Subcutaneous vacuoles with suppuration and granulomas: a histological clue to atypical mycobacterial infection.

An 83-year-old woman was referred to the Dermatology department with a papular eruption on her left arm, occurring below the scar site of a malignant melanoma in situ, which had been excised 6 months previously. On physical examination, multiple, tender, violaceous papules and nodules inferior to the scar were noted, with central pustules in some of the lesions.

Immunological indicators of coeliac disease activity are not altered by long-term oats challenge.

Coeliac disease is a gluten-sensitive enteropathy that develops in genetically susceptible individuals. The disease exhibits many features of an autoimmune disorder. These include the production of highly specific anti-endomysial autoantibodies directed against the enzyme tissue transglutaminase. It is well accepted that wheat-, barley- and rye-based foods should be excluded in the gluten-free diet. Although several studies report that oats ingestion is safe in this diet, the potential toxicity of oats remains controversial. In the current study, 46 coeliac patients ingested oats for 1 year and were investigated for a potential immunogenic or toxic effect. Stringent clinical monitoring of these patients was performed and none experienced adverse effects, despite ingestion of a mean of 286 g of oats each week. Routine histological analysis of intestinal biopsies showed improvement or no change in 95% of the samples examined. Furthermore, tissue transglutaminase expression in biopsy samples, determined quantitatively using the IN Cell Analyzer, was unchanged. Employing immunohistochemistry, oats ingestion was not associated with changes in intraepithelial lymphocyte numbers or with enterocyte proliferation as assessed by Ki-67 staining. Finally, despite the potential for tissue transglutaminase to interact with oats, neither endomysial nor tissue transglutaminase antibodies were generated in any of the patients throughout the study. To conclude, this study reaffirms the lack of oats immunogenicity and toxicity to coeliac patients. It also suggests that the antigenic stimulus caused by wheat exposure differs fundamentally from that caused by oats.

Evaluation of soluble CD163 as a marker of inflammation in psoriasis.

A reliable biomarker of disease activity in psoriasis would be helpful for management, especially if this gave early information on treatment efficacy. This study investigated whether serum levels of soluble (s)CD163 correlated with psoriasis activity as assessed by the Psoriasis Area and Severity Index (PASI). CD163, a glycoprotein molecule expressed on macrophages and dendritic cells, is cleaved from the surface of these cells in some inflammatory diseases, and sCD163 levels have been shown to correlate with disease activity in other disorders. In this study, levels of sCD163 did not correlate with PASI in the patients (P = 0.56). Five patients had moderately increased PASI (12.6-20.3) but their sCD163 levels were within the normal range. From this study, it seems that sCD163 levels do not correlate with the inflammatory process in the skin of patients with psoriasis and thus sCD163 is not likely to be a useful biomarker for this disease.

HLA-D region-associated determinants serve as targets for human cell-mediated lysis.

Effector cells for cell-mediated lysis (CML) were generated by in vitro culture of lymphocytes from selected donors with X-irradiated cells from unrelated subjects who were HLA-D homozygous and matched to the responders for the antigens of the HLA-A and HLA-B regions. By using chromium labeled monocytes as target cells, cytotoxicity was found to correlate with presence of HLA-D region antigens matching those of the stimulating cells. Such CML reactions apparently directed at products of HLA-D, were inhibited by addition of unlabeled monocytes or B lymphocytes. These unlabeled cells had to be matched for HLA-D with the stimulating cells used to generate the effector populations. The results suggested that products of HLA-D, perhaps the DR antigens, were recognized by cytotoxic lymphocytes.

Cutaneous Fusarium solani infection in childhood acute lymphoblastic leukaemia.

Cutaneous involvement is often an initial presentation of infection with Fusarium species, which occurs more commonly in immunocompromised hosts and may be either localized or widespread. Skin lesions typically appear as red or grey macules, which may develop central ulceration and black eschar. Secondary dissemination to extracutaneous organs may occur in immunocompromised hosts, especially those with prolonged and severe neutropenia. We describe a case of widespread cutaneous involvement after infection with Fusarium solani in childhood acute lymphoblastic leukaemia that responded successfully to treatment with prolonged liposomal amphotericin B.

Autoantibody facilitated cleavage of C1-inhibitor in autoimmune angioedema.

C1-inhibitor (C1-Inh) is an important inhibitor of the inflammatory response and deficiency of this inhibitor, which may be hereditary or acquired, is associated with recurrent episodes of edema. Recently, an autoimmune form of angioedema has been described that is associated with functional deficiency of C1-Inh and an autoantibody that impedes C1-Inh function. In this report we describe the isolation of C1-Inh from the monocytes and plasma of a patient with autoimmune angioedema and demonstrate that the patient's monocytes secrete structurally and functionally normal C1-Inh, but show that this protein circulates in the patient's plasma in an inactive, structurally altered form. Furthermore, using analytic gel electrophoresis techniques it is demonstrated that the patient's autoantibody facilitates cleavage of normal C1-Inh, by its target proteases, to the same species of C1-Inh that is found circulating in the patient's plasma. This autoantibody facilitated cleavage of normal C1-Inh is apparently a consequence of destabilization of protease/inhibitor complexes. These findings contribute to our understanding of protease/C1-Inh interactions and document important observations on pathogenic mechanisms in autoimmune disease.

Barley and rye prolamins induce an mRNA interferon-gamma response in coeliac mucosa.

BACKGROUND: In coeliac disease, wheat, barley and rye are traditionally excluded in the gluten-free diet. However, few studies have examined the small intestinal immune response to barley and rye. AIM: To investigate the immunogenicity of barley and rye prolamins (hordein and secalin respectively) in comparison with wheat gliadin. METHODS: Duodenal biopsies from 22 coeliac patients and 23 disease controls were cultured for 4 h with gliadin, hordein or secalin and compared with culture medium alone. Proinflammatory cytokines, interferon-gamma and interleukin-2, were quantified by TaqMan polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Hordein caused the greatest increase in interferon-gamma mRNA in coeliac patients (median: 3.3-fold) in comparison with control subjects (median: 0.28-fold, P < 0.085). Secalin and gliadin induced similar levels of interferon-gamma mRNA with median fold-changes of 3.4 and 2.8, respectively, in coeliac patients in comparison with 1.6- and 1.1-fold increases in control subjects (P < 0.294 and P < 0.105, respectively). The median fold-changes for interleukin-2 mRNA did not differ between coeliac patients and controls. Cytokine protein was not upregulated. CONCLUSION: The findings of this study provide evidence that barley and rye cause immune activation in the mucosa of coeliac patients and support the practice that barley and rye should be excluded from the gluten-free diet.

Serum levels of soluble CD163 correlate with the inflammatory process in coeliac disease.

BACKGROUND: In coeliac disease, following the introduction of a gluten-free diet, monitoring mucosal disease activity requires repeated small intestinal biopsies. If a test measuring a circulating inflammatory marker was available, this would be clinically valuable. AIM: To determine if levels of soluble CD163, a scavenger receptor shed by tissue macrophages, correlated with the inflammatory lesion in coeliac disease. METHODS: Serum samples were collected from 131 patients with untreated coeliac disease, 40 patients with treated coeliac disease, 92 non-coeliac disease control subjects and 131 healthy controls. A capture enzyme linked immunosorbance assay was established to measure levels of soluble CD163 in sera. The extent of the histological lesion in coeliac biopsies was assessed using a Marsh grading system. RESULTS: Levels of CD163 in untreated coeliac subjects were significantly elevated when compared with the treated coeliac patients, the disease control group and the healthy control subjects (P < 0.0001 in each instance). Moreover, coeliac patients with the most marked histological lesion (Marsh 3) had significantly higher levels of soluble CD163 than patients with Marsh grade 2 lesions (P < 0.0004), with grade 1 lesions (P < 0.0001) and grade 0 lesions (P < 0.0001). CONCLUSIONS: Measurement of soluble CD163 may be a useful method of monitoring the inflammatory lesion in coeliac disease.

Leukocyte function-associated antigen 1 (LFA-1) and CD44 are signalling molecules for cytoskeleton-dependent morphological changes in activated T cells.

Signaling through the leukocyte function-associated antigen 1 (LFA-1) molecule has previously been shown to induce homotypic aggregation in T cells and to induce cytoskeletal changes in T lymphoma cells. In this study we describe the induction of a dendritic phenotype associated with cytoskeletal rearrangement in activated human peripheral blood T cells stimulated with monoclonal antibody SPV-L7 to LFA-1 alpha. Maximal expression of this phenotype required 72 h preactivation with phorbol myristate acetate and expression was abolished using the protein kinase C inhibitor staurosporine. Monoclonal antibody to CD18, the beta-chain of LFA-1, did not induce this phenotype. Monoclonal antibody MEM 83 to presumably a discrete epitope on LFA-1 alpha did not induce this phenotype but induced homotypic aggregation. However, a monoclonal antibody to CD44 induced a similar phenotype in activated lymphocytes. Induction of both homotypic in activated lymphocytes. Induction of both homotypic aggregation and the dendritic phenotype was abolished by preincubation with soluble intracellular adhesion molecule 1 (ICAM-1). Cytoskeletal inhibitors prevented the morphological changes in SPV-L7-activated lymphocytes. Preincubation with tyrosine kinase inhibitor, protein kinase C inhibitors, and inhibitors of new protein synthesis also prevented these morphological changes. These data suggest that discrete epitopes on LFA-1 alpha may be capable of inducing discrete signals either for homotypic aggregation or for a dendritic phenotype. As both LFA-1 and CD44 are involved in the migration of lymphocytes through high endothelial venules, these data could suggest that these molecules transduce signals resulting in cytoskeletal modification necessary for lymphocyte transmigration.

Allergy in Irish adults: a survey of referrals and outcomes at a major centre.

BACKGROUND: There is an increasing demand for specialist public allergy services across Ireland. Little data exist on the patterns of allergic disease in Irish adults. The limited resources available require innovative strategies to ensure quality care delivery. AIMS: This study aimed to review the types of allergy referrals and diagnostic outcomes at a major Irish centre, and to establish an efficient method of communication with non-specialist practitioners. METHODS: Demographic data, referral characteristics and diagnostic outcomes from one hundred consecutive new allergy referrals were identified. Additionally, communications to a pilot email service were reviewed over a 12-month period and user satisfaction assessed. RESULTS: Requests for the investigation of food allergy accounted for 71% of referrals. Despite this, the main diagnostic outcome in this cohort was a non-allergic condition, chronic spontaneous urticaria (56%). immunoglobulin E (IgE)-mediated food allergy was definitively diagnosed in only 9% of patients, with the majority of these presenting with anaphylaxis. The allergy advice email service received 43 requests for assistance over 12 months, mainly for help in the interpretation of an allergy clinical history. Feedback on the email service was universally positive. CONCLUSIONS: The majority of patients in this cohort did not have IgE-mediated allergic disease. Increased awareness of the features that differentiate allergy from non-allergic conditions such as food intolerance or chronic spontaneous urticaria is required. The allergy advice email service should be developed further to play a key role in education and care delivery in partnership primary care.

Three possible laboratory indexes of disease activity in multiple sclerosis.

In a search for an objective measure of disease activity in MS, we studied three laboratory indexes in 15 patients over 12 months, relating them to the occurrence of relapse and the development of increased disability. Relapse was associated with detection of myelin basic protein (MBP) in the CSF (p less than 0.01), but not with decreased numbers of peripheral blood T lymphocytes. Persistently low T-cell numbers and more frequent detection of MBP in remission were associated with increased disability (p less than 0.01). There were no associations between other CSF abnormalities and either relapse or increased disability.

Collagenase and Dispase enzymes disrupt lymphocyte surface molecules.

Collagenase and Dispase enzymes are often used to disaggregate tissue. In this study, we examined by flow cytometry the effects of these enzyme preparations on peripheral blood lymphocyte surface marker expression. Peripheral blood mononuclear cells (PBMC) were obtained from seven healthy volunteers. Cells were incubated with collagenase (128 U/ml, type 1 A) for 3 h and overnight and with Dispase (1.6 mg/ml, grade II) for 15 min, 30 min, 1 h, 3 h and overnight. The intensity of expression of CD3, CD4, CD8, alpha beta and gamma delta T cell receptors was decreased by 25-40% in all cases, while CD4+ and CD8+ lymphocyte populations were undetectable after treatment with Dispase. Moreover, reappearance of these surface molecules did not occur following the incubation of cells in culture medium for 3 h. The results of this study emphasise the importance of considering the influence of isolation procedures on cell characteristics.

Removal of endogenous peroxidase activity from cryostat sections for immunoperoxidase visualisation of monoclonal antibodies.

The interpretation of immunoperoxidase staining of frozen tissue sections can be severely limited by the presence of endogenous peroxidase enzymes in the tissue. Previously recommended methods of reducing this enzyme activity were examined, but were found to be unsatisfactory when applied to the small intestine. A method, which effectively eliminates this background staining but which does not interfere with the binding of monoclonal antibodies to various tissue components, is described. This method may prove useful in immunoperoxidase studies of other tissues in which high levels of endogenous peroxidase are present.

Flow cytometric analysis of surface major histocompatibility complex class II expression on human epithelial cells prepared from small intestinal biopsies.

A technique for preparing viable, single cell suspensions of the epithelial layer of small intestinal tissue obtained endoscopically is described. Constant agitation of four biopsies for 60 min in the presence of chelating and reducing agents gave yields of 1.2-6.7 x 10(6) cells, of which 11-30% were intraepithelial lymphocytes (IEL). Passage through a nylon wool column removed dead cells. This preparation was suitable for flow cytometric analysis. Using this technique, surface MHC class II molecule expression was studied in 14 patients with normal small intestinal mucosa. Fluorescence labelling of these cells showed strong HLA-DR expression by epithelial cells (EC), DP was expressed less strongly, while little DQ expression could be detected. This technique demonstrates that small intestinal biopsies taken during routine endoscopy can yield adequate numbers of viable epithelial cells to perform flow cytometric analysis.

Heterogeneous recognition of beta 2-glycoprotein I by antibodies from antiphospholipid syndrome patients.

Beta 2-glycoprotein I plays a pivotal role in the binding of antiphospholipid antibodies to phospholipid in patients with antiphospholipid syndrome. In this study the nature of the epitopes on beta 2-glycoprotein I (beta2-GPI) recognised by sera from antiphospholipid syndrome (APS) patients (n = 15) was investigated and compared to rabbit polyclonal and mouse monoclonal anti-beta2-GPI antibodies. beta2-GPI was only recognised when bound to a high affinity binding support. The antigenic epitope on beta2-GPI recognised by all APS patients was also dependent on disulphide bond integrity. Digestion of beta2-GPI with elastase rapidly destroyed the epitope(s) on beta2-GPI recognised by antibodies in 91% of APS patients. The main cleavage occurred at tryptophan316-lysine317 in the fifth domain. Digestion with staphylococcal V8 protease resulted in a 50% reduction in antibody binding in 81% of patients and the cleavage sites mainly involved the first domain of the molecule. There was considerable variability in the recognition of six different species of beta2-GPI by serum from APS patients. The epitopes on beta2-GPI bound by APS sera appear conformationally determined in all patients but are quite heterogeneous in the regions of beta2-GPI that are recognised.

Possible in vivo modulation of Leu 2a expression on suppressor T cells in active multiple sclerosis.

Reduced numbers of suppressor T lymphocytes were identified by monoclonal antibodies in the peripheral blood of patients with multiple sclerosis (MS) in acute relapse. In vitro culture of cells from these patients resulted in a significant increase in the number of cells identified with the suppressor T cell marker Leu 2a but not OKT 8. The expression of other T cell antigens (Leu 4 and Leu 3a) remained unchanged. This change in Leu 2a expression did not occur when cells from healthy controls were similarly treated.