bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytotoxic activity of Vβ8+ T cells in Crohn's disease: the role of bacterial superantigens
Authors: Baca-Estrada M E , Wong D K H , Croitoru K
Published: July 2010

In Crohn's disease, disease-related stimuli could alter the T cell receptor (TCR) repertoire. To examine the possibility that changes in function may occur in T cell subsets without obvious changes in expression of TCR, we analysed the TCR repertoire of cytotoxic T lymphocytes in Crohn's disease peripheral blood. Furthermore, we examined the effect of bacterial superantigens, staphylococcal enterotoxin B (SEB) and E (SEE) on the cytotoxic function of T cell subsets bearing different TCR V genes using MoAbs specific for CD3 and TCR V gene products in a redirected cytotoxicity assay. There was no difference between patients and controls in the cytotoxicity measured in concanavalin A (Con A)-stimulated peripheral blood mononuclear cells (PBMC) with anti-CD3 or with six of seven anti-TCR V gene MoAbs. However, the cytotoxicity of Vβ8 T cells was decreased in Crohn's disease patients. This was not due to a decrease in total or CD8+ T cells expressing Vβ8. Furthermore, in normal subjects, PBMC stimulation with SEE and SEB selectively expanded and increased the cytotoxicity of Vβ8 and Vβ12 T cells, respectively. In Crohn's disease, although SEB stimulation increased the number and cytolytic function of the Vβ12 subset, SEE stimulation failed to increase cytolytic activity of Vβ8+ T cells in spite of the expansion of Vβ8+ T cells. These results suggest that the changes in cytotoxic function observed in Vβ8 T cells in Crohn's patients may reflect previous exposure to a Vβ8-selective superantigen.
Antibodies to theCryptococcus neoformanscapsular glucuronoxylomannan are ubiquitous in serum from HIV+ and HIV− individuals
Authors: Deshaw M , Pirofski L-A
Published: July 2010

Murine MoAbs to the Cryptococcus neoformans capsular glucuronoxylomannan (GXM) polysaccharide are protective in mice in vivo and in vitro. The prevalence of protective anti-GXM antibodies in human serum is unknown. To provide further insight into the human antibody response to C. neoformans we determined the prevalence, isotype, and IgG subclass utilization of human anti-GXM antibodies in HIV+ and HIV− sera by a sensitive antigen capture ELISA assay. One hundred and twenty-three sera from the Bronx Municipal Hospital Centre serum bank were studied retrospectively. Seventy were from HIV+ individuals, 10 with a history of cryptococcal meningitis (CM), and 53 were from HIV− individuals. Serum GXM determinations were also performed on 61 HIV+ sera. Our results demonstrated that anti-GXM IgG, IgA, and IgM are ubiquitous in both HIV+ (including those with CM), and HIV− sera. Anti-GXM IgA titres and total serum IgA concentration were elevated in HIV+ sera. Anti-GXM IgG antibodies were almost exclusively isotype-restricted to the IgG2 subclass. Our data also demonstrated elevations of anti-bovine serum albumin (BSA) titres in HIV+ sera. Taken together, our findings confirm hypergammaglobulinaemia and expansion of anti-protein (BSA) antibodies in HIV+ individuals and isotype restriction of human anti-carbohydrate (GXM) antibodies to the IgG2 subclass. Our report of ubiquitous anti-GXM antibodies of the IgG and IgA isotypes suggests that anti-GXM antibodies exist before HIV infection.
Anti-inflammatory activity of salmeterol: down-regulation of cytokine production
Authors: Sekut L , Champion B R , Page K , Jr J A MENIUS , Connolly K M
Published: July 2010

Elevation of intracellular cAMP levels has been shown previously to inhibit cytokine secretion by various cell types in vitro. Since salmeterol is a β2-agonist which activates adenylate cyclase, its ability to inhibit cytokine production was evaluated. Though salmeterol, and the related drug albuterol, did not inhibit IL-1β production in vitro, both drugs did inhibit tumour necrosis factor-alpha (TNF-α) secretion by lipopolysaccharide (LPS)-activated THP-1 cells with similar IC50S of approximately 0·1μm. This inhibition was effectively reversed by the β2-antagonist oxprenolol, indicating that the inhibition was mediated through the β2-adrenergic receptor. A strikingly different reactivity profile was seen with T cells. Salmeterol was able to inhibit the activation of both mouse and human T cells, as measured by proliferation and IL-2 secretion in response to anti-CD3 antibody, whereas albuterol was completely inactive in these assays. This T cell inhibition by salmeterol was about 10-fold less potent than that for TNF-α production, and was not reversed by a β2-antagonist, indicating that a different mechanism was involved in the effect of salmeterol on T cells. Paralleling the TNF-a inhibitory activity in vitro, oral dosing of salmeterol and albuterol inhibited LPS-induced increase in murine serum TNF level in vivo, with ED50s of approximately 0·1 mg/kg. This inhibition could be abrogated by dosing orally with the β-blocker propranolol. The long-acting pharmacological profile of salmeterol was apparent in that it maintained its efficacy for 3h, while albuterol had a much shorter duration of action. Salmeterol also had some protective effects in the galactosamine/LPS model of endotoxic shock, which is dependent upon TNF-α production. Though salmeterol inhibited serum TNF-α levels by up to 94% in this assay, it protected less than 50% of the animals from the lethal effects of the LPS/galactosamine mixture. This observation suggests that functional levels of TNF-α localized in tissues may not be accurately reflected by serum levels.
Co-Stimulation of T cells via CD28 inhibits human IgE production; reversal by Pertussis toxin
Authors: Van Der Pouw-Kraan C T M , Rensink H J A M , Rappuoli R , Aarden L A
Published: July 2010

In lymphocyte cultures, IgE production was achieved by stimulating T cells with anti-CD2 and IL-2. Here we show that anti-CD28, in the presence or absence of IL-2, reduces this IgE production approximately 10-fold. This inhibition of IgE production was almost completely reversed by Pertussis toxin (PT). PT had no effect on IgE production when the cells were stimulated in the absence of anti-CD28. No major effects of PT were found on IgM production. PT had no effect on purified B cells, stimulated with IL-4 and anti-CD40. In the presence of saturating amounts of rIL-4 similar results were obtained, albeit the absolute amounts of IgE produced were higher in all situations. Furthermore, PT-induced IgE production was still dependent on IL-4, as was evident from experiments in which anti-IL-4 was added to the culture. The IgE enhancing effect was dependent on the adenosine diphosphate (ADP)-ribosyltransferase activity of PT, because a mutant molecule lacking this activity was not able to restore anti-CD28-induced inhibition of IgE synthesis. Thus, we show that co-stimulation with anti-CD28 causes an inhibition of T cell-dependent IgE production by B cells, which inhibition can be specifically overcome by PT. An analysis of the molecular pathways underlying this phenomenon may contribute to our understanding of the regulation of IgE synthesis in (patho)physiological conditions.
Animal models in ANCA-vasculitis
Authors: Kettritz R , Yang Jia Jin , Kinjoh K , Jennette J C , Falk R J
Published: July 2010

The animal models described to date suggest pathogenicity of ANCA. Unfortunately, none of the models unequivocally proves that ANCA are pathogenic. Further search for a suitable animal model to document or exclude causal significance of ANCA in vasculitis is needed.
Expression of IL-10, IL-4 and interferon-gamma in unstimulated and mitogen-stimulated peripheral blood lymphocytes from HIV-seropositive patients
Authors: Diaz-Mitoma F , Kumar A , Karimi S , Kryworuchko M , Daftarian M P , Creery W D , Filion L G , Cameron W
Published: July 2010

Infection of immune cells with HIV induces dysregulation of cytokines which may play a vital role in HIV pathogenesis. We analysed the expression of T helper type I (Th1) (interferon-gamma (IFN-γ)) and Th2 (IL-4, IL-10) type cytokines in peripheral blood lymphocytes (PBL) from HIV patients. The semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that IFN-γ mRNA in unstimulated PBL was significantly decreased and IL-10 mRNA was significantly upregulated in patients with < 400 CD4+ T cells/mm3 (n= 30) as compared to patients with > 400 CD4+ T cells/mm3 (n= 6) and normal controls (n= 16). In addition. IL-10 mRNA levels were inversely associated with IFN-γ expression. Similar results were obtained by measuring IL-10 production in the supernatants of PBL cultured in vitro without stimulation by employing an enzyme immunosorbent assay (ELISA). However, the levels of IL-4 and IFN-7 produced by unstimulated PBL were undetectable by ELISA. Mitogen stimulation of PBL revealed two groups of HIV individuals based on IL-10 production. PBL from one set of individuals produced low levels of IL-10 (low IL-10 producers) whereas the other group produced IL-10 comparable lo that of normal controls (IL-10 producers). Production of IL-4 was significantly reduced in HIV+ individuals with<400 CD4+ T cells/mm3 as compared to the normal controls. However, ability to produce IFN-γ by mitogen-stimulated total PBL and CD4+ purified cells was not impaired in HIV+ individuals. These results suggest that unstimulaied and mitogen-stimulated PBL of HIV+ individuals exhibit dysregulation of Th2 type cytokines which may play a role in HIV immunopathogenesis.
Demonstration of virus-specific CD8+ memory T cells in measles-seropositive individuals by in vitro peptide stimulation
Authors: Nanan R , Carstens C , Kreth H W
Published: July 2010

CD8+ cytotoxic T lymphocytes (CTL) in measles virus infection have been difficult to investigate due to the strong immunosuppressive effects exhibited by infectious measles virus in vitro. In order to circumvent immunosuppression we used predicted peptide epitopes to induce measles virus-specific CTL, This was done by screening the structural proteins of measles virus for HLA-A2.1 peptide-binding motifs with valine in position 2 and leucine in position 9. Synthetic peptides np210 218. np226-234, and np340-348 from the nucleoprotein, peptide hp29-37 from the haemagglutinin protein, and peptide pp 519-527 from the polymerase protein were synthesized and used to expand measles virus-specific CD8+ CTL in vitro. Induction of CTL with synthetic peptides was restricted to HLA-A2-positive peripheral blood mononuclear cells (PBMC) from measles-seropositive individuals. We conclude that this method is a useful tool to demonstrate memory CD8+ CTL in measles-seropositive adults and to evaluate the role of structural proteins in CTL responses against measles.
T cell clones from a non-leprosy exposed subject recognize theMycobacterium leprae18-kD protein
Authors: Adams E , Basten A , Prestidge R , Britton W J
Published: July 2010

Although Mycobacterium leprae shares many protein antigens with other mycobacterial species, there is a degree of specificity in the T cell response to the organism. This is evident in the failure of cross-protection between mycobacterial species and the specific unresponsiveness to M. leprae in lepromatous leprosy patients. The antigenic basis of this specificity is unresolved, but the M. leprae 18-kD protein is one candidate because of its restricted distribution and the isolation of M. leprae-specific T cell clones reactive with the protein from M. leprae-vaccinated subjects. In the course of analysing the human T cell repertoire to mycobacteria we have isolated further CD4+ T cell clones reactive with this protein from a subject who had never been exposed to M. leprae. These clones did not respond to other mycobacteria, including M. tuberculosis and M. bovis (BCG). In addition, they were unreactive with the M. tuberculosis 16-kD protein which has recently been shown to have limited amino acid identity with the M. leprae 18-kD protein. Both clones reacted with peptide 38 50 from the M. leprae 18-kD protein, the T cell response to which a restricted by HLA-DR4. Although homologues for the gene encoding the M. leprae 18-kD antigen have been identified in M. avium and M. intracellulare, the clones failed to respond to preparations of M. avium. Both clones secreted interferon-gamma (IFN-γ) and tumour necrosis factor-beta (TNF-β) and were cytolytic against autologous targets pulsed with peptide 38-50 or the 18-kD protein. The nature of the antigen which stimulates this apparently “M. leprae-specific” response is unknown. Nevertheless the recognition of the 18-kD protein by individuals not exposed to leprosy indicates that this protein may not be suitable as a reagent to distinguish between infection with M. leprae and other pathogenic mycobacteria.
Cysteine protease of the nematodeNippostrongylus brasiliensispreferentially evokes an IgE/IgG1 antibody response in rats
Authors: Kamata I , Yamada M , Uchikawa R , Matsuda S , Arizono N
Published: July 2010

Some cysteine proteases such as papain and those of mites and schistosomes have potent allergenic properties. To clarify the allergenicity of nematode cysteine proteases, the enzyme was purified from the intestinal nematode Nippostrongylus brasiliensis using cation exchange chromatography and gel filtration chromatography. The purified protease, of 16 kD and pi 8-5, showed maximum enzyme activity at pH 55 and substrate preference for Z-Phe-Arg-MCA. The specific inhibitors of cysteine protease leupeptin, iodoacetic acid, and E-64. completely suppressed the activity, indicating that the purified enzyme belongs to the cysteine protease family. Cysteine protease activity was found not only in somatic extract, but also in the excretory-secretory (ES) product of the nematode. When anti-cysteine protease immunoglobulin isotypes were examined in sera from rats infected with N. brasiliensis. a high level of IgG1 and a lower level of IgE antibody were detected. Depletion of IgG antibodies from the sera using protein G affinity columns resulted in a marked increase in reactivity of anti-cysteine protease IgE with the antigen, possibly due to the removal of competing IgG antibodies. In contrast to IgE and lgG1. production of anti-cysteine protease IgG2a was negligible. These results indicate that the nematode cysteine protease preferentially evokes an IgE/IgG1 antibody response.
Endothelial cells and renal epithelial cells do not express the Wegener's autoantigen, proteinase 3
Authors: King WJ , Adu. D , Daha MR , Brooks CJ , Radford DJ , Pall AA , Savage COS
Published: July 2010

Proteinase 3 (PR3) is the major antigen for autoantibodies (C-ANCA) against cytoplasmic components of neutrophils which are strongly associated with Wegener's granulomatosis (WG). Recent data that PR3 may be expressed by renal tubular epithelial cells and endothelial cells suggest potential for a direct pathogenic effect against these cells by C-ANCA or cytoxic T lymphocytes. Using a semi-quantitative polymerase chain reaction (PCR), ELISA and indirect immunofluorescence staining we studied endothelial and epithelial cell PR3 expression. By PCR, no PR3 expression was found in human umbilical vein endothelial cells (HUVEC) either untreated, or when treated with interferon-gamma (IFN-γ) (200 U/ml, 6h, 24h), IL-1 (20U/ml, 6h), tumour neurosis factor-alpha. (TNF-α) (200U/ml. 0. 1.2, 4, 6h) or IFN-γ+ TNF-a (6h); iliac vein and artery endothelial cells did not express PR3 either. In contrast. PR3 was detected in HL60 cells and neutrophils by PCR. expression being confirmed by sequence analysis. Three PR3 MoAbs showed no binding to unstimulated or TNF-α-stimulated HUVEC either by ELISA or by indirect immunofluorescence staining. The epithelial cell line A549 expressed PR3 when assayed by PCR. However, three renal epithelial cell lines (two tubular and one glomerular) showed little or no PR3 expression by PCR or ELISA. These studies fail to demonstrate evidence for PR3 expression by endothelial cells, even when using the highly sensitive PCR assay. Whilst PR3 expression by A549 cells is intriguing, the relevance of this in the pathology of WG is doubtful considering the negligible expression by renal epithelial cell lines.
Signalling via CD28 of human naive neonatal T lymphocytes
Authors: Hassan J , O'neill S , O'neill L A J , Pattison U , Reen D J
Published: July 2010

Accessory molecules play a crucial role in the development of the T cell response to antigenic challenge. We have examined the role of CD28 in modulating the‘naive’ neonatal T cell response to anti-CD2-mediated activation. To compare the role of CD28, neonatal and adult T cells were stimulated with a pair of mitogenic anti-CD2 antibodies in the presence or absence or anti-CD28 MoAb. With anti-CD2 alone, neonatal T cells proliferated slightly but produced no detectable IL-2, whereas adult T cells proliferated vigorously, with significant IL-2 production. Costimulation with anti-CD28 MoAb greatly enhanced the proliferative response of neonatal T cells to levels equivalent to those of adult T cells, whereas adult T cells showed only slight increases. Although IL-2 secretion was increased in the presence of anti-CD28 MoAb. neonatal T cell IL-2 production remained lower than in adults. In contrast, enhancement of IL-2 mRNA expression in neonates was similar to adult levels. Anti-CD28 MoAb costimulation increased NFkB levels in neonates, albeit to levels lower than that of adults. The cellular mechanism governing the diminished proliferative response of neonatal T lymphocytes to anti-CD2 may therefore be due to decreased NFkB induction, reduced IL-2 mRNA expression and deficient IL-2 production. Although anti-CD28 MoAb costimulation enhances all of the above signals, NFkB and IL-2 levels remain lower than in adults, suggesting the need for further activation requirements in the neonate.
Glycosylphosphatidylinositol (GPI)-anchored surface antigens in the allogeneic activation of T cells
Authors: Schubert J , Stroehmann A , Scholz C , Schmidt R E
Published: July 2010

GPI-linked surface molecules have recently been described as structures with an activation potential for human T lymphocytes. To study the role of these molecules in T cell activation we analysed GPI-deficient or normal T cells from patients with paroxysmal nocturnal haemoglobinuria (PNH). On activation with allogeneic Epstein-Barr virus (EBV)-transformed B cell lines GPI-deficient freshly separated T cells or continuously growing T cell lines exhibited a significantly lower proliferation or cytokine production compared with their normal counterparts. In contrast, stimulation via the T cell receptor-associated CD3 structure resulted in a comparable response. There was no difference in activation of normal T lymphocytes when GPI-deficient B cells were used as stimulators compared with normal B cells obtained from the same PNH patient. We conclude from these data that GPI deficiency in PNH leads to a functional deficiency of GPI-deficient T cells. In contrast, no difference in activation of T lymphocytes for GPI-deficient cells on the stimulator cell level was observed.
Delineation of producing ability of IgG and IgA subclasses by naive B cells in newborn infants and adult individuals
Authors: Yachie A , Konno A , Ohta K , Wada T , Seki H , Taniguchi N , Miyawaki T
Published: July 2010

Neonatal B cells with the naive (sIgD+) phenotype are able to generate IgG- and IgA-producing cells as well as IgM production in the presence of memory CD4+ T cells expressing L-selectin (CD62L) in pokeweed mitogen-stimulated cultures. We used this system to examine comparatively the ability of naive B cells to produce IgG and IgA subclasses in newborn infants and adult individuals. Naive B cells were enriched from both donors on the basis of sIgD positivity, and memory (CD45RO+) CD4+ T cells with CD62L expression were isolated from adults. We here demonstrate some differences in profiles of IgG and IgA subclass production between neonatal and adult naive B cells. In neonatal B cells, IgG1 and IgG3 were predominantly produced, but IgG2 and IgG4 production was virtually absent. Similar to neonatal B cells, adult naive B cells produced mainly IgGq and IgG3, although memory (sIgD’) B cells from adults secreted all of the IgG subclasses. It should be noted that low but detectable levels of IgG2 and IgG4 were found in adults’naive B cell cultures. Although IgA produced by neonatal B cells was exclusively IgA1, IgA2-secreting cells were identifiable in adult naive B cells. The results suggest that further class switch of naive B cells to IgG2, IgG4 and IgA2 in addition to IgG1 and IgG3 may be controlled by their own age-dependent maturation process.
The acute phase protein response in patients receiving subcutaneous IL-6
Authors: Banks R E , Forbes M A , Storr M , Higginson J , Thompson D , Raynes J , Illingworth J M , Perren T J , Selby P J , Whicher J T
Published: July 2010

IL-6, tumour necrosis factor-α (TNE-α) and IL-1 are thought to be the key mediators of the acute phase response although much of the evidence is based on in vitro studies. It is not clear to what extent each of the acute phase proteins are regulated in vivo by each of these cytokines. The aim of this study was to examine the effects of IL-6 treatment in eight patients with cancer on the concentrations of an extensive range of positive and negative acute phase proteins. It was part of a larger investigation to assess the value of IL-6 in the management of chemotherapy-induced thrombocytopenia. IL-6 was administered by a daily subcutaneous injection for 7 days at a dose level of 1, 3. or 10 μg/kg/day. Increases in the positive acute phase proteins, serum amyloid A. C-reactive protein. α1-acid glycoprotein, α1-antichymotrypsin, haptoglobin, α1-antitrypsin, fibrinogen, complement component C3, and caeruloptasmin, were observed, with the greatest incremental changes and fastest responses being seen for C-reactive protein and serum amyloid A protein. The negative acute phase proteins transferrin, transthyretin and retinol binding protein all fell to a nadir within 48-96 h after the first IL-6 injection. Increases in complement component C4 were only found in two patients, which may be related to the increase in circulating TNF-α concentrations found only in these patients. This study has therefore shown that IL-6 is capable of causing changes in the majority of acute phase proteins in vivo. Although secondary induction of TNF-α was not observed in the majority of patients examined, it is still possible however that other cytokines involved in regulation of the acute phase response, such as IL-1, may have been induced and contributed to the overall response.
Modulation of dendritic cell development by immunoglobulin G in control subjects and multiple sclerosis patients
Authors: Ohkuma K , Sasaki T , Kamei S , Okuda S , Nakano H , Hamamoto T , Fujihara K , Nakashima I , Misu T , Itoyama Y
Published: July 2010

Intravenous immunoglobulin (IVIg) preparations are reportedly effective in inhibiting the relapse of multiple sclerosis (MS), but few reports have investigated the effect of IVIg on dendritic cells (DCs), which are thought to be involved in such relapses. In the system that uses monokines to differentiate DCs from peripheral blood monocytes (Mo-DCs), we investigated the effect of immunoglobulin G (IgG) on these antigen-presenting cells. Using monocytes derived from healthy volunteers, IgG partially inhibited the expression of CD1a, a marker of immature DCs (imDCs), and CD40 and CD80, which are markers associated with T cell activation. In contrast, IgG enhanced the expression of CD83, a marker of mature DCs (mDCs). Furthermore, IgG markedly inhibited the expression of CD49d [very late activation antigen (VLA)-4 α4-integrin], the adhesion molecule required for mDCs to cross the blood–brain barrier. We obtained similar results on all the aforementioned cell surface molecules investigated in both healthy controls and MS patients. In addition, IgG treatment of cells from both healthy controls and MS patients inhibited the production of interleukin (IL)-12, a cytokine associated with mDC differentiation, but did not inhibit the production of IL-10. These results suggested the possibility that IgG treatment, apart from its known ability to regulate inflammation, may help to prevent relapses of MS by controlling DC maturation, consequently inhibiting invasion of immune cells into the central nervous system and affecting the cytokine profile.
Stimulation of monocytes is a pathway involved in systemic inflammatory response following haemorrhagic shock resuscitation: the effect of hypoxaemic resuscitation
Authors: Douzinas E E , Livaditi O , Tasoulis M-K , Pelekanou A , Giamarellos-Bourboulis E J
Published: July 2010

The present study was designed to investigate whether serum of animals subjected to hypoxaemic resuscitation from haemorrhagic shock may be a weak stimulant for monocytes or not. Twenty rabbits were subjected to haemorrhagic shock after blood exsanguination; resuscitation was performed by infusion of the shed blood in eight rabbits under normoxaemic conditions (NormoxRes) and in 12 under hypoxaemic conditions (HypoxRes); seven rabbits were subjected to sham operation. Malondialdehyde (MDA) and tumour necrosis factor (TNF)-α were estimated in serum at serial time intervals; the serum was applied for stimulation of U937 monocytes with or without the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580. Expression of triggering receptor expressed on myeloid cells-1 (TREM-1) on U937 was also assessed by flow cytometric analysis. Death supervened in four animals of the NormoxRes (50%) and in one animal of the HypoxRes group (8·33%, P: 0·032). Serum levels of TNF-α and MDA were higher in NormoxRes compared to HypoxRes animals. Expression of TREM-1 on U937 monocytes was similar after stimulation with serum sampled from both groups. Concentrations of interleukin (IL)-1β, IL-6 and IL-8 of monocyte supernatants were higher after stimulation with serum of NormoxRes than HypoxRes rabbits. Production of cytokines after stimulation with serum was decreased significantly after addition of SB203580. It is concluded that stimulation of monocytes may contribute to the generation of the systemic inflammatory response during reperfusion after ischaemia. Lower stimulation of the p38 MAPK-mediated production of IL-1β, IL-6 and IL-8 by monocytes may be implicated as an explanation for the benefits shown for the host when resuscitation is performed under hypoxaemic conditions.
Systemic and local interferon-gamma production following Mycobacterium ulcerans infection
Authors: Schipper H S , Rutgers B , Huitema M G , Etuaful S N , Westenbrink B D , Limburg P C , Timens W , Van Der Werf T S
Published: July 2010

Buruli ulcer disease (BUD) is an emerging predominantly tropical disease caused by Mycobacterium ulcerans. The initial pre-ulcerative skin lesion often breaks down into an ulcer with undermined edges. Healing is common but may require considerable time, and scarring often results in functional limitations. Considerable evidence has now emerged that patients with early BUD cannot mount a sufficient protective T helper 1 (Th1) cell response to M. ulcerans, but uncertainty remains as to whether immune protection is restored over time. This study investigates the Th1 cell response of patients with various stages of BUD on mycobacterial antigens. We measured interferon (IFN)-γ levels after ex vivo whole blood stimulation with tuberculin purified protein derivative (PPD), and compared the Th1 cell response of individuals with pre-ulcerative, ulcerative and healed BUD as well as healthy controls. Moreover, the systemic Th1 cell response was related to histopathological features in the various stages of surgically resected BUD lesions. We show that patients with ulcerative and healed BUD produce significantly higher IFN-γ levels after mycobacterial ex vivo whole blood stimulation than healthy controls, and that patients with a granulomatous tissue response produce higher IFN-γ levels than individuals without. We therefore suggest that the mounted Th1 cell response in ulcerative BUD patients might be related to their histopathological tissue response.
A comparison of ex vivo cytokine production in venous and capillary blood
Authors: Eriksson M , Sartono E , Martins C L , Balé C , Garly M-L , Whittle H , Aaby P , Pedersen B K , Yazdanbakhsh M , Erikstrup C , Benn C S
Published: July 2010

We performed a randomized study of the immunological effects of an early measles vaccine given at 4·5 months of age and aimed to obtain venous samples from the infants at baseline and 6 weeks later. If this was not feasible, a capillary sample was obtained. We analysed baseline samples from the first 50 children enrolled in the study to investigate the potential differences in ex vivo cytokine production between venous blood and capillary blood. We also obtained paired venous and capillary blood samples from 11 adult volunteers. Whole blood was stimulated with lipopolysaccharide (LPS) [a Toll-like receptor (TLR)-4 ligand], (S)-(2, 3-bis (palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys4-OH, trihydrochloride (PAM3Cys) (a TLR-2 ligand), phytohaemagglutinin (PHA) or purified protein derivative (PPD). Cytokine concentrations in the supernatants were assessed by a multiplexed assay and were compared between venous and capillary samples in both infants and adults. The production of both the pro- and the anti-inflammatory cytokines, tumour necrosis factor (TNF)-α and interleukin (IL)-10, was higher in cultures of capillary blood compared with venous blood. This was found in non-stimulated control samples as well as in blood stimulated with PAM3Cys and PPD. Adults produced more IL-5 in venous blood than in capillary blood upon PHA stimulation. We found no other difference in the levels of IL-5 or IFN-γ between venous and capillary blood. In capillary blood we found sex differences in response to PHA but this was not the case in venous blood. We found significant differences in the production of cytokines between venous and capillary blood. Such differences should be taken into account when setting up immuno-epidemiological studies.
Exploring the feasibility of selective depletion of T lymphocyte subsets by whole blood immunoadsorption cytapheresis
Authors: Belak M , Valeri C R , Wright D G
Published: July 2010

Normal turnover of T lymphocytes is slow relative to other blood cells. Consequently, the physical removal of circulating leucocytes by thoracic duct drainage, repeated leukapheresis or blood filtration results in T cell depletion and immunosuppression. However, clinical use of such procedures is impractical compared with immunosuppressive drugs or radiation. None the less, immunosuppression by physical depletion of T cells, avoiding the systemic toxicities of drugs and radiation, might have clinical advantages if immunophenotypically distinct T cell subsets could be depleted selectively. Recent advances in targeted plasma protein apheresis using adsorbent macrobead columns prompted us to determine whether analogous techniques might permit CD4+ T lymphocytes to be removed selectively from whole blood. To explore this possibility, we linked murine anti-human-CD4 and isotype-identical control monoclonal antibodies (mAbs) to agarose, polyacrylamide and polystyrene macrobeads (150–350 μm) and then evaluated the selectivity, specificity and efficiency of macrobead columns to remove CD4+ T cells from anti-coagulated whole blood at varying mAb densities and flow rates. We also examined saturation kinetics and Fc-oriention versus random coupling of mAbs to macrobeads. Sepharose 6MB macrobead (250–350 μm) columns proved to be most effective, selectively removing up to 98% of CD4+ T cells from whole blood. Moreover, depletion efficiency and selectivity were retained when these columns were reused after elution of adherent CD4+ cells. These studies indicate that selective depletion of T lymphocyte subsets by whole blood immunoadsorption apheresis using mAb-linked macrobead columns may be feasible on a clinical scale. It is possible that such apheresis techniques could achieve targeted forms of immunosuppression not possible with drugs or radiation.
A 10% ready-to-use intravenous human immunoglobulin offers potential economic advantages over a lyophilized product in the treatment of primary immunodeficiency
Authors: Kallenberg C G M
Published: July 2010

Intravenous immunoglobulin (IVIg) replacement therapy is the standard of care for patients with primary humoral immunodeficiencies. This study evaluated differences in infusion time between a 10% IVIg ready-to-use solution and a 6% IVIg lyophilized product and addressed potential cost implications. After receiving in-hospital treatment with 6% IVIg lyophilized solution for at least 6 months, 14 patients with humoral immunodeficiency without anti-IgA antibodies received five successive infusions with 10% IVIg ready-to-use solution. Data on infusion times were collected during the last two infusions of each IVIg product when maximum infusion rates had been reached. The median infusion time was reduced from 104·4 min with the 6% IVIg lyophilized solution to 51·0 min with the 10% IVIg ready-to-use solution (51% reduction), with corresponding median maximum infusion rates of 4·1 ml/kg/h and 5·9 ml/kg/h, respectively. Median gammaglobulin (IgG) trough levels were 7·1 g/l for the 6% IVIg lyophilized solution and 7·9 g/l for the 10% IVIg ready-to-use solution. Fewer adverse events were observed after infusing with 10% IVIg ready-to-use solution compared with 6% IVIg lyophilized preparation. We conclude that the 10% IVIg ready-to-use solution was well tolerated by most patients and reduced the median infusion time by 51% compared with a 6% lyophilized preparation of IVIg. The reduced bed occupancy and nursing time associated with a reduced infusion time, together with the elimination of a reconstitution step, were estimated to provide a cost-saving of €59·42 per patient per infusion. Thus, this product has the potential to reduce overall costs of IVIg treatment. Reduced infusion time is also likely to improve patients' quality of life.
Selective expansion of T cell receptor (TCR) V beta 6 in tonsillar and peripheral blood T cells and its induction byin vitrostimulation withHaemophilus parainfluenzaein patients with IgA nephropathy
Authors: Nozawa H , Takahara M , Yoshizaki T , Goto T , Bandoh N , Harabuchi Y
Published: July 2010

IgA nephropathy (IgAN), the most common form of primary glomerulonephritis, is recognized as a disease that often becomes worse during acute tonsillitis. Although many reports have shown that tonsillectomy is an effective treatment for IgAN patients, the immunological evidence has not yet been investigated fully. In this study, we compared the expression of T cell receptor (TCR) V beta families in tonsillar T cells of IgAN patients to those of non-IgAN patients. The reverse transcription–polymerase chain reaction (RT–PCR) and flow cytometric analyses showed that the TCR V beta 6 was used more frequently in tonsillar T cells of IgAN patients than in those of non-IgAN patients (P < 0·01 each). Similarly, the proportions of TCR V beta 6-positive cells in peripheral blood T cells were significantly higher in IgAN patients than in non-IgAN patients (P < 0·05). After tonsillectomy, the proportions decreased in IgAN patients (P < 0·05), but did not in non-IgAN patients. Furthermore, in vitro stimulation with Haemophilus parainfluenzae antigen, which is reported to deposit in the glomerular mesangium of IgAN, enhanced expression of TCR V beta 6 in tonsillar T cells from both IgAN and non-IgAN patients. These results suggest that TCR V beta 6-positive tonsillar T cells might be activated by H. parainfluenzae, move into the kidney through blood circulation and induce glomerulonephritis.
Neutrophil secretion products regulate anti-bacterial activity in monocytes and macrophages
Authors: Soehnlein O , Kenne E , Rotzius P , Eriksson E E , Lindbom L
Published: July 2010

Macrophages represent a multi-functional cell type in innate immunity that contributes to bacterial clearance by recognition, phagocytosis and killing. In acute inflammation, infiltrating neutrophils release a wide array of preformed granule proteins which interfere functionally with their environment. Here, we present a novel role for neutrophil-derived granule proteins in the anti-microbial activity of macrophages. Neutrophil secretion obtained by antibody cross-linking of the integrin subunit CD18 (X-link secretion) or by treatment with N-Formyl-Met-Leu-Phe (fMLP secretion) induced a several-fold increase in bacterial phagocytosis by monocytes and macrophages. This response was associated with a rapid activation of the monocytes and macrophages as depicted by an increase in cytosolic free Ca2+. Interestingly, fMLP secretion had a more pronounced effect on monocytes than the X-link secretion, while the opposite was observed for macrophages. In addition, polymorphonuclear cells (PMN) secretion caused a strong enhancement of intracellular reactive oxygen species (ROS) formation compared to incubation with bacteria. Thus, secretion of neutrophil granule proteins activates macrophages to increase the phagocytosis of bacteria and to enhance intracellular ROS formation, indicating pronounced intracellular bacterial killing. Both mechanisms attribute novel microbicidal properties to PMN granule proteins, suggesting their potential use in anti-microbial therapy.
Glabridin, a functional compound of liquorice, attenuates colonic inflammation in mice with dextran sulphate sodium-induced colitis
Authors: Kwon H-S , Oh S-M , Kim J-K
Published: July 2010

Inflammatory bowel disease (IBD) is characterized by detrimental immune reactivity in the gut, and the imbalance between proinflammatory and anti-inflammatory reactivity. The aims of this study were to determine whether oral administration of glabridin, a functional component of liquorice, could ameliorate dextran sulphate sodium (DSS)-induced colitis, as well as to understand the possible underlying mechanisms. Acute experimental colitis was induced in BALB/c mice by treatment with 5% DSS for 7 days. Glabridin (10 or 50 mg/kg/day) was given for 7 days. Treatment with glabridin significantly attenuated mortality, loss of body weight, shortening of the colon and severe clinical symptoms. This was associated with a remarkable amelioration of the disruption of the colonic architecture, a significant reduction in colonic myeloperoxidase (MPO) activity and the production of inflammatory mediators such as nitric oxide (NO), prostaglandin (PG) E2, and proinflammatory cytokines. These results suggest that glabridin-mediated anti-inflammatory action on colorectal sites may be a useful therapeutic approach to IBD.
Maternal and neonatal lipopolysaccharide and Fas responses are altered by antenatal risk factors for sepsis
Authors: Molloy E J , O'Neill A J , Grantham-Sloan J J , Webb D W , Watson R W G
Published: July 2010

The diagnosis of neonatal sepsis is difficult, resulting in unnecessary treatment to minimize morbidity and mortality. We hypothesized that exposure to antenatal risk factors for sepsis alters the perinatal neutrophil phenotype. The study setting was a tertiary referral university-affiliated maternity and neonatal hospital. Neutrophils from adults, normal neonates, neonates with antenatal sepsis risk factors and their respective maternal samples were incubated alone, with agonistic Fas antibody or with lipopolysaccharide (LPS). Surface receptor CD11b expression and the percentage apoptosis (persistent inflammatory response) were assessed using flow cytometry. Both mothers and asymptomatic neonates exposed to maternal sepsis risk factors had increased spontaneous neutrophil apoptosis compared to their respective controls. Infants with sepsis were LPS and Fas hyporesponsive. Maternal neutrophils had a delay in apoptosis in all groups with enhanced LPS and Fas responses associated with neonatal sepsis. CD11b expression was not altered significantly between groups. Maternal neutrophil function is altered in neonatal sepsis and may have a diagnostic role. Neonatal sepsis was associated with LPS hyporesponsiveness, potentially increasing susceptibility to infection.
Levamisole enhances immune response by affecting the activation and maturation of human monocyte-derived dendritic cells
Authors: Chen L-Y , Lin Y-L , Chiang B-L
Published: July 2010

Levamisole is a synthetic phenylimidazolthiazole that was first introduced in 1966 as an anti-helmintic agent. Current studies have been focused upon its effect on immune response and on cancer treatment. We examined the molecular mechanisms of levamisole in the activation and maturation of human monocyte-derived dendritic cells (DC) and human T cells. Treatment of DC with levamisole increased the presentation of CD80, CD86, CD83 and human leucocyte antigen D-related (HLA-DR) molecules on the cell membrane, as well as the production of interleukin (IL)-12 p40 and IL-10. Levamisole-treated human DC also enhanced T cell activation towards type 1 T helper immune response by inducing interferon-γ secretion. Neutralization with antibodies against Toll-like receptor (TLR)-2 inhibited levamisole-induced production of IL-12 p40 and IL-10, suggesting a vital role for TLR-2 in signalling DC upon incubation with levamisole. The inhibition of nuclear factor-κB, extracellular signal-regulated kinases 1/2 or c-Jun N-terminal kinases pathways also prevented the effects of levamisole on DC in producing IL-12 p40 or IL-10. Taken together, levamisole could enhance immune response towards T helper 1 development through the activation of dendritic cells or T cell aspects.
Development of time-resolved immunofluorometric assays for vascular endothelial growth factor and application on plasma of patients with gastric tumours
Authors: Sheng S L , Bao S H , Huang G , Wang L M
Published: July 2010

A highly sensitive and accurate time-resolved immunofluorometric assay (TR-IFMA) has been developed, for the first time, to measure plasma vascular endothelial growth factor (VEGF) in patients with gastric tumours. A monoclonal anti-hVEGF antibody and a biotinylated anti-hVEGF antibody were used to develop a non-competitive ‘sandwich’-type assay. Fluorescence can be measured by a time-resolved fluorometer after binding of europium (Eu)3+-labelled streptavidin to the biotinylated immunoglobulin. Plasma VEGF concentrations were measured by TR-IFMA in 92 healthy controls, in 36 benign stomach disease patients and in 92 gastric cancer patients before surgery. The association between plasma VEGF levels and clinicopathological features was evaluated. A standard curve for VEGF TR-IFMA has been developed with good sensitivity (0·37 pg/ml). Accuracy studies, specificity, parallelism and precision data were determined and all were found to be satisfactory. The validity of the VEGF assay was confirmed by the good correlation between the results obtained by TR-IFMA and commercial enzyme-linked immunosorbent assay (ELISA) (ELISA result = 1·862 + 0·953 (TR-IFMA result), r = 0·944]. The plasma levels of VEGF are higher in gastric cancer patients than in healthy controls. VEGF levels were associated significantly with the presence of distant metastases, as well as invasion depth of the tumour and tumour stage, but not with tumour location, tumour histology, differentiation or the presence of lymph node metastases. At the cut-off of 217·79 pg/ml, the diagnostic sensitivity, specificity and accuracy of the TR-IFMA were 40·2%, 93·7% and 69·9%, respectively. A highly sensitive and reliable TR-IFMA for VEGF has been developed. The determination of plasma VEGF levels may be clinically useful.
Serial lung function tests in primary immune deficiency
Authors: Rich A L , Le Jeune I R , McDermott L , Kinnear W J M
Published: July 2010

Pulmonary complications are common in patients with primary immune deficiency (PID). The aim of this study was to assess the usefulness of lung function tests (LFTs) in the management of these patients, and in particular to see if carbon monoxide transfer factor (TLCO) is needed in addition to spirometry. We studied 20 patients (11 female) with PID in a tertiary referral clinic, with a mean age of 47·6 years. Serial LFTs, spanning a mean of 101 months, were correlated with immunoglobulin levels and antibiotic usage. Seven patients showed a decline in forced expiratory volume in 1 second over the period of the study. An additional five patients showed a decline in TLCO. Of these 12 patients, two had no radiographic evidence of lung disease. Higher levels of immunoglobulin were associated with slower decline in LFTs (P < 0·05). The analysis of antibiotic usage and LFTs failed to show a statistically significant effect, although there was a trend towards a slower rate of decline with greater use of antibiotics. LFTs decline slowly in patients with PID. Annual testing (both spirometry and transfer factor) is useful in the assessment of these patients, and should not be confined to those with radiological evidence of lung disease.
Long-term interleukin-10 presence induces the development of a novel, monocyte-derived cell type
Authors: Schoenbein C , Docke W-D , Wolk K , Belbe G , Hoflich C , Jung M , Grutz G , Sterry W , Volk H-D , Asadullah K , Sabat R
Published: July 2010

Interleukin (IL)-10 is one of the most crucial immunoregulatory cytokines. Its short-term effects have been analysed extensively, but little is known about its long-term effects. This is of considerable importance, as high systemic IL-10 levels are present for long periods in patients with persistent viral infections, certain cancers and in critical care patients. Our study investigated the effects of the long-term presence of IL-10 on human peripheral blood monocytes. In vitro, IL-10 treatment of these cells for 7 days induced the development of a novel cell type characterized by unique phenotypical and functional characteristics. These cells showed high HLA-DR expression and low expression of CD86 and other co-stimulatory molecules on their surface. The mRNA levels of both HLA-DR and CD86 were high, but no intracellular accumulation of CD86 protein was observed. With respect to its function, these cells showed strongly diminished tumour necrosis factor-α production following lipopolysaccharide stimulation, strongly diminished allogenic CD4+ T cell stimulatory capacity, and even induced a hyporesponsive state in CD4+ T cells. The phenotype remained stable despite the removal of IL-10. In vivo, we found monocytic cells from patients exhibiting this phenotype after long-term IL-10 exposure. These results complement our knowledge further about the biological effects of IL-10 and may provide an explanation for the sustained immunodeficiency in cases of the persistent presence of systemic IL-10.