Changes in student physical health behaviour: an opportunity to turn the concept of a Healthy University into a reality.

AIMS:: Previous studies have reported a high prevalence of unhealthy behaviours in the student population, which the Healthy University concept is now seeking to address, by taking a settings approach to health promotion. This study investigated how far students are already seeking to make changes to improve their health behaviour while in a university setting, to help inform the development of Healthy Universities. METHODS:: Data on student health behaviour change, health indicators and demographics were gathered from 550 students attending two London universities, via an online questionnaire released through the student union email system at one university and through iPads at a student centre at the other. RESULTS:: In total, 84% of respondents reported making changes to try to become healthier while at university, primarily for proactive health reasons rather than reacting to a perceived health or weight issue. Universities and student unions were reported as influencing behaviour change by only five students. Compared with previous studies, a higher proportion of respondents were pursuing healthier lifestyles, including only 11% reporting they smoked. There were some statistically significant demographic differences as regards alcohol consumption, physical activity, the types of food students were seeking to avoid and the reasons for this. CONCLUSION:: The findings provide a novel perspective on student health behaviour and suggest that the traditional stereotype of a hedonistic student lifestyle freed from family constraints may need to be reassessed. Universities and student unions appear to have a significant opportunity to build on a more health conscious cohort of students, employing targeted approaches where appropriate, to encourage positive health behaviour change and make the Healthy Universities concept a reality, with important public health implications.

Aberrant dendritic cell function conditions Th2-cell polarization in allergic rhinitis.

BACKGROUND: Myeloid (m) and plasmacytoid (p) dendritic cells (DCs) regulate immune responses to allergens, whereas it remains unclear whether abnormal DC function characterizes patients with airway allergy and whether putative dysfunction exists only in target organs. To evaluate DC function from patients with allergic rhinitis (AR), we assessed nasal, cutaneous as well as blood DCs after in vivo and in vitro allergen challenge, respectively. METHODS: DCs were immunostained in nasal and skin tissues, and cytokine expression was assessed by dual immunofluorescence. Cytokine production and regulation of cocultured peripheral CD4+ T cells were assayed by ELISA. RESULTS: In AR patients, local allergen challenge resulted in increases in pDC and mDC numbers at 8 h in the nasal mucosa and at 8-48 h in the skin. Defects in IL-10 and IFN-α were observed in both organs from AR. Blood mDCs from AR exhibited reduced IL-10 and IL-12 expression. The capacity of activated pDCs from AR to produce IFN-α and to trigger IL-10 by allogeneic CD4(+) T cells was diminished, whereas mDCs from these patients supported Th2- and Th17-cell differentiation. CONCLUSION: In allergic rhinitis, DCs are altered not only locally but also in the systemic circulation. mDCs and pDCs increased in airway and skin tissues exposed to the allergen and displayed reduced production of IL-10 and 'type 1 signals' (IL-12, IFN-α) both locally and in blood. Functional studies showed that this results in preferential Th2/Th17-cell polarization and impaired generation by blood DCs of IL-10+ T cells, linking systemic DC dysfunction and biased T-cell responses.

Local receptor revision and class switching to IgE in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (NP) and allergic rhinitis (AR) is characterized by local Th2 inflammation and up-regulation of IgE; however, IgE in NP is 'polyclonal' and allergen specific, whereas IgE in AR is 'oligoclonal' and allergen specific. Germinal center (GC) reactions occur in AR, while only the formation of GC-like structures in NP is described. The aim of this study was to investigate the involvement of local IgE production, class switch recombination, and receptor revision in NP. METHODS: We compared the levels of local IgE, germline gene transcripts, and mature Ig mRNA expression, recombination activating gene (RAG1 and RAG2), key markers of Th2 inflammation, and GC reactions in NP tissue vs AR and control tissue. Nasal mucosa was immunostained for the co-expression of RAG1 and RAG2 in B cells, plasma cells, and T cells, using dual or triple immunofluorescence (IF). RESULTS: In NP, local IgE level and key markers of local class switching are increased compared with AR and normal controls (NC). In NP, switch circle transcripts reveal ongoing local class switch recombination to IgE. Up to 30% of B cells, plasma cells, and T cells in nasal polyps re-express both RAG1 and RAG2, required for receptor revision. RAG1 and RAG2 mRNA concentrations are increased in NP and correlated with the magnitude of inflammation and the presence of S. aureus enterotoxin (superantigen)-specific IgE in the nasal polyp mucosa. CONCLUSION: Our results provide the first evidence of local receptor revision and class switching to IgE, and B-cell differentiation into IgE-secreting plasma cells in NP.

Reduced T-bet in addition to enhanced STAT6 and GATA3 expressing T cells contribute to human allergen-induced late responses.

BACKGROUND: T-bet and GATA-3 are transcriptional factors involved in Th1 and Th2 cell differentiation, although their concomitant roles at protein levels in target organs during human allergic disease have not been assessed. OBJECTIVES: We investigated the expression of T-bet and GATA-3 in nasal and cutaneous models of Th2 (grass-pollen allergen) and a cutaneous model of Th1 (PPD) responses in man. METHODS: Nasal biopsies were obtained at 8 h and skin biopsies at 8 and 48 h after allergen and PPD challenges, respectively, from 10 allergic rhinitics and 6 non-atopic controls. T cells were assessed using immunofluorescence microscopy. RESULTS: There were increases in CD3(+)STAT6(+)cells (P = 0.01 for nose and skin) and CD3(+)GATA3(+)cells (P = 0.03 for skin) in response to allergen compared with diluent in allergics. When compared with non-atopics after allergen challenge the difference between the two groups was also significant for CD3(+)STAT6(+) (P = 0.001 and 0.03) and for CD3(+)GATA3(+)cells (P = 0.04 and 0.001) for nose and skin respectively. Following PPD challenge CD3(+)STAT4(+)cells and CD3(+)T-bet(+)cells increased in both groups compared with diluent (P = 0.02 and 0.03 for both TFs), whereas only CD3(+)T-bet(+) cells were significantly greater in non-atopics compared with allergics (P = 0.04). The ratio of GATA3(+):T-bet(+) T cells in allergen-induced responses was significantly greater in the allergics (P = 0.008 and 0.01 nose and skin respectively), whereas the ratio of T-bet:GATA3(+)T cells was significantly higher in the non-atopics during PPD-induced responses (P = 0.003). CONCLUSIONS AND CLINICAL RELEVANCE: Dysregulation of Th1 transcription may contribute to heightened expression of STAT6 and GATA3 leading to exaggerated Th2-driven manifestations of allergic disease.

CC chemokine receptor 4 (CCR4) in human allergen-induced late nasal responses.

BACKGROUND: CC Chemokine receptor 4 (CCR4) is preferentially expressed on Th2 lymphocytes. CCR4-mediated inflammation may be important in the pathology of allergic rhinitis. Disruption of CCR4 - ligand interaction may abrogate allergen-induced inflammation. METHODS: Sixteen allergic rhinitics and six nonatopic individuals underwent both allergen and control (diluent) nasal challenges. Symptom scores and peak nasal inspiratory flow were recorded. Nasal biopsies were taken at 8 h post challenge. Sections were immunostained and examined by light or dual immunofluorescence microscopy for eosinophils, T-lymphocytes, CCR4(+)CD3(+) and CXCR3(+)CD3(+) cells and examined by in situ hybridization for CCR4, IL-4 and IFN-gamma mRNA(+) cells. Peripheral blood mononuclear cells were obtained from peripheral blood of nine normal donors and the CCR4(+)CD4(+) cells assessed for actin polymerization in response to the CCR4 ligand macrophage-derived chemokine (MDC/CCL22) and the influence of a CCR4 antagonist tested. RESULTS: Allergic rhinitics had increased early and late phase symptoms after allergen challenge compared to diluent; nonatopics did not respond to either challenge. Eosinophils, but not total numbers of CD3(+) T cells, were increased in rhinitics following allergen challenge. In rhinitics, there was an increase in CCR4(+)CD3(+) protein-positive cells relative to CXCR3(+)CD3(+) cells; CCR4 mRNA+ cells were increased and IL-4 increased to a greater extent than IFN-gamma. CCR4(+)CD4(+) T cells responded to MDC in vitro, and this response was inhibited by the selective CCR4 antagonist. CONCLUSION: Lymphocyte CCR4 expression is closely associated with induction of human allergen-induced late nasal responses. Blocking CCR4-ligand interaction may provide a novel therapeutic approach in allergic disease.

Sublingual grass pollen immunotherapy is associated with increases in sublingual Foxp3-expressing cells and elevated allergen-specific immunoglobulin G4, immunoglobulin A and serum inhibitory activity for immunoglobulin E-facilitated allergen binding to B cells.

BACKGROUND: The mechanisms of sublingual immunotherapy (SLIT) are less well understood than those of subcutaneous immunotherapy (SCIT). OBJECTIVES: To determine the effects of grass-pollen SLIT on oral mucosal immune cells, local regulatory cytokines, serum allergen-specific antibody subclasses and B cell IgE-facilitated allergen binding (IgE-FAB). METHODS: Biopsies from the sublingual mucosa of up to 14 SLIT-treated atopics, nine placebo-treated atopics and eight normal controls were examined for myeloid dendritic cells (mDCs) (CD1c), plasmacytoid dendritic cells (CD303), mast cells (AA1), T cells (CD3) and Foxp3 using immunofluorescence microscopy. IL-10 and TGF-beta mRNA expression were identified by in situ hybridization. Allergen-specific IgG and IgA subclasses and serum inhibitory activity for binding of allergen-IgE complexes to B cells (IgE-FAB) were measured before, during and on the completion of SLIT. RESULTS: Foxp3(+) cells were increased in the oral epithelium of SLIT- vs. placebo-treated atopics (P=0.04). Greater numbers of subepithelial mDCs were present in placebo-treated, but not in SLIT-treated, atopics compared with normal controls (P=0.05). There were fewer subepithelial mast cells and greater epithelial T cells in SLIT- compared with placebo-treated atopics (P=0.1 for both). IgG(1) and IgG(4) were increased following SLIT (P<0.001). Peak seasonal IgA(1) and IgA(2) were increased during SLIT (P<0.05). There was a time-dependent increase in serum inhibitory activity for IgE-FAB in SLIT-treated atopics. CONCLUSIONS: SLIT with grass pollen extract is associated with increased Foxp3(+) cells in the sublingual epithelium and systemic humoral changes as observed previously for SCIT.

Grass pollen sublingual immunotherapy for seasonal rhinoconjunctivitis: a randomized controlled trial.

BACKGROUND: Previous studies suggest that sublingual immunotherapy (SLIT) represents a safer alternative to injection immunotherapy but equivalent efficacy is yet to be confirmed. OBJECTIVE: To evaluate the efficacy and safety of SLIT in grass pollen-induced seasonal rhinoconjunctivitis. METHODS: A randomized, placebo-controlled trial in 56 adults over 18 months. Outcome measures included diary scores of seasonal symptoms and medication use, overall assessments, conjunctival and intradermal provocation tests and serum antibody measurements. To investigate possible mechanisms, sublingual biopsies were taken for measurement of local T cells, antigen-presenting cells and IL-12 mRNA expression. RESULTS: There were no significant differences between the immunotherapy (IT) and placebo groups for diary symptom scores (P = 0.48) or rescue medication (P = 0.19). The patients' overall assessment of hayfever severity compared with previous years showed a highly significant improvement in favour of the IT group (P < 0.02). After treatment the late skin response was smaller (P = 0.003) and the ratio of serum allergen-specific IgG4/IgE was higher (P = 0.05) in the IT group. Both of these variables correlated with the clinical response to SLIT. There were no differences between groups in either the sublingual epithelium or lamina propria for numbers of CD3+ cells (epithelium: P = 0.9, lamina propria: P = 0.2), CD1a+ cells (P = 0.3, P = 0.25), CD68+ cells (P = 0.9, P = 1.0) or IL-12 mRNA+ cells (P = 0.6, P = 0.4). Local side-effects were minor and there were no serious treatment-related adverse events. CONCLUSION: Grass pollen sublingual immunotherapy was well tolerated. Although there was no significant change in diary scores, the improvement in overall assessments, which correlated with inhibition of the late skin response and increases in serum IgG4 : IgE ratio, indicates the need for larger, dose-ranging studies.

Basophil recruitment and IL-4 production during human allergen-induced late asthma.

BACKGROUND: Basophils represent an important source of inflammatory mediators and cytokines after IgE-dependent activation in human beings. OBJECTIVE: To assess the role of basophils in allergic asthma, we measured the number of basophils in the bronchial mucosa and their capacity to express IL-4 mRNA and protein during allergen-induced late asthmatic responses. METHODS: Fiberoptic bronchoscopic bronchial biopsies were obtained at 24 hours from sites of segmental bronchial allergen challenge and control sites in 19 patients with atopic asthma and 6 nonatopic healthy volunteers. Basophil numbers were assessed by immunohistochemistry through use of mAb 2D7. IL-4 mRNA--positive cells were detected through use of in situ hybridization and colocalized to basophils through use of sequential immunohistochemistry/in situ hybridization. IL-4 protein was detected and colocalized to basophils through use of dual immunohistochemistry. RESULTS: After allergen challenge, there was an increase in the median number of 2D7-positive basophils per square millimeter in the bronchial mucosa in patients with asthma (0.9 cells/mm(2) at baseline to 8.8 cells/mm(2) after challenge; P =.002), which also was significantly higher than what was seen in nonasthmatic controls (P =.01). Similarly, IL-4 mRNA--positive cells were increased at 24 hours in patients with asthma (1.4 to 14) in comparison with controls (0 to 0; P =.02). Colocalization studies revealed that 15% and 41% of the basophil population in patients with asthma after allergen-challenge expressed, respectively, IL-4 mRNA and protein. Conversely, 19% of IL-4 mRNA-positive cells and 72% of IL-4 protein--positive cells were accounted for by basophils. CONCLUSION: After allergen provocation in sensitive patients with atopic asthma, basophils are recruited to the bronchial mucosa and express IL-4 mRNA and protein, which might contribute to local IgE synthesis and/or tissue eosinophilia or other aspects of allergic inflammation during late responses and ongoing asthma.

Grass pollen immunotherapy: symptomatic improvement correlates with reductions in eosinophils and IL-5 mRNA expression in the nasal mucosa during the pollen season.

BACKGROUND: Tissue eosinophilia and infiltration by T(H)2-type T cells are characteristic features of allergic rhinitis both after allergen challenge and during natural allergen exposure. Specific immunotherapy inhibits allergen-induced nasal eosinophilia. OBJECTIVES: We sought to assess, in the context of a randomized trial, the relationships between symptomatic improvement after immunotherapy and eosinophil numbers and IL-5 expression in the nasal mucosa during the pollen season. METHODS: Nasal biopsy specimens were taken from 37 adults with severe summer hay fever at baseline (out of season) and at peak season after 2 years of treatment with a depot grass pollen extract or placebo. Biopsy specimens were processed for immunohistochemistry by using mAbs against eosinophils (EG2), T cells (CD3), and IL-2 receptor-positive cells (CD25), as well as for in situ hybridization by using a sulfur 35-labeled antisense riboprobe directed against IL-5. RESULTS: Immunotherapy significantly reduced symptoms (49%, P =.01) and medication requirements (80%, P =.007) compared with placebo. There was a 400% increase (P =.004) in eosinophils during the pollen season in placebo-treated patients, which was inhibited in the immunotherapy group (20% increase, P =.04 between groups). Seasonal increases were also observed for CD25(+) cells (P =.002), CD3(+) cells (P =.02), and IL-5 mRNA-expressing cells (P =.03) in the placebo group but not in the immunotherapy group. A significant correlation was observed between eosinophils and IL-5 expression (r = 0.5, P <.05). Both eosinophils (r = 0.6, P <.02) and IL-5 (r = 0.6, P <.02) correlated with symptoms after immunotherapy. CONCLUSION: Improvement in symptoms after grass pollen immunotherapy may result, at least in part, from inhibition of IL-5-dependent tissue eosinophilia during the pollen season.

Cytokine expression during allergen-induced late nasal responses: IL-4 and IL-5 mRNA is expressed early (at 6 h) predominantly by eosinophils.

The production of TH2-type cytokines [interleukin-4 (IL-4) and IL-5] and tissue eosinophilia are characteristic features of allergic diseases. It was previously reported that at 24 h after allergen provocation, CD3+ T-lymphocytes were the principal cell source of IL-4 and IL-5 mRNA transcripts in both atopic asthma and rhinitis. To investigate whether IL-4 and IL-5 mRNA are expressed earlier during late nasal responses and if so, which cell(s) are responsible. Nasal biopsies were obtained at 6 h after nasal allergen challenge and following a control challenge with the allergen diluent. Sections were immunostained for T-lymphocytes (CD3+, CD4+) and eosinophils (EG2+). In situ hybridization was used to detect the number of cells expressing messenger RNA (mRNA) for IL-4 and IL-5. In patients with allergic rhinitis, eosinophils (EG2+ cells P = 0. 006) but not T- cells (CD3+ cells) increased in the nasal mucosa at 6 h after allergen challenge. The number of cells expressing IL-4 mRNA (P = 0.01) and IL-5 mRNA (P = 0.05) also increased at 6 h. Co-localization studies showed that 76% of IL-4 mRNA+ cells and 77% of IL-5 mRNA+ cells were eosinophils, whereas at this time point, T-cells and mast cells accounted for

Increased expression of IL-16 immunoreactivity in bronchial mucosa after segmental allergen challenge in patients with asthma.

BACKGROUND: We have previously shown increased expression of the CD4(+) cell chemoattractant IL-16 in bronchial mucosa of patients with asthma. We investigated the effects of allergen challenge on airway IL-16 expression. METHODS: We investigated the expression of IL-16 immunoreactivity in bronchial biopsy samples obtained from atopic asthmatic subjects (n = 19) and normal subjects (n = 6) 24 hours after segmental allergen challenge. Control biopsy samples were obtained either at baseline or after diluent challenge. IL-16 expression was correlated to numbers of CD4(+) cells, CD25(+) cells, and activated eosinophils. IL-16 bioactivity was assessed in bronchoalveolar fluid obtained from patients with asthma. RESULTS: IL-16 expression was higher in control biopsy specimens obtained from subjects with asthma compared with normal subjects (P<.05). In patients with asthma, numbers of IL-16 immunoreactive cells were significantly higher in biopsy specimens obtained after allergen challenge compared with control biopsy specimens (P<.001). Allergen provocation was associated with release of IL-16 in bronchoalveolar fluid in patients with asthma. In normal subjects, there was no difference in the number of IL-16-immunoreactive cells in biopsy specimens obtained after allergen challenge compared with biopsy specimens obtained after diluent challenge. Allergen challenge was associated with an increase in the numbers of EG2(+) eosinophils in patients with asthma but not in normal subjects. IL-16 expression correlated with the numbers of CD4(+) cells and CD25(+) cells after allergen challenge in asthmatic subjects with a provocative concentration required to decrease the FEV(1) by 20% of its baseline value (PC(20)FEV(1)) < 4 mg/mL. IL-16-immunoreactive cells were identified mainly as T cells and eosinophils in asthmatic subjects after allergen challenge. CONCLUSION: Endobronchial allergen provocation in atopic asthmatic patients resulted in increased airway expression of IL-16 and release of bioactive IL-16 in airways. IL-16 may contribute to the immunoregulation of the inflammatory infiltrate in the airways in response to antigen.

T cells from human allergen-induced late asthmatic responses express IL-12 receptor beta 2 subunit mRNA and respond to IL-12 in vitro.

IL-12 suppresses proallergic Th2-type cytokine production and induces Th1-type cytokine production by peripheral blood T cells from subjects with allergic disease. The objective of the present study was to examine the relevance of these findings to target organ T cell responses in human asthma. Bronchoalveolar lavage (BAL) and PBMC were collected from atopic asthmatics 24 h after fiberoptic allergen challenge of a segmental bronchus. BAL T cells and PBMC were cultured with allergen in the presence of recombinant IL-12 or IFN-gamma, and cytokines were measured in culture supernatants after 6 days. IL-5 production by BAL T cells and PBMC was inhibited by IL-12 and, to a lesser extent, by IFN-gamma. IL-12 also induced IFN-gamma production by BAL T cells and PBMC. The effects of IL-12 nor IFN-gamma on IL-5 production could not be reversed by neutralizing anti-IFN-gamma or anti-IL-12 mAbs, respectively. Thus, the effect of neither IL-12 nor IFN-gamma appeared to be mediated through induction of the other cytokine. In situ hybridization revealed that approximately one-third of BAL T cells expressed mRNA transcripts encoding the IL-12R beta 2 subunit following allergen challenge. Thus, human T cells obtained from BAL during asthmatic late responses, like T cells in the peripheral circulation, remain susceptible to immunomodulation by IL-12. These findings raise the possibility that IL-12 may hold therapeutic potential in allergic diseases such as asthma.

Long-term clinical efficacy of grass-pollen immunotherapy.

BACKGROUND: Pollen immunotherapy is effective in selected patients with IgE-mediated seasonal allergic rhinitis, although it is questionable whether there is long-term benefit after the discontinuation of treatment. METHODS: We conducted a randomized, double-blind, placebo-controlled trial of the discontinuation of immunotherapy for grass-pollen allergy in patients in whom three to four years of this treatment had previously been shown to be effective. During the three years of this trial, primary outcome measures were scores for seasonal symptoms and the use of rescue medication. Objective measures included the immediate conjunctival response and the immediate and late skin responses to allergen challenge. Cutaneous-biopsy specimens obtained 24 hours after intradermal allergen challenge were examined for T-cell infiltration and the presence of cytokine-producing T helper cells (TH2 cells) (as evidenced by the presence of interleukin-4 messenger RNA). A matched group of patients with hay fever who had not received immunotherapy was followed as a control for the natural course of the disease. RESULTS: Scores for seasonal symptoms and the use of rescue antiallergic medication, which included short courses of prednisolone, remained low after the discontinuation of immunotherapy, and there was no significant difference between patients who continued immunotherapy and those who discontinued it. Symptom scores in both treatment groups (median areas under the curve in 1995, 921 for continuation of immunotherapy and 504 for discontinuation of immunotherapy; P=0.60) were markedly lower than those in the group that had not received immunotherapy (median value in 1995, 2863). Although there was a tendency for immediate sensitivity to allergen to return late after discontinuation, there was a sustained reduction in the late skin response and associated CD3+ T-cell infiltration and interleukin-4 messenger RNA expression. CONCLUSIONS: Immunotherapy for grass-pollen allergy for three to four years induces prolonged clinical remission accompanied by a persistent alteration in immunologic reactivity.

Tolerance and latent cellular rejection in long-term liver transplant recipients.

Tolerance develops in a proportion of long-term liver transplant recipients but currently cannot be identified before an attempt at withdrawal from immunosuppression therapy. In the present study, we have examined the immunophenotypic characteristics of the cellular infiltrate in portal tracts and lobules as observed in liver biopsy specimens in relation to the outcome of subsequent withdrawal from immunosuppression therapy. Cryostat biopsy specimens from 27 long-term recipients before drug withdrawal, and from 10 patients with recent transplants who were having acute rejection, were analyzed. Immunohistochemical staining was performed for CD3+ (pan T cell), CD8+ (cytotoxic), CD4+ (helper), CD45RO+ (memory), CD45RA+ (naive), CD56+ (natural killer), CD68+ (macrophage), and CD8+ perforin+ cells. Fewer CD8+ and CD3+ cells were present in the lobular areas of biopsy specimens from patients who were successfully withdrawn from immunosuppression therapy (n = 6) compared with biopsy specimens from patients with nontolerant grafts (n = 9; 15 vs. 23 cells/high-power field [hpf] [P < .01] and 16 vs. 26 cells/hpf [P < .03], respectively) or biopsy specimens obtained during acute rejection (15 vs. 31 cells/hpf [P < .01] and 16 vs. 32 cells/hpf [P < .01]). Cell frequencies in the biopsy specimens of nontolerant long-term patients were similar to those found with acute rejection. Immunophenotyping the lobular inflammation within long-term liver allografts assists in identifying those patients in whom drug withdrawal is likely to be unsuccessful and in whom it is postulated a form of inactive, latent cellular rejection exists.

Mycobacterial DNA not detected in liver sections from patients with primary biliary cirrhosis.

BACKGROUND/AIMS: Recent studies in primary biliary cirrhosis have reported the detection of serum antibodies against Mycobacterium gordonae and of mycobacterial DNA in liver sections. The aim of this study was to investigate whether mycobacterial DNA is present in liver biopsy material in primary biliary cirrhosis. METHODS: Archival liver biopsy specimens from 11 patients with primary biliary cirrhosis (10 female, mean age 52 years) and 11 patients with autoimmune hepatitis (10 female, mean age 53 years) were identified. Positive control tissue comprised five archival lymph node specimens from patients with tuberculous lymphadenopathy, three of which had stained positive on ZN staining, and also a liver biopsy specimen from a patient with tuberculous hepatitis (ZN positive). Fixed sections were deparaffinised and DNA was extracted by mechanical disruption with glass beads. DNA was purified by use of diatoms and lysis in guanidinium thiocyanate in a technique previously validated for archival DNA. Primers were directed to amplify a partial 16S ribosomal RNA gene yielding the species-specific character for mycobacteria, and also to amplify the constitutively-expressed human gene GAPDH. RESULTS: The polymerase chain reaction was shown to be capable of detecting 1 fg of M. gordonae DNA in 'spiked' samples, equivalent to 1-5 bacterial cells. No mycobacterial DNA was detected in liver biopsy samples from either the primary biliary cirrhosis or autoimmune hepatitis groups. Of the tuberculous control sections, mycobacterial DNA was detected in four of five lymph nodes and the liver biopsy specimen. GAPDH amplification was detected in all tested samples from liver disease and tuberculous control samples. CONCLUSION: These data do not support a role for mycobacteria in the aetiology of primary biliary cirrhosis.

Granulocyte/macrophage-colony stimulating factor in allergen-induced rhinitis: cellular localization, relation to tissue eosinophilia and influence of topical corticosteroid.

BACKGROUND: Allergen-induced late nasal responses are associated with recruitment of T lymphocytes and eosinophils, and preferential messenger RNA (mRNA) expression of 'TH2-type' cytokines. We previously showed that topical steroid inhibited the late response and associated tissue eosinophilia. In this study we tested the hypothesis that granulocyte/macrophage-colony stimulating factor (GM-CSF) may contribute to late-responses and tissue eosinophilia and is inhibitable by topical corticosteroid. METHODS: Nasal biopsies were taken before and 24 h after nasal allergen provocation following 6 weeks of treatment with either a nasal corticosteroid spray (fluticasone propionate) or a matched placebo nasal spray twice daily. Cryostat sections were processed by immunohistochemistry and in situ hybridization to assess cytokine mRNA expression for GM-CSF. RESULTS: Increases in T lymphocytes and eosinophils were seen in the nasal mucosa after allergen challenge (p = 0.01) which were accompanied by a 5-fold increase in cells expressing mRNA for GM-CSF (p = 0.01). Double immunohistochemistry/in situ hybridization demonstrated that the majority of GM-CSF mRNA+ cells were co-localized to CD68+ (40%), or T cells (40%) with a lesser contribution from eosinophils (<20%). Topical steroid treatment was accompanied by a decrease in both the CD3+ and major basic protein (MBP+) cells expressing GM-CSF mRNA (p = 0.01) with a corresponding proportionate increase in the % of macrophages expressing GM-CSF. CONCLUSIONS: The results indicate that after allergen provocation, eosinophils are recruited to the nasal mucosa and that, at least in part, this may be due to GM-CSF. Topical nasal corticosteroid inhibits late responses and the associated eosinophilia, possibly indirectly by decreasing GM-CSF from T lymphocytes or reducing autocrine production of GM-CSF from eosinophils.

Usefulness and limitation of phylogenetic analysis for hepatitis C virus core region: application to isolates from Egyptian and Yemeni patients.

We report here the nucleotide sequences of the core region of HCV isolates from Egyptian and Yemeni patients and the method for classifying these HCV isolates by phylogenetic analysis. Sequence comparison suggested that the genotypes of these isolates were the same. Preliminary phylogenetic analysis of the HCV core region indicated that the genotypes of both isolates were 1c. However, an additional phylogenetic tree of the HCV core region constructed using a greater number of HCV isolates than that used in the preliminary analysis and on the basis of alignment of nucleotide sequences in an appropriate length indicated that the genotypes of these isolates were 4 and not 1c. For a more detailed analysis, the nucleotide sequences of the HCV E1 region as well as the core region for the same Yemeni patient were determined. A phylogenetic tree of the E1 region confirmed that the genotype of the HCV isolate from the Yemeni patient was 4. These data indicate that even when classifying HCV isolates using phylogenetic analysis, the misclassification would occur if care is not taken regarding the number and sequence lengths of the isolates included in the analysis.

Serum intercellular adhesion molecule-1 levels in chronic hepatitis C: association with disease activity and response to interferon alpha.

Soluble intercellular adhesion molecule-1 (sICAM-1) is probably released from a variety of cells, including leukocytes and endothelial cells at sites of inflammation or in the circulation, and serum levels may therefore be used to give an indication of immune activation and inflammatory processes. In the present study, an ELISA was used to measure serum ICAM-1 levels in 43 patients with chronic hepatitis C and these were correlated with histological changes in the liver and the response to interferon alpha treatment. Serum ICAM-1 levels were significantly higher in patients with chronic hepatitis C infection than in normal subjects and correlated positively with the grade of histological activity, in particular the degree of portal, periportal, and lobular inflammation, but not with the presence of lymphoid aggregates. There was also a weak but significant positive correlation between sICAM-1 and serum aspartate aminotransferase activities, and sICAM-1 levels were substantially greater in patients with than those without cirrhosis. Serum ICAM-1 levels fell significantly in 11 responders out of 19 patients treated with interferon alpha, whereas levels remained unchanged in the non-responder group. sICAM-1 levels correlate with the clinical status of patients with chronic hepatitis C infection and fall with successful interferon treatment.

Intrahepatic expression of hepatitis C virus antigens in chronic liver disease.

Localization of hepatitis C virus (HCV) antigens was studied in fresh frozen and formalin-fixed, paraffin-embedded liver tissue by immunoperoxidase using monoclonal antibodies to nucleocapsid protein and polyclonal human immunoglobulin G purified from plasma containing antibodies to structural and non-structural antigens of hepatitis C virus. The results observed using monoclonal antibody to HCV core were similar to those of polyclonal IgG against HCV antigens in the majority of cases and both correlated well with HCV status as defined by 'nested' polymerase chain reaction. HCV antigens were detected in both hepatocytes and mononuclear cells. Using polyclonal human IgG, a small proportion of biliary epithelial cells were also positive in 6/29 patients. In most of the specimens examined, relatively few cells (1-5 per cent) were found to be positive for HCV antigens. The cryostat sections, using polyclonal IgG against HCV antigens, exhibited greater immunohistochemical staining, suggesting that the fixation and processing of the tissue may be a major factor in the conservation and the outcome of HCV antigen(s) findings. However, the results using monoclonal antibodies may reflect the specificity of antigen expression.

Nucleotide sequence of the core region of hepatitis C virus in Pakistan and Bangladesh and the geographic characterisation of hepatitis C virus in south Asia.

A large number of complete and partial hepatitis C virus (HCV) sequences have been reported and classified into several genotypes, although none have been reported from South Asia. We have determined and evaluated partial sequences in the core region of HCV obtained from patients with chronic hepatitis in Pakistan and Bangladesh. Nucleotide sequences from these viruses show significant homology with the Japanese HCV-TR isolate (91.7%-97.9%) and low homology with other Japanese, American, and UK isolates including HCV-1, HC-J4, HC-J6, HC-J8, and E-b1 (79.3%-86.2%). The homologies of their deduced amino acids sequence with HCV-1, HC-J4, HC-J6, HC-J8, E-b1, and HCV-TR were 84.3%-89.8%, 85.0-87.9%, 84.1%-86.9%, 84.3%-87.0%, 90.2%-93.1%, and 89.8%-93.5%, respectively. These results suggest that our clones might be classified into the same genotype as HCV-TR. Further analysis using molecular evolutionary methods strongly supported the classification of these sequences with the HCV-TR genotype. Moreover, we could not detect any isolates which were closely related to our clones or HCV-TR in countries outside the South Asian area. These data further support the association of HCV genotypes with distinct geographic regions.