The Influence of Cytomegalovirus on Expression of HLA-G and its Ligand KIR2DL4 by Human Peripheral Blood Leucocyte Subsets.

HLA-G is a non-classical class I HLA antigen, normally expressed in high levels only on extravillous cytotrophoblast. It has immunosuppressive properties in pregnancy and has also been found to be upregulated on leucocytes in viral infection. In this study, proportions of all leucocyte subsets expressing HLA-G were found to be low in healthy subjects positive or negative for cytomegalovirus (CMV). Significantly greater proportions of CD4+ CD69+ and CD56+ T cells expressed HLA-G compared to other T cells. However, following stimulation with CMV antigens or intact CMV, proportions of CD4+, CD8+, CD69+ and CD56+ T cells, and also B cells expressing HLA-G, were significantly increased in CMV+ subjects. Despite some subjects having alleles of HLA-G associated with high levels of expression, no relationship was found between HLA-G genotype and expression levels. Purified B cells from CMV+ subjects stimulated in mixed culture with CMV antigens showed significantly increased HLA-G mRNA expression by real-time polymerase chain reaction. Serum levels of soluble HLA-G were similar in CMV- and CMV+ subjects but levels in culture supernatants were significantly higher in cells from CMV+ than from CMV- subjects stimulated with CMV antigens. The HLA-G ligand KIR2DL4 was mainly expressed on NK cells and CD56+ T cells with no differences between CMV+ and CMV- subjects. Following stimulation with IL-2, an increase in the proportion of CD56+ T cells positive for KIR2DL4 was found, together with a significant decrease in CD56dimCD16+ NK cells. The results show that CMV influences HLA-G expression in healthy subjects and may contribute to viral immune evasion.

Weakly acidic pH reduces inflammatory cytokine expression in airway epithelial cells.

BACKGROUND: Aspiration lung disease (ALD) is a common cause of respiratory morbidity in children and adults with severe neurodisability (sND). Recent studies suggest that chronic microaspiration of gastric contents is associated with mild rather than low, airway acidification. We investigated inflammatory responses to infection by airway epithelial cells (AECs) exposed to weakly acidic media. METHODS: Using pH measurements from children with sND at high risk of ALD as a guide, we incubated AECs in weakly acidic (pH5.5-7.4) media alone; in combination with lipopolysaccharide (LPS); or prior to LPS stimulation at normal pH. Interleukin (IL) -6 and IL-8 expression were measured. RESULTS: IL-6/8 expression in AECs simultaneously exposed to weakly acidic media and LPS for 4 h was reduced with no effect on cell viability. Pre-incubation of AECs at weakly acidic pH also reduced subsequent LPS-induced cytokine expression. Suppression of inflammation was greatest at lower pHs (pH 5.5-6.0) for prolonged periods (16/24 h), but this also adversely affected cell viability. CONCLUSION: AEC inflammatory responses to bacterial stimuli is markedly reduced in a mildly acidic environment.

Respiratory syncytial virus infection of airway epithelial cells, in vivo and in vitro, supports pulmonary antibody responses by inducing expression of the B cell differentiation factor BAFF.

BACKGROUND: The mechanisms regulating antibody expression within the human lung during airway infection are largely unknown. In this study, our objectives were to determine if infection with respiratory syncytial virus (RSV) upregulates expression of the B cell differentiation factors A proliferation inducing ligand (APRIL) and B cell activating factor of the TNF family (BAFF), if this is a common feature of viral airway infection, and how this is regulated in human airway epithelial cells. METHODS: We measured BAFF and APRIL protein expression in bronchoalveolar lavage (BAL) fluid from infants with severe RSV disease, and healthy control children, and in nasopharyngeal aspirates from preschool children with other single respiratory viral infections. We also measured mRNA expression in bronchial brushings from RSV-infected infants, and in RSV-infected paediatric primary airway epithelial cell cultures (pAEC). Beas-2B cell cultures were used to examine mechanisms regulating BAFF expression. RESULTS: BAFF protein and mRNA were elevated (in marked contrast with APRIL) in BAL and bronchial brushings, respectively, from RSV-infected infants. BAFF protein was also found in upper airway secretions from children with human metapneumovirus, H1N1, bocavirus, rhinovirus, RSV and Mycoplasma pneumoniae infection. BAFF mRNA and protein were expressed following in vitro RSV infection of both pAEC and Beas-2B cultures, with mRNA expression peaking 12-h postinfection. BAFF induction was blocked by addition of a neutralising anti-interferon-ß antibody or palivizumab. CONCLUSIONS: BAFF, produced through an interferon-ß-dependent process, is a consistent feature of airway infection, and suggests a role for the airway epithelia in supporting protective antibody and B cell responses in the lung.

Primary airway epithelial cultures from children are highly permissive to respiratory syncytial virus infection.

BACKGROUND: Respiratory syncytial virus (RSV) infection of airway epithelial cells (AECs) is an important initial event in RSV bronchiolitis. AEC immunological responses are thought to be critical in driving the subsequent inflammation in the airway. This study examined viral replication, cytotoxicity and cytokine production in cultures of primary AECs from children compared with responses to RSV infection in an immortalised epithelial cell line and to those from infants with RSV bronchiolitis. METHODS: RSV replication, proinflammatory cytokine responses and cytotoxicity in RSV-infected primary AEC cultures derived from bronchial brushings from the lungs of children were compared with those seen in BEAS-2B cultures, as well as AECs and bronchoalveolar lavage fluid collected from children with and without RSV bronchiolitis. RESULTS: Viral replication, cytotoxicity and inflammatory cytokine production were greater in primary AEC cultures than in BEAS-2B cells. Different response patterns were observed, with RSV infection of primary AEC cultures causing distinct peaks of viral replication and matched cytotoxic responses. Some primary AEC culture immunological responses, such as interleukin 8, were similar in magnitude to those seen in clinical samples from the lungs of children with RSV bronchiolitis. Although variable amounts of RSV were detected by PCR in freshly isolated primary AECs, RSV was not detected by immunocytochemistry. CONCLUSION: This is one of the first studies to examine comprehensively the responses to RSV infection in primary AEC cultures from children and shows marked differences from those of a commercially available immortalised human cell line but reassuring similarities to results found in vivo. This suggests that future work investigating responses of AECs to RSV infection should use primary AEC cultures.

Neutrophil TLR4 expression is reduced in the airways of infants with severe bronchiolitis.

BACKGROUND: In respiratory syncytial virus (RSV) bronchiolitis, neutrophils account for >80% of cells recovered from the airways in bronchoalveolar lavage (BAL) fluid. This study investigated neutrophil activation and Toll-like receptor (TLR) expression in the blood and lungs of infants with severe RSV bronchiolitis. METHODS: BAL fluid and (blood) samples were collected from 24 (16) preterm and 23 (15) term infants ventilated with RSV bronchiolitis, and 12 (8) control infants. Protein levels and mRNA expression of CD11b, myeloperoxidase (MPO) and TLRs 2, 4, 7, 8 and 9 were measured in neutrophils. RESULTS: Blood neutrophils had more CD11b in preterm and term infants with RSV bronchiolitis than control infants (p<0.025) but similar amounts of MPO. BAL fluid neutrophils from infants with RSV bronchiolitis had greater amounts of CD11b and MPO than blood neutrophils and BAL fluid neutrophils from controls (p<0.01). Blood neutrophils from term infants with RSV bronchiolitis had less total TLR4 protein than preterm infants with RSV bronchiolitis (p = 0.005), and both had less than controls (p<0.04). Total TLR4 for each group was greater in BAL fluid neutrophils than in blood neutrophils. Blood neutrophils from preterm infants with RSV bronchiolitis had greater TLR4 mRNA expression than term infants with RSV bronchiolitis (p = 0.005) who had similar expression to controls (p = 0.625). CONCLUSIONS: In infants with severe RSV bronchiolitis, neutrophil activation starts in the blood and progresses as they are recruited into the airways. Total neutrophil TLR4 remains low in both compartments. TLR4 mRNA expression is unimpaired. This suggests that neutrophil TLR4 expression is deficient in these infants, which may explain why they develop severe RSV bronchiolitis.

The complement regulatory proteins CD55 (decay accelerating factor) and CD59 are expressed on the inner acrosomal membrane of human spermatozoa as well as CD46 (membrane cofactor protein).

The complement regulatory proteins CD55 and CD59 are expressed on the plasma membrane of human spermatozoa, whereas CD46 is only on the inner acrosomal membrane (IAM) which becomes surfaced exposed after the acrosome reaction when sperm assume fertilisation-competence. CD55 & CD59, two glycosylphosphatidylinositol (GPI)-anchored proteins, have been detected previously in some studies also in the acrosomal region of chemically fixed spermatozoa but never demonstrated at this site on unfixed spermatozoa. Dual labelling immunofluorescence and confocal microscopy on fresh unfixed spermatozoa, with minimal subsequent time to fixation, has shown CD55 to be markedly expressed on the IAM, more than on the plasma membrane. However, unlike for CD46, CD55 displayed patchy staining over the acrosome, with some variation between individual spermatozoa. All IAM-associated CD55 was localised within GM1-containing lipid rafts. CD59 was expressed also on the IAM, but in a pronounced granular pattern with more variation observed from one spermatozoa to another. Both CD55 & CD59 were released from the IAM by PI-PLC, demonstrating them to be GPI-anchored. Analysis of acrosome-reacted spermatozoal CD55 by Western blotting revealed a novel single 55 kDa protein lacking significant oligosaccharides susceptible to glycosidases. Antibody-induced membrane rafting and release of CD55 & CD59 in vitro may have influenced previous results. Significant coexpression of CD55 & CD46 on the IAM suggests some functional cooperation at this site.

Flow cytometric measurement of phagocytosis reveals a role for C3b in metal particle uptake by phagocytes.

A methodology for the quick and efficient study of phagocytosis has been developed. It uses the flow cytometer to exploit the change in size and granularity that occurs in cells upon the ingestion of particulate material. The numbers of cells that have phagocytosed particles can be calculated from the distinct shift in regions that occurs. The method also allows the factors governing phagocytosis to be studied in detail through the use of blocking agents or antibodies. Blood-derived monocytes were studied to investigate the role of complement in metal particle phagocytosis to further understand aseptic loosening. Factor C3b was found to be fundamental to the opsonization and phagocytosis of metal particles by monocytes.

Differential regulation of mast cell cytokines by both dexamethasone and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580.

Activated mast cells generate multiple cytokines but it is not known if these can be differentially regulated by pharmacological agents. We report here that the glucocorticoid dexamethasone (DEX) preferentially inhibited Ag-induced expression of IL-4 and IL-6 mRNA relative to TNF-alpha mRNA in RBL-2H3 cells. Likewise, the drug more readily inhibited release of IL-4 than TNF-alpha protein. SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK), enhanced Ag-induced TNF-alpha mRNA expression without affecting IL-4 or IL-6 mRNA. At the protein level, SB203580 exerted little effect on TNF-alpha release but inhibited IL-4 release; notably, the ratio of TNF-alpha : IL-4 increased markedly with the concentration of SB203580, confirming the differential regulation of these cytokines. PD98059, an inhibitor of MAPK kinase (MEK), a component of the p44/42 MAPK pathway, partially inhibited Ag-induced expression of mRNA for all three cytokines while cyclosporin A inhibited Ag-induced IL-4 and IL-6 mRNA more readily than TNF-alpha mRNA. Ag activation of the cells led to phosphorylation of p38 and p44/42 MAPK but this was not influenced by DEX. In conclusion, mast cell cytokines can be differentially regulated pre- and post-translationally by DEX and SB203580 but there does not appear to be a direct mechanistic link between the actions of these two drugs.

Pro- and anti-inflammatory responses in respiratory syncytial virus bronchiolitis.

Respiratory syncytial virus (RSV) bronchiolitis is an important cause of severe respiratory disease in infants. This study aimed to characterise changes in pulmonary pro- and anti-inflammatory responses in infants with RSV bronchiolitis over the course of the illness. On the day of intubation (Day 1) and the day of extubation (Day X), nonbronchoscopic bronchoalveolar lavage was performed on term and preterm infants ventilated for RSV bronchiolitis and on control infants on Day 1. Tumour necrosis factor (TNF)-alpha, soluble TNF receptor (sTNFR) and interleukin (IL)-6 messenger ribonucleic acid (mRNA) and protein were measured. Twenty-four infants, born at term and 23 infants born preterm with RSV bronchiolitis and 10 controls were recruited. TNF-alpha and IL-6 mRNA and protein in infants with bronchiolitis were greater than the control group on Day 1. In preterm infants, who were ventilated for longer than term infants, TNF-alpha and IL-6 proteins decreased between Day 1 and Day X. Concentrations of sTNFRs differed between groups on Day 1, but levels did not change between Day 1 and Day X. Large amounts of tumour necrosis factor-alpha and interleukin-6 in the respiratory syncytial virus-infected lung suggest important roles for these cytokines in the pathogenesis of respiratory syncytial virus bronchiolitis. The decrease in tumour necrosis factor-alpha and interleukin-6 protein in preterm infants may reflect the prolonged clinical course seen in these infants.

The ratio of 2nd to 4th digit length: a proxy for transactivation activity of the androgen receptor gene?

The androgen receptor gene (AR) contains a domain which includes a variable number of CAG sequences and alleles with low numbers of CAG repeats show high transactivation activity when complexed with testosterone. The ratio of 2nd and 4th digit length (2D:4D) is negatively correlated with phenotypic effects of testosterone. Low numbers of CAG repeats and low 2D:4D are both associated with high sperm numbers and protection against breast cancer. This suggests that CAG number and 2D:4D are correlated i.e. low CAG number and low 2D:4D indicate high activation of androgen-responsive genes. Findings from AR studies predict that low 2D:4D will be associated with prostate and hepatocellular cancer, urolithiasis, ADHD, ankylosing spondylitis, spontaneous abortion, and polycystic ovaries, while high 2D:4D will be associated with motor neuron diseases and endometrial cancer. Findings from 2D:4D studies predict that short CAG length will be common in autism and Asperger's syndrome, while high numbers of CAG repeats will be found in men who are prone to early myocardial infarction.

A study of tissue interface membranes from revision accord knee arthroplasty: the role of T lymphocytes.

Despite four decades of advances in the design of orthopaedic devices aseptic loosening remains a major cause for the revision of total joint arthroplasty. This study used the techniques of immunohistochemistry and reverse transcription polymerase chain reaction to identify the inflammatory cell types, cytokines and chemokines within the interface tissue surrounding failed Accord Knee prostheses. Many T cells were identified within the tissue: however, the classical marker of activation, CD25 was expressed on very few cells. Molecular analysis failed to detect the synthesis of either Th1 or Th2 cytokines. These results suggest that the T cells are being actively recruited to the site of inflammation along the chemokine gradients but are not participating in a classical immune response.

Penicillin conjugates to interferon-gamma and reduces its activity: a novel drug-cytokine interaction.

Beta-lactam antibiotics are the class of drug most frequently associated with IgE-mediated allergy but the mechanisms underlying this response are poorly understood. IFN-gamma is a key cytokine in immunity with regulatory actions on monocytes, NK cells, epithelial cells, and T and B lymphocytes. IFN-gamma promotes Th1 responses and inhibits Th2- and IgE-mediated responses. In this study we show, by Western blotting, that the prototype beta-lactam benzylpenicillin (BP) conjugates to human IFN-gamma but not to IL-4. The interaction of BP with IFN-gamma inhibited the cytokine's detection by immunoassay and impaired its activity, as assessed in three different assays: upregulation of MHC molecules on monocytes plus induction of nitric oxide synthesis and expression of monocyte chemoattractant protein-1 mRNA by epithelial cells. This is the first reported example of a direct drug-cytokine interaction and suggests a mechanism by which penicillin may disrupt IFN-gamma-dependent immune responses and promote allergy.

Eotaxin and eotaxin receptor (CCR3) expression in Sephadex particle-induced rat lung inflammation.

The beta chemokine eotaxin is a potent eosinophil activator and chemoattractant. We examined immunohistochemically eotaxin protein expression in a range of normal rat tissues and in rat lung during Sephadex particle-induced pulmonary inflammation. The time course of eotaxin expression in lung at various time points after Sephadex administration was related to the appearance of eosinophils in the bronchoalveolar lavage fluid and tissue distribution of eotaxin receptor (CCR3) positive cells. Results showed that eotaxin protein was constitutively expressed by both lung airway epithelial cells and gut epithelial cells in normal tissues in the absence of inflammation. During Sephadex induced pulmonary inflammation, eotaxin expression increased in alveolar macrophages prior to the major increase in eosinophil numbers which reached a peak at 72 h. The pattern of eotaxin pulmonary expression and the location of CCR3 receptor positive cells suggest a chemoattractant gradient resulting in migration firstly into the tissue and subsequently through the airway epithelium into the airways. Treatment of rats with the glucocorticoid dexamethasone or the immunosuppressant cyclosporin A reduced eosinophil entry into lung tissue and airways but had no apparent effect on eotaxin expression in vivo, indicating that both these drugs inhibit eosinophil recruitment either by an eotaxin-independent mechanism, or by targetting factors that synergise with eotaxin, or an event post eotaxin expression.

Human decidualized endometrial T lymphocytes do not substantially down-regulate CD3zeta.

PROBLEM: The T-cell antigen receptor (TCR) has been reported to be down-regulated on T-cells in the decidualized endometrium in early pregnancy. METHOD OF STUDY: The expression of CD3zeta, a component of the TCR complex, has been investigated in human first-trimester decidual T-cells using flow cytometric analysis of permeabilized cells. RESULTS: Levels of CD3zeta expression were significantly lower in decidual than in peripheral T-cells from non-pregnant women, as assessed by mean fluorescence intensity (4.2 vs. 5.5, logarithmic scale, P < 0.05). However, when decidual and peripheral T-cells from the same subjects were analyzed (n = 10), mean levels of CD3zeta were slightly, but not significantly, lower in decidual than in peripheral T-cells (P > 0.1). CD3zeta was not substantially down-regulated systemically as mean cytoplasmic CD3zeta levels did not differ significantly between peripheral blood T-cells from pregnant women and non-pregnant controls (P > 0.2). CD8+ cells outnumber CD4+ cells in decidua, but neither the proportions of these two T-cell subsets positive for cytoplasmic CD3zeta nor the mean levels of CD3zeta were significantly different. CONCLUSIONS: These results indicate that human decidual T-cells do not greatly down-regulate CD3zeta, but it is unclear if a small decrease in mean levels may be sufficient to compromise their capacity for activation.

Molecular characterisation of a putative endogenous retrovirus cDNA isolated from human placental tissue.

The human genome comprises of abundant DNA sequences related to endogenous retroviruses (ERV) and a variety of solitary long terminal repeats (LTRs). Substantial numbers of intact retroviral particles have been detected by electron microscopy in normal human placental villous tissue particularly in syncytiotrophoblast. Understanding the molecular structure, organisation and distribution of these ERV sequences may lead to elucidation of their possible dual function at the foetal-maternal interface; proliferation and differentiation of cytotrophoblast and induction of local pregnancy-associated immune suppression thus allowing survival of the foetal allograft. In this study, antibody probes were used to screen a human placental expression library and cDNA clones isolated were characterized by polymerase chain reaction, Southern blot hybridisation, DNA cloning and partial nucleotide sequencing. A specific 1.7kb-cDNA clone was isolated from a human placental expression library. Further characterisation showed this clone represents a single copy gene, approximately 9-10kb and did not hybridise to the env region of ERV3 human endogenous retrovirus. The 1.7kb-cDNA clone may represent a provirus co-expressed with cellular sequences.

Molecular Characterisation of a Putative Endogenous Retrovirus cDNA Isolated from Human Placental Tissue

The human genome comprises of abundant DNA sequences related to endogenous retroviruses (ERV) and a variety of solitary long terminal repeats (LTRs). Substantial numbers of intact retroviral particles have been detected by electron microscopy in normal human placental villous tissue particularly in syncytiotrophoblast. Understanding the molecular structure; organisation and distribution of these ERV sequences may lead to elucidation of their possible dual function at the foetal-maternal interface; proliferation and differentiation of cytotrophoblast and induction of local pregnancy-associated immune suppression thus allowing survival of the foetal allograft. In this study; antibody probes were used to screen a human placental expression library and cDNA clones isolated were characterized by polymerase chain reaction; Southern blot hybridisation; DNA cloning and partial nucleotide sequencing. A specific 1.7kb-cDNA clone was isolated from a human placental expression library. Further characterisation showed this clone represents a single copy gene; approximately 9-10kb and did not hybridise to the env region of ERV3 human endogenous retrovirus. The 1.7kb-cDNA clone may represent a provirus co-expressed with cellular sequences

A novel isoform of human membrane cofactor protein (CD46) mRNA generated by intron retention.

The reverse transcription polymerase chain reaction (RT-PCR) with primers specific for each of the 14 exons of the human complement regulatory protein membrane cofactor protein (MCP;CD46) has been utilized to determine MCP mRNA transcript expression in peripheral blood mononuclear cells (PBMC). An additional transcript of a larger size than predicted was consistently detected in reactions with a sense primer for exon 7, that encodes the first alternatively spliced serine-threonine-rich region (ST-A), together with an antisense exon 12 primer, RT-PCR with primers for other exons both 5' and 3' of exon 7 further showed that these MCP transcripts contain additional sequences immediately both 5' and 3' to the exon 7-encoded sequence. Comparison of genomic DNA with cDNA by PCR, in combination with sequence analysis, demonstrated the presence of the complete invariant sequences of both introns adjacent to exon 7, i.e. intron 6 (411 bp) and intron 7 (127 bp). RT-PCR using primers specific for the intron 6 sequence, together with Southern and Northern blotting using an intron 6-specific probe, confirmed retention of this intron within a novel 4.8-kb mRNA transcript in human PBMC. Due to the presence of a stop codon within intron 6, translation would result in a novel truncated MCP isoform (MCPi) containing the four invariant short consensus repeat (SCR) regions and a unique C-terminal 39 amino acid transmembrane and cytoplasmic tail region that may promote endoplasmic reticulum retention.

TNFB gene polymorphism in insulin-dependent diabetes mellitus: association with HLA-DR alleles.

A polymorphism of the TNF-beta gene can be detected by restriction digestion of a PCR product with NcoI. In this study we look at the risk associated with this polymorphism in a study of 69 insulin-dependent diabetes patients and 119 healthy controls. The risk was further characterized by comparison to the HLA type of the individual, since the TNF polymorphism is in linkage disequilibrium with HLA genes.

Human eotaxin induces alpha 4 and beta 2 integrin-dependent eosinophil accumulation in rat skin in vivo: delayed generation of eotaxin in response to IL-4.

The CC chemokine eotaxin, originally purified from bronchoalveolar lavage fluid of sensitized guinea pigs following allergen challenge, is a potent eosinophil-selective chemoattractant. In the present study, we have used (111)In-eosinophils and human eotaxin to characterize the profile of chemokine-induced eosinophil accumulation in vivo in rat skin. Intradermally injected eotaxin caused a dose-dependent accumulation of (111)In-eosinophils. Time course studies indicated that the response was rapid, since all the accumulation occurred within the first 1 to 2 h of eotaxin injection. The i.v. administration of anti-intercellular adhesion molecule-1, anti-vascular cell adhesion molecule-1, or anti-alpha4 integrin mAbs significantly inhibited the eosinophil accumulation induced by 100 pmol of human eotaxin by 73, 43, and 67%, respectively. Further, when (111)In-eosinophils were pretreated in vitro with anti-alpha4 integrin or anti-beta2 integrin mAbs, or with a combination of both mAbs, eotaxin-induced responses in vivo were reduced by 52, 49, and 68%, respectively. Eosinophil accumulation induced by intradermal IL-4, but not that induced by TNF-alpha or leukotriene B4, appeared to be mediated in part by endogenously generated eotaxin. Anti-eotaxin Abs significantly inhibited (54%) the later phases (24-28 h) but not the early phase (0-4 h) of the response to IL-4. This was consistent with eotaxin mRNA expression peaking at 18 h after IL-4 injection. Our findings show that human eotaxin is a potent inducer of eosinophil accumulation in vivo, this response being dependent on alpha4 integrin/vascular cell adhesion molecule-1 and beta2 integrin/intercellular adhesion molecule-1 adhesion pathways. Further, the eosinophil accumulation in response to IL-4 is partly mediated by endogenously generated eotaxin.