Probiotic Bifidobacterium lactis, anti-oxidant vitamin E/C and anti-inflammatory dha attenuate lung inflammation due to pm2.5 exposure in mice.

The incidence of asthma and allergic diseases of the airways is constantly increasing, both in the industrialised and developing countries, due to harmful and excessive quantities of air pollution. Although some studies have shown an effect of dietary supplementation of specific nutrients (especially with anti-oxidant and anti-inflammatory properties) in reducing airways inflammatory response, the results are not yet conclusive and the science is still at its infancy. Our hypothesis is that combining such nutrients could provide more benefits than using them alone. The aim of the research project proposed here is to investigate whether specific combinations of nutrients (docosahexanoic acid, vitamin C and E, and Bifidobacterium lactis strain BB-12®, included in an engineered diet) can act synergistically to reduce inflammation given by high level of air pollution. Beside the role of docosahexanoic acid, vitamins C and E on airways inflammatory disease, no study examined the effect of the supplementation of this probiotic strain in pathological conditions caused by air pollution so far. Herein we used a well-established in vivo model for the study of pollution effects, which consists in female BALB/c mice receiving by pharyngeal aspiration either a sham or a particulate matter with diameter <2.5 µm (PM 2.5) containing aerosol. Before treatment, mice were fed either a chow or a supplemented diet. By performing histological analyses and gene expression profiles on lung sections and serum measurement of the cytokine interleukin 10, we found that a specific combination of all the aforementioned nutrients rather than nutrients alone had a synergistic protective effect against PM2.5-induced inflammation. In conclusion, our study support that a supplemental nutritional intervention based on a combination of the probiotic B. lactis BB-12, the anti-oxidant vitamin C and E, and the anti-inflammatory docosahexanoic acid represents a rational option for alleviating air pollution-related lung inflammation.

Down syndrome, autoimmunity and T regulatory cells.

Autoimmune diseases are more represented in Down syndrome (DS) individuals compared to chromosomally normal people. Natural T regulatory cells (nT(reg) ) have been considered to be primary in the role of controlling the intensity and targets of the immune response. We have investigated the phenotypical and functional alteration of nT(reg) in a group of DS people. The phenotypical characteristic of T(reg) cells of 29 DS was analysed and compared with an age-matched healthy control group. The inhibitory potential of CD4(+) CD25(high) CD127(low) T regulatory cells was evaluated on autologous CD4(+) CD25(-) T cell proliferation in response to activation with a mytogenic pan-stimulus (anti-CD2, anti-CD3 and anti-CD28 antibodies). The CD4(+) CD25(high) cells in the DS and control groups were 2·692±0·3808%, n=29 and 1·246±0·119, n=29%, respectively (P=0.0007), with a percentage of forkhead box protein 3 (FoxP3)-expressing cells of 79·21±3·376%, n=29 and 59·75±4·496%, respectively (P=0.0015). CD4(+) CD25(+) FoxP3(+) cells were increased in peripheral blood from DS subjects (DS mean 5·231±0·6065% n=29, control mean 3·076±0·3140% n=29). The majority of CD4(+) CD25(high) were CD127(low) and expressed a high percentage of FoxP3 (natural T(reg) phenotype). While the proliferative capacity of DS T cells was not altered significantly compared to normal individuals, a reduced inhibitory potential of T(reg) compared to healthy controls was clearly observed (mean healthy control inhibition in T(eff) : T(reg) 1:1 co-culture: 58·9%±4·157%, n=10 versus mean DS inhibition in T(eff) :T(reg) 1:1 co-culture: 39·8±4·788%, n=10, P=0.0075; mean healthy control inhibition in T(eff) : T(reg) 1:0·5 co-culture: 45·10±5·858%, n=10 versus DS inhibition in T(eff) : T(reg) 1:0·5 co-culture: 24·10±5·517%, n=10, P=0.0177). DS people present an over-expressed peripheral nT(reg) population with a defective inhibitory activity that may partially explain the increased frequency of autoimmune disease.

MHC immunoevasins: protecting the pathogen reservoir in infection.

Alteration of antigen recognition by T cells as result of insufficient major histocompatibility complex (MHC)-dependent antigen-presenting function has been observed in many cases of infections, particularly in in vitro systems. To hide themselves from an efficient immune response, pathogens may act on MHC-related functions at three levels: (i) by limiting the number of potential antigens that can be presented to naïve T cells; (ii) by synthesizing proteins which directly affect MHC cell-surface expression; and (iii) by altering the normal intracellular pathway of peptide loading on MHC. Here, we review examples of pathogens' action on each single step of MHC function and we suggest that the result of these often synergistic actions is both a limitation of the priming of naïve T cells and, more importantly, a protection of the pathogen's reservoir from the attack of primed T cells. The above mechanisms may also generate a skewing effect on immune effector mechanisms, which helps preserving the reservoir of infection from sterilization by the immune system.

Preservation of clonal heterogeneity of the Pneumocystis carinii-specific CD4 T cell repertoire in HIV infected, asymptomatic individuals.

The loss of CD4 lymphocytes in HIV disease associates with opportunistic infections. Since diverse CD4 T cell clones respond to an opportunistic pathogen, we asked whether CD4 depletion deletes selected clones in the repertoire (vertical depletion) or it affects all clones by reducing the cell number in each progeny without affecting the overall number of clones (horizontal depletion). Understanding this point may help explain the mode of CD4 depletion and the mode of immunoreconstitution after therapy. Therefore we examined the CD4 T cell repertoire specific for Pneumocystis carinii, a relevant opportunistic pathogen in AIDS, in HIV-infected, asymptomatic individuals. We identified two patients of 36 asymptomatics for lack of proliferation to P. carinii, suggesting selective depletion of specific CD4 cells. To investigate clonal heterogeneity of P. carinii-responsive CD4 lymphocytes, specific CD4 T cell lines were generated and studied by TCR BV gene family usage and CDR3 length analysis (spectratyping). Clonal heterogeneity was similar in antigen-specific CD4 lines generated from P. carinii non-responding HIV seropositives and from controls. Thus, despite undetectable response to the pathogen, residual specific cells probably prevent overt infection and, when expanded in vitro, exhibit a clonal diversity similar to normal controls. These findings suggest a horizontal, rather than vertical, depletion in these asymptomatic patients.

Analysis of the antigen specific T cell repertoires in HIV infection.

In addition to HIV infection, several acquired immunodeficiencies lead to depletion of CD4 lymphocytes. These include immunosuppression resulting from high dose cancer chemotherapy or induced to control graft rejection, as well as in autoimmune diseases. The consequence of this depletion is an increased susceptibility to opportunistic infections or the inability to control primary infection in the case of HIV infection. In all instances a full or partial immunoreconstitution is desirable. In order to monitor the cellular immune state of a patient, rational information cannot be simply derived from phenotypic quantification of T lymphocytes. Instead loss or recovery of CD4 cells should be monitored by defining the specificity, the function and the clonality of the relevant cell population. Several methods are now available for this type of investigation. Here we describe an approach for the definition of clonal heterogeneity of antigen specific CD4 lymphocytes, a parameter that may help monitor loss or reconstitution in acquired immunodeficiencies. As examples of antigen specific CD4 T cell responses we focused on Pneumocystis carinii and on cytomegalovirus, as prototypic opportunistic pathogens which are responsible for severe infections in AIDS and in other immunosuppressive conditions which arise for instance following transplantation. Specific CD4 T cell lines were generated from normal controls and from seropositives in order to select antigen specific lymphocytes. The cells were subsequently analyzed for clonal diversity according to TCR BV gene family usage and according to TCR CDR3 size heterogeneity (spectratyping).

The MHC class II transactivator: prey and hunter in infectious diseases.

The MHC class II transcriptional activator (CIITA) is the major regulator of expression of MHC class II genes. Thus, CIITA plays a fundamental role in the regulation of the immune response. Here, we discuss our findings on the dual role of CIITA during infections, as the target (prey) for certain pathogens but the host effector (hunter) against other pathogens, including HIV-1. This dual role is placed in an evolutionary context as a rather peculiar example of a strategy used by pathogens to evade host defenses and a counteraction of the host to minimize the survival and spread of the pathogen.

Tat protein is an HIV-1-encoded beta-chemokine homolog that promotes migration and up-regulates CCR3 expression on human Fc epsilon RI+ cells.

Human basophils and mast cells express the chemokine receptor CCR3, which binds the chemokines eotaxin and RANTES. HIV-1 Tat protein is a potent chemoattractant for basophils and lung mast cells obtained from healthy individuals seronegative for Abs to HIV-1 and HIV-2. Tat protein induced a rapid and transient Ca(2+) influx in basophils and mast cells, analogous to beta-chemokines. Tat protein neither induced histamine release from human basophils and mast cells nor increased IL-3-stimulated histamine secretion from basophils. The chemotactic activity of Tat protein was blocked by preincubation of FcepsilonRI(+) cells with anti-CCR3 Ab. Preincubation of Tat with a mAb anti-Tat (aa 1-86) blocked the migration induced by Tat. In contrast, a mAb specific for the basic region (aa 46-60) did not inhibit the chemotactic effect of Tat protein. Tat protein or eotaxin desensitized basophils to a subsequent challenge with the autologous or the heterologous stimulus. Preincubation of basophils with Tat protein up-regulated the level of CCR3 mRNA and the surface expression of the CCR3 receptor. Tat protein is the first identified HIV-1-encoded beta-chemokine homologue that influences the directional migration of human FcepsilonRI(+) cells and the expression of surface receptor CCR3 on these cells.

Highly stable oligomerization forms of HIV-1 Tat detected by monoclonal antibodies and requirement of monomeric forms for the transactivating function on the HIV-1 LTR.

The use of newly generated murine monoclonal antibodies directed against distinct epitopes of a functionally active, chemically synthesized HIV-1 Tat protein has permitted the identification of several molecular forms including monomers, dimers and trimers. Dimers and trimers are particularly stable and resistant to strong reducing conditions. Through epitope mapping it has been possible to demonstrate that the major immunodominant epitope is contained within the basic region of the Tat protein and is lost after oligomerization of the molecule. In contrast, N-terminal, C-terminal and conformation-dependent epitopes are still accessible to mAb specific recognition after Tat oligomerization. Moreover, by using a quantitative HIV-LTR transactivation assay depending upon exogenous Tat, we could extrapolate the amount of functional Tat produced by cell lines stably transfected with the viral transactivator. More importantly, we could show that only the monomeric form of exogenous Tat is the relevant functional form acting in cells harbouring the HIV-1 LTR promoter.

HIV-1 Tat mutants in the cysteine-rich region downregulate HLA class II expression in T lymphocytic and macrophage cell lines.

Human macrophage and T cell lines were stably transfected with HIV-1 wild-type Tat or Tat mutants in the cysteine-rich region displaying trans-dominant negative effects on HIV-1 life cycle. The expression of HLA class I and class II molecules was not affected by wild-type Tat. Tat mutants, instead, profoundly down-regulated in a dose-dependent fashion the expression of class II, but not of class I, in both cell types by acting at the transcriptional level. Down-regulation was manifested on constitutive and IFN-gamma-induced class II gene expression and did not correlate with reduced transcription of the AIR-1 gene product CIITA, the major transcriptional activator of class II genes, indicating that Tat mutants did not act by inhibiting AIR-1 gene expression. Class II down-modulation had important functional implications in macrophages, as both antigen processing and presenting capacity were inhibited. These results represent the first evidence that a modified HIV-1 Tat product can act as a potent immunosuppressor by inhibiting the HLA class II expression necessary for triggering both cellular and humoral responses against pathogens. The use of these HIV-1 Tat mutants also discloses new opportunities to investigate the molecular mechanisms underlying the coordinate HLA class II gene transcription.

Distinct regulation of HLA class II and class I cell surface expression in the THP-1 macrophage cell line after bacterial phagocytosis.

Expression of HLA and CD1b molecules was investigated in the THP-1 macrophage cell line within 2 weeks following phagocytosis of mycobacteria or Escherichia coli. During the first 2-3 days, cell surface expression of HLA class II and CD1b was drastically down-modulated, whereas HLA class I expression was up-modulated. In the following days both HLA class II and CD1b expression first returned to normal, then increased and finally returned to normal with kinetics similar to that observed for the steadily increased HLA class I. The initial down-modulation of HLA class II and CD1b cell surface antigens was absolutely dependent on phagocytosis of bacteria. Further studies indicated that initial HLA class II cell surface down-modulation (1) was not due to reduced transcription or biosynthesis of mature HLA class II heterodimers, (2) was only partially, if at all, rescued by treatment with IFN-gamma, although both mRNA and corresponding intracellular proteins increased up to sixfold with respect to untreated cells, and (3) resulted in failure of THP-1 cells to process and present mycobacterial antigens to HLA-DR-restricted antigen-specific T cell lines. The existence of a transient block of transport of mature HLA class II heterodimers to the cell surface in the first days after phagocytosis of bacteria may have negative and positive consequences: it decreases APC function early but it may increase it later by favoring optimal loading of bacterial antigens in cellular compartments at high concentration of antigen-presenting molecules.

Double-stranded deoxyribonucleic acid binds to HLA class II molecules and inhibits HLA class II-mediated antigen presentation.

CD4+ T cells proliferating in response to purified double-stranded deoxyribonucleic acid (dsDNA) have been recently demonstrated in peripheral blood mononuclear cells of patients with systemic lupus erythematosus. Their activation was inhibited by anti-HLA class II (HLA-II) monoclonal antibodies; thus, the existence of a molecular interaction between dsDNA and HLA-II is conceivable. In this report we show that dsDNA specifically bind to HLA-II. After preincubating cells with purified dsDNA or synthetic oligonucleotides, dsDNA was detected on the cell membrane and in the lysates of HLA-II+ but not of isogenic HLA-II- cell lines. We demonstrate that dsDNA binding inhibits that of a specific peptide to HLA-II. Mixed lymphocyte reaction and antigen-specific T cell proliferation were inhibited by the preincubation of stimulator cells or antigen-presenting cells with dsDNA. These results suggest the existence of a novel mechanism of down-modulation of the CD4+ T cell function generated by lack of stimulation due to the HLA-II presenting molecules being "occupied" by dsDNA.

HLA class II expression in uninducible hepatocarcinoma cells after transfection of AIR-1 gene product CIITA: acquisition of antigen processing and presentation capacity.

The AIR-1-encoded CIITA transcriptional activator is crucial for both constitutive and IFN-gamma-induced MHC class II gene transcription. We show here that the MHC class II negative phenotype of the human hepatocarcinoma cell lines Alexander and HepG2 remains unmodified after treatment with IFN-gamma, although MHC class I expression is up-modulated. This correlates with absence of CIITA mature transcripts. Transfection of an expressible CIITA cDNA in Alexander cells resulted in a very high cell surface expression of all three human class II subsets, HLA-DR, -DP and -DQ, indicating that normally observed induction of CIITA expression by IFN-gamma is probably blocked, in the hepatocarcinoma cell lines, at the level of CIITA transcription and not at the level of IFN-gamma receptor binding and signal transduction mechanisms. To assess whether MHC class II expression on CIITA-transfected Alexander cells could have functional relevance, we tested their capacity to present antigenic peptides to an HLA-DR-restricted T cell line specific for a peptide of Mycobacterium tuberculosis Ag85 protein. It was found that the transfected cells could not only present the exogenously supplemented peptide but also process Ag85 protein to generate the specific epitope recognized by the HLA-DR-restricted T cell line. Similar results were obtained with CIITA-transfected CFPAC-1 pancreatic adenocarcinoma cells, which differed from Alexander cells in that they were inducible by IFN-gamma. These results suggest new strategies to act on CIITA for increasing the potential of a tumor cell to present putative tumor Ags to the immune system.

Human T-cell leukemia virus type II directly acts on CD34+ hematopoietic precursors by increasing their survival potential. envelope-associated HLA class II molecules reverse this effect.

The role of human T-cell leukemia virus type II (HTLV-II) in human lymphoproliferative and hematopoietic abnormalities in which the retrovirus can be isolated is still elusive. Here we show that the C344 T-cell-derived lymphotropic HTLV-II type IIa Mo strain acts directly on CD34+ hematopoietic precursors by rescuing them from apoptosis induced by interleukin-3 (IL-3) deprivation. This effect is viral strain-specific, as it is not observed with the B-lymphotropic HTLV-II type IIb Gu strain, it does not require infection of the hematopoietic precursors, and, interestingly, it is strongly dependent on the infected cellular host from which the virus was derived. Indeed, growth adaptation of the Mo strain to the permissive B-cell line, BJAB, renders the virus no longer capable of mediating the antiapoptotic effect. However, pretreatment of the BJAB-adapted Mo strain with antibodies specific for HLA class II, but not class I, histocompatibility antigens restores the antiapoptotic potential of the virus. These results constitute the first evidence that HTLV-II retrovirus can directly influence the homeostasis of human progenitors, without infecting them, and that this crucial activity is strongly inhibited by the presence of host-derived envelope-associated HLA class II antigens.

A girl with diabetes and severe combined immunodeficiency from adenosine deaminase deficiency.

We present a girl with severe combined immunodeficiency (SCID) from adenosine deaminase (ADA) deficiency who developed insulin dependent diabetes mellitus (IDDM). This combination of features has not been previously reported. Because HLA typing (DQbeta-57 Asp/Asp and DQalpha-52 Ser/Ser) showed no alleles usually associated with IDDM, and ICA were repeatedly negative even after treatment with PEG-ADA and gene transplant, hypotheses on the pathogenesis of diabetes mellitus in this patient are discussed.

Functional significance of polymorphism among MHC class II gene promoters.

The functional significance of polymorphism among MHC class II promoters in man and mouse is here reviewed, mainly in terms of the hypothesis of differential expression. The hypothesis proposes that differences between antigen-presenting cells in MHC class II expression exert a co-dominant effect on the Th1-Th2 cytokine balance, such that class II molecules of one type come to control to a greater extent the production of one group of cytokines, and those of another type the production of the alternative group. The survey deals with the influence of signal strength and antigen-presenting cell type on T-cell subset differentiation; functional differences between MHC class II molecules not obviously related to determinant selection; disease protection mediated by HLA alleles; mechanisms possibly responsible for allotypic and isotypic bias; overdominance (heterozygous advantage) in selection for expression of class II alleles; MHC class II promoter structure and function; inter-locus and inter-allele variability within human MHC class II gene upstream regulatory regions; a comparison of these polymorphisms in mouse and man; read-out of class II promoter function; and a comparison with expression of MHC class I. We conclude that the evidence that this variation is functionally active (i.e. controls expression) is increasing, but is not yet compelling. The crucial test still to come, we suggest, is whether or not the biological effects attributable to this polymorphism will line up with molecular studies on expression.

APC gene mutations and allelic losses in sporadic ampullary tumours: evidence of genetic difference from tumours associated with familial adenomatous polyposis.

We explored APC gene mutations and chromosome 5q21 allelic losses (5qLOH) in 18 neoplasms of the papilla of Vater, including 6 early-stage tumours (3 adenomas, 3 carcinomas) and 12 advanced-stage cancers. Eleven PCR-amplified polymorphic sequences were used to analyse 5qLOH. APC mutations were investigated both by an in vitro APC-protein truncation test and by single-strand conformation polymorphism analysis. Mutations in the Ki-ras, N-ras and p53 genes were also assessed. We found: 5qLOH in 8 of 16 cases (50%), including 1 adenoma, 3 early- and 4 advanced-stage cancers; APC mutations in 2 adenomas and 1 advanced-stage carcinoma; Ki- or N-ras mutations in 3 adenomas and 3 advanced-stage cancers; p53 mutations in 2 early-stage and 7 advanced-stage adenocarcinomas. Our results suggest that 5qLOH, APC mutations and ras mutations are present at early stages, whereas p53 inactivation is associated with progression of malignancy in a large proportion of cases. These data indicate that sporadic ampullary tumours differ from those occurring in familial adenomatous polyposis in the frequency (17% vs. 64%) as well as in the site of APC somatic mutations, suggesting a different molecular pathogenesis in the 2 conditions.

Expression of MHC class I and class II antigens in pancreatic adenocarcinomas.

The antigens encoded by the major histocompatibility complex (MHC) are cell surface glycoproteins that play a fundamental role in the regulation of the immune response. Anomalous MHC expression in tumor cells has been viewed as an important feature to escape tumor recognition by immune cells. Low or absent MHC class I expression as well as ectopic MHC class II expression have been often observed to correlate with high grade malignancy and metastatic potential in a variety of human cancers. To date, very little investigation of MHC (HLA in man) class I and class II expression in human pancreatic cancer has been reported. We investigated this aspect on frozen sections of 8 pancreatic adenocarcinomas and 18 established in vitro cell lines. HLA class I was expressed in all but two cancers whereas de novo HLA class II expression was detected in 3 of 8 cancers. Interestingly, a hierarchy in the expression of the various subsets of HLA class II was found with HLA- DR > -DP > -DQ. Results on cell lines strongly resembled the ones obtained in cancer tissues. However, a peculiar feature was observed in certain cell lines. HLA class II antigens were expressed in only a few cell lines and in some of them a mixed population of positive and negative cells was found. Sorting and cloning of the two populations confirmed the existence of tumor cell clones with stable and distinct HLA class II phenotype. Taken together, these results indicate the cellular heterogeneity of pancreatic cancer cells with regard to the qualitative and quantitative expression of major histocompatibility complex genes, and may provide new insights for a better understanding of the tumorhost relationships in this extremely severe form of neoplasia.