Th2 responses in GD was suggested by the enrichment in IL-4 production after antigen-specific expansion of two oligoclonal T cell lines. These data show that both type 1 and type 2 cytokines are produced in the thyroid glands affected by autoimmunity and that the difference between diseases may be the effect of a functionally dominant population at a given time. This in vivo chronically activated antigen-specific population, producing type 1 or type 2 cytokines locally, may be responsible for the effect finally leading to one of the disease states.

Self-reactive cytotoxic gamma delta T lymphocytes in Graves' disease specifically recognize thyroid epithelial cells.

In this paper we report the isolation of a self-reactive cytotoxic gamma delta T cell line, 158RE.2, that originates from the T lymphocyte population infiltrating the thyroid gland of a patient with Graves' disease. Functional data using this cell line demonstrate that gamma delta T cells expanded in the thyroid tissue specifically recognize a ligand expressed by thyroid epithelial cells and cell lines of endocrine epithelial origin. The TCR expressed by these gamma delta T cells--V gamma I/V delta 5--is unusual in peripheral blood lymphocytes, and its specificity is clearly different from that observed in a high percentage of gamma delta T cells from PBL, which express the common TCR V gamma 9/V delta 2. The V gamma I/V delta 5 receptor is involved in the recognition of the ligand expressed by the thyroid cells, but not in the NK-like activity also displayed by 158RE.2. These cells express CD8 alpha alpha dimers, which participate in the thyroid ligand recognition but not in the NK-like activity. The epithelial cell recognition is not restricted by classical MHC class I or class II molecules, although the CD8 alpha alpha participation in the recognition suggests the involvement of nonclassical MHC molecules. These are the first data to be presented on self-reacting gamma delta T cells in human epithelium.

Characterization of neural cell adhesion molecule (NCAM) expression in thyroid follicular cells: induction by cytokines and over-expression in autoimmune glands.

NCAM (CD56) is a cell surface glycoprotein of the immunoglobulin superfamily expressed on neuroendocrine and natural killer (NK) cells which has considerable molecular heterogeneity due to differential splicing and post-translational modifications. NCAM has been detected in the thyroid follicular cells (thyrocytes) immunohistologically. We report here the molecular form, the modulation by cytokines and the levels of expression in thyroid pathology. By using a panel of MoAbs to NCAM on Western blots from thyrocyte extract we have determined that these cells express the 140- and 180-kD forms of NCAM. Exposure of primary cultures of thyrocytes to interferon-gamma (IFN-gamma), and even more, to the combination of IFN-gamma plus tumour necrosis factor-alpha (TNF-alpha) induced a clear increase in the expression of NCAM as assessed by FACS analysis. NCAM expression in thyrocytes was assessed by immunofluorescence in 59 surgical specimens of thyroid glands, and was found increased in 11/17 (64%) of Graves', in 5/25 (20%) of multinodular goitre (MNG) and in occasional adenoma glands. No correlation was found with the expression of HLA class I, class II or the degree of lymphocytic infiltration scored in adjacent sections, but it was often seen in areas infiltrated by macrophages. In conclusion, NCAM is an adhesion molecule whose expression is clearly increased in thyrocytes in autoimmune glands, probably as a consequence of exposure to cytokines locally released. Since one of the forms of NCAM expressed by thyrocytes has the capability to generate intracellular signal it may play a role in normal thyroid function. In addition, NCAM may facilitate the recognition of thyrocytes by lymphocytes, particularly by NK CD56+ lymphocytes.

Expression of intercellular adhesion molecule-1 in thyroid follicular cells in autoimmune, non-autoimmune and neoplastic diseases of the thyroid gland: discordance with HLA.

The presence of intercellular adhesion molecule-1 (ICAM-1) on epithelial cells facilitates their recognition by specific T lymphocytes. To assess the possible role of ICAM-1 in the recognition of thyroid follicular cells by T cells in thyroid autoimmune disease, we investigated the expression of ICAM-1 in thyrocytes from thyroid glands affected by Graves' disease, in glands with non-autoimmune pathology and normal glands using immunofluorescence staining on cryostat sections and on dispersed cell preparations. Sequential tissue sections from glands affected by Graves' disease (n = 15), multinodular goitre (MNG, n = 26), benign nodules (n = 11), primary carcinomas (n = 12) and control thyroid glands (n = 5) were stained for ICAM-1, HLA class I, HLA class II, CD3 and thyroid peroxidase (TPO). Weak and patchy ICAM-1 expression was found in the thyrocytes of 4/15 (27%) Graves' disease and of 1/26 (4%) multinodular goitre glands. In contrast, ICAM-1 expression was detected in the thyrocytes of 5/11 (45%) benign nodules and of 8/12 (67%) thyroid carcinomas in which it was sometimes strong. Thyrocytes in the five control glands were negative. These results correlated well with flow cytometry data from 23 of these glands which showed that ICAM-1 expression in thyrocytes from Graves' patients was, when present, 'dull', while in some malignant thyrocytes it was 'bright'. In preparations of thyrocytes from Graves' disease glands we found a striking discordance between the high levels of expression of HLA class I and HLA class II and the low expression of ICAM-1. This is surprising since in vitro the expression of these three molecules is equally induced by IFN-gamma and TNF-alpha. These results suggest that additional factors are involved in the induction of the inappropriate HLA class II expression observed in the thyrocytes of glands affected by Graves' disease.

Autoantibodies in chronic delta virus infection recognize a common protein of 46 kD in rat forestomach basal cell layer and stellate thymic epithelial cells.

Chronic hepatitis delta virus infection is associated with the presence of autoantibodies to rat forestomach and thymus in approximately 60% of patients' sera. We have characterized the antigen against which these autoantibodies are directed as a protein of 46 kD by immunoblotting studies on rat forestomach and thymus extracts. Normal human sera or sera from patients with other hepatic or non-hepatic autoimmune disorders did not bind to this protein. The immunoblot assay was more sensitive than immunofluorescence. Maximal titre was 1:10,000 versus 1:5120. By techniques of elution of specific antibodies from immunoblots, our results showed that the same antigen was present in both tissues. This antigen did not share common epitopes with hepatitis delta virus (HDV). Patients' sera depleted of basal cell layer and thymic stellate epithelial cell antibodies by absorption with the corresponding tissue extract maintained the HDV antibody titres. The autoimmune phenomena observed in patients with HDV infection seems to be a colateral process induced by the replication of delta virus in the host.