Differential binding and internalization of Clostridium difficile toxin A by human peripheral blood monocytes, neutrophils and lymphocytes.

Colitis due to Clostridium difficile infection is mediated by secreted toxins A and B and is characterized by infiltration by cells from the systemic circulation. The aim of our study was to investigate interactions between fluorescently labelled toxin A and peripheral blood monocytes, neutrophils and lymphocytes. Purified toxin A was labelled with Alexa Fluor® 488 (toxin A(488)) and incubated with isolated human peripheral blood mononuclear cells or washed whole blood cells for varying time intervals at either 37 or 4 °C/ice. The ability of trypan blue to quench cell surface-associated (but not cytoplasmic) fluorescence was also investigated. At 37 °C, toxin A(488) -associated fluorescence in monocytes peaked at 1 h (majority internalized), with subsequent loss associated with cell death. In contrast to monocytes, binding of toxin A(488) in neutrophils was greater on ice than at 37 °C. Studies using trypan blue suggested that over 3 h at 37 °C, most of the toxin A(488)-associated fluorescence in neutrophils remained at the cell surface. Over 48 h (37 °C and ice/4 °C), there was minimal toxin A(488)-associated fluorescence in lymphocytes. These studies suggest major differences in interactions between toxin A and circulating cells that infiltrate the mucosa during colonic inflammation in C. difficile infection.

Proteolytic cleavage of CD25, the alpha subunit of the human T cell interleukin 2 receptor, by Der p 1, a major mite allergen with cysteine protease activity.

Recent reports have indicated that the cysteine protease activity of Der p 1 may play a significant role in its ability to elicit IgE antibody responses, mainly through cleavage of membrane CD23 on B cells and interleukin (IL)-4 synthesis and secretion from mast cells and basophils. Here we demonstrate for the first time that Der p 1 also cleaves the alpha subunit of the IL-2 receptor (IL-2R or CD25) from the surface of human peripheral blood T cells and, as a result, these cells show markedly diminished proliferation and interferon gamma secretion in response to potent stimulation by anti-CD3 antibody. Given that the IL-2R is pivotal for the propagation of Th1 cells, its cleavage by Der p 1 may consequently bias the immune response towards Th2 cells, thereby creating an allergic microenvironment.

The cysteine protease activity of the major dust mite allergen Der p 1 selectively enhances the immunoglobulin E antibody response.

The house dust mite Dermatophagoides pteronyssinus allergen Der p 1 is the most immunodominant allergen involved in the expression of dust mite-specific immunoglobulin (Ig)E-mediated hypersensitivity. The reason for this potent IgE-eliciting property of Der p 1 remains unknown, but there is mounting in vitro evidence linking the allergenicity of Der p 1 to its cysteine protease activity. Here we demonstrate for the first time that immunization of mice with proteolytically active Der p 1 results in a significant enhancement in total IgE and Der p 1-specific IgE synthesis compared with animals immunized with Der p 1 that was irreversibly blocked with the cysteine protease inhibitor E-64. We conclude that the proteolytic activity of Der p 1 is a major contributor to its allergenicity.

Technical validation of an autoantibody test for lung cancer.

BACKGROUND: Publications on autoantibodies to tumour-associated antigens (TAAs) have failed to show either calibration or reproducibility data. The validation of a panel of six TAAs to which autoantibodies have been described is reported here. MATERIALS AND METHODS: Three separate groups of patients with newly diagnosed lung cancer were identified, along with control individuals, and their samples used to validate an enzyme-linked immunosorbant assay. Precision, linearity, assay reproducibility and antigen batch reproducibility were all assessed. RESULTS: For between-replicate error, samples with higher signals gave coefficients of variation (CVs) in the range 7%-15%. CVs for between-plate variation were only 1%-2% higher. For between-run error, CVs were in the range 15%-28%. In linearity studies, the slope was close to 1.0 and correlation coefficient values were generally >0.8. The sensitivity and specificity of individual batches of antigen varied slightly between groups of patients; however, the sensitivity and specificity of the panel of antigens as a whole remained constant. The validity of the calibration system was demonstrated. CONCLUSIONS: A calibrated six-panel assay of TAAs has been validated for identifying nearly 40% of primary lung cancers via a peripheral blood test. Levels of reproducibility, precision and linearity would be acceptable for an assay used in a regulated clinical setting.

Differential protein expression by dendritic cells from atopic and non-atopic individuals after stimulation by the major house dust mite allergen Der p 1.

BACKGROUND: Dendritic cells (DCs) are sentinels of the immune system and are known to play a key role in allergic responses. However, it is not clear how DCs that have been exposed to an allergen support Th2 type immune responses. It is possible that DCs from atopic individuals are inherently programmed to support allergic disease, or it is the exposure of dendritic cells to allergens that is key to the development of allergic sensitisation. METHODS: We used 2D gel electrophoresis and MALDI mass spectrometry to compare the proteome of DCs from atopic and non-atopic individuals in both the resting state and after stimulation with the major house dust mite allergen Der p 1. RESULTS: Our data show that unstimulated DCs from atopic and non-atopic individuals are very similar at the whole cell proteome level, showing few differentially expressed proteins. However, upon stimulation with Der p 1, a number of additional proteins are differentially expressed, and of these several were of potential relevance to Th2 cell differentiation and the allergic response, including GTP-binding regulatory protein Gi alpha-2, frabin and cathepsin D. CONCLUSION: Whilst there are inherent differences between DCs from atopic and non-atopic individuals, it seems that exposure to allergen plays a key role in differential expression of proteins by these key immune cells. Further studies should now focus on establishing the biological relevance of these proteins as biomarkers in house dust mite allergy and their role in allergen induced Th2 cell differentiation.

An immunoglobulin E-reactive chimeric human immunoglobulin G1 anti-idiotype inhibits basophil degranulation through cross-linking of FcepsilonRI with FcgammaRIIb.

BACKGROUND: IgE binds to mast cells and basophils via its high-affinity receptor, FcepsilonRI, and cross-linking of FcepsilonRI-bound IgE molecules by allergen leads to the release of allergic mediators characteristic of type I hypersensitivity reactions. Previous work has shown that cross-linking of FcepsilonRI with FcgammaRIIb, an ITIM-containing IgG receptor, leads to inhibition of basophil triggering. 2G10, a chimeric human IgG1 anti-idiotype, has broad reactivity with human IgE and as such has the potential to bind simultaneously to FcepsilonRI-bound IgE, via its Fab regions, and the negative regulatory receptor, FcgammaRIIb, via its Fc region. OBJECTIVE: To assess the ability of human 2G10 to inhibit anti-IgE and allergen-driven basophil degranulation through cross-linking of FcepsilonRI-bound IgE with FcgammaRIIb. METHODS: 2G10 was assessed for its ability to bind to FcgammaRIIb on transfected cells and on purified basophils. In the basophil degranulation assay, basophils were purified from peripheral blood of atopic individuals and activated with either anti-IgE or the house dust mite allergen Der p 1, in the presence or absence of human 2G10. Basophil activation was quantified by analysis of CD63 and CD203c expression on the cell surface, and IL-4 expression intracellularly, using flow cytometery. RESULTS: Human 2G10 was able to bind to FcgammaRIIb on transfected cells and on purified basophils, and induce a dose-dependent inhibition of both anti-IgE and Der p 1-driven degranulation of basophils. CONCLUSION: The inhibition of basophil degranulation by the human IgG1 anti-idiotype 2G10 highlights the therapeutic potential of IgE-reactive IgG antibodies in restoring basophil integrity through recruitment of the inhibitory receptor FcgammaRIIb.

Enhanced cell-mediated tumor killing in patients immunized with human monoclonal antiidiotypic antibody 105AD7.

A human antiidiotypic monoclonal antibody (105AD7) has been shown to induce antitumor cellular responses in animals and appears to prolong survival in patients with metastatic colorectal cancer without associated toxicity. Proliferative leukocyte responses to the targeted tumor antigen gp72 were observed in these patients and plasma interleukin 2 levels were increased following immunization. Autologous tumor tissue was not available in these patients, so antitumor cytotoxicity could not be measured. This issue has now been addressed in an adjuvant clinical study in primary rectal cancer patients. Six patients with rectal cancer were immunized preoperatively with 105AD7. Peripheral blood lymphocytes taken prior to immunization were tested against tumor cells extracted from biopsies also obtained prior to immunization or from natural killer (NK)-sensitive target cells. Cryopreserved lymphocytes taken before and after tumor immunization, fresh peripheral blood lymphocytes taken immediately prior to surgery, and lymphocytes from tumor-draining lymph nodes were tested against autologous cells from the resected specimen or NK-sensitive target cells. Significant killing of autologous tumor cells, which was not due to NK activity, was seen with cryopreserved lymphocytes or lymph node cells of three patients at 1-2 weeks postimmunization with 105AD7 but not on pretreatment biopsies. Enhanced NK activity was seen 2-3 weeks postimmunization in 3 of 6 patients. These results indicate that 105AD7 human monoclonal antibody immunization enhances cytotoxicity in rectal cancer patients by specific and nonspecific effector mechanisms.

Immunocytochemical study of Peyer's patches follicular-associated epithelium in the marsupial, Didelphis albiventris.

The lack of probes defining leukocyte subpopulations has restricted ontogenetic studies of the opossum gut. We report for the first time the organization of the gut cellular immune components using species cross-reactive antibodies. Mouse monoclonal antibodies against human HLA-DR were used together with immunocytochemistry to demonstrate MHC class II-like antigens in the opossum Peyer's patches (PP). Positive staining was obtained in the M cell and enterocytes comprising the follicular-associated epithelium (FAE). Rabbit polyclonal antibody against human CD3 stained opossum thymocytes and T-cell dependent areas of spleen, lymph node, and PP interfollicular zones, but failed to stain intraepithelial lymphocytes in the FAE. In contrast rabbit polyclonal antibody against human IgA stained B-cell immunocytes and plasma cells present in the M-cell lateral invaginations. It is surmised that B-cell activation could occur in the opossum M-cell niches by thymus independent antigens, bypassing T-helper-cell function.

Immunoregulatory T cells in B cell chronic lymphocytic leukaemia: evidence of a unique phenotype a flow cytometric study.

CD4+ T cells of 57 patients with B cell chronic lymphocytic leukaemia (CLL) were analysed for expression of CD45RA and CD29. The majority of CLL patients (33 cases) showed a novel coexpression of these markers on a significant proportion of CD4+ T cells; however, analysis of a single blood sample revealed no apparent prognostic value associated with the markers. Expression of CD45RA and CD29 correlated with parameters of immune function consistent with the known attributes of these markers.

Phenotypic abnormality of T cells in B cell non-Hodgkin's lymphoma.

CD4+ T cells of patients with B cell non-Hodgkin's lymphoma (NHL) were analysed for expression of CD45RA and CD29. It was found that CD45RA expression was significantly lower, and CD29 expression significantly higher, in lymphoma patients compared to normal controls. Moreover absolute numbers of CD4+ T cells were significantly lower in NHL patients, due to selective depletion of CD4+ CD45RA+ cells.

Immunophenotyping of leukaemias by flow cytometry and APAAP: a two-year comparative analysis in hospital practice.

Using a panel of monoclonal antibodies for the immunophenotyping of haematological malignancies, we have made a direct comparison of the usefulness of indirect immunofluorescence using flow cytometry (IFC) alongside the alkaline phosphatase-anti-alkaline phosphatase (APAAP) immunoenzyme method. Analysis of 400 consecutive patient samples (blood and bone marrow) over a 2-year period, resulted in unequivocal phenotyping agreement by both methods in 98 per cent of cases. The positive results accounting for 2 per cent discordance were obtained by the APAAP method. The value of the technique in the clinical management of patients with leukaemia is documented together with guidelines for clinical laboratories.

Immunohistochemical localization of murine alpha 1-pregnancy-associated protein (alpha 1-PAP) in pregnant mice: relationship between serum alpha 1-PAP levels and incidence of positive cells.

Using an electroimmunoassay, the murine pregnancy-associated protein alpha 1-PAP was detected in the sera of virgin MF1 but not C57BL/10 female mice. During pregnancy, alpha 1-PAP levels rose in both strains, although concentrations were higher in the latter and in both fell towards term. Using the unlabelled peroxidase anti-peroxidase (PAP) staining procedure, alpha 1-PAP was detected within mononuclear leukocytes, the majority resembling macrophages, in the small and large intestinal mucosae, Peyer's patches and hepatocytes of virgin MF1 but not C57BL/10 females. During pregnancy, alpha 1-PAP positive cells were observed in each of these sites and in the decidua and placenta of both strains. Quantitative studies revealed that the incidence of alpha 1-PAP positive cells in the gut and associated lymphoid tissues reflected the circulating levels of the protein. In the placenta, the frequency and intensity of alpha 1-PAP positive staining was also reduced towards parturition. In contrast, hepatocyte staining remained constant throughout gestation in both strains. Our observations suggest that there may be at least two types of alpha 1-PAP synthesis operative and that circulating levels of the protein in female mice are influenced by strain, pregnancy and stage of gestation. These findings are discussed in relation to the cell types involved, their contribution to serum levels and the possible role of alpha 1-PAP in fetal allograft survival.

Atrophoderma of Pasini and Pierini. An immunopathologic case study.

Atrophoderma of Pasini and Pierini (APP) is a rare and distinctive form of dermal atrophy of uncertain origin. In only one previous report have immunopathologic methods been used to study a case of atrophoderma of Pasini and Pierini, and on the basis of the results obtained it was concluded that immunologic mechanisms were relevant to the pathogenesis of the condition. A detailed investigation of a case of atrophoderma of Pasini and Pierini was conducted using immunofluorescence and immunoperoxidase techniques. The epidermal Langerhans cells were abundant and expressed polyclonal immunoglobulin M on the cell-surface membrane. Biopsy of the same lesion was repeated 6 months later and revealed staining for immunoglobulins A and M and also for C3. This pattern of staining could not be reproduced in a range of other atrophic or scarring cutaneous lesions. Immunophenotypic analysis of the mild perivascular mononuclear cell infiltrate revealed an aberrant T-cell phenotype of uncertain significance.

Longitudinal study of circulating gastric antibodies in pernicious anaemia.

Temporal changes in gastric antibody response were investigated in 113 (51 men, 62 women) patients with confirmed pernicious anaemia. Their ages ranged from 31-92 years (mean (SD 13.2) 66). At diagnosis, parietal cell antibody and intrinsic factor antibody were detected in 90.9% and 39.1% of all patients, respectively. When the tests were repeated after a mean follow up of 70 months (range 14-137), parietal cell antibody and intrinsic factor antibody were positive in 82.8% and 58.7%, respectively. There was a definite but not significant trend for the organ specific parietal cell antibody to disappear; intrinsic factor antibody became more positive. These results may indicate that with progressive parietal cell destruction, the antigen is no longer available to sustain an immunological response. On the other hand, this hypothesis does not explain the increased prevalence of intrinsic factor antibody which is also a product of parietal cells.

Prevalence of Epstein-Barr virus in the cervix.

Cervical smears from 327 women were examined using the polymerase chain reaction (PCR) targeted to a sequence in the Bam H1 W region of the Epstein-Barr virus (EBV) to determine the prevalence of the virus in the cervix. EBV was detected in 131 (40%) of the 327 women. Of the 235 women with normal cytology, 98 (42%) were positive. Of the 92 women with dyskariotic smears, 33 (36%) were positive.

Autoantibody to nerve tissue in a patient with a peripheral neuropathy and an IgG paraprotein.

The antibody activity of a benign IgG lambda paraprotein to nerve tissue in a case of peripheral neuropathy has been investigated using immunohistochemical methods on tyrpsin-treated, formalin-fixed, paraffin-embedded tissue. IgG lambda was found in th sural nerve biopsy of the patient. Specific binding of the purified IgG lambda paraprotein and its isolated F(ab')2 fragment to homologous nerve and brain tissue was demonstrated. Similar activity was not demonstrable on fresh frozen cryostat sections. The results suggest that tests for autoantibodies to nerve tissue in neuropathological disorders should not be confined to fresh frozen tissue substrates.

Intra-abdominal sepsis: an immunocytochemical study of the small intestine mucosa.

AIM: To investigate immunocytochemical changes in intestinal tissues from patients with intra-abdominal sepsis, and to relate the changes to the possibility of enhanced bacterial adhesion and translocation. METHODS: Tissues from 17 patients suffering from intra-abdominal sepsis and from controls were sectioned and stained immunocytochemically for IgA, IgM, secretory component, J chain, and HLA-DR. Differences in the distribution and characteristics of positively staining cells between the patient groups were assessed. RESULTS: Patients with intra-abdominal sepsis had noticeable reductions in numbers of IgA and IgM plasma cells, reduced J chain staining, and had little immunoglobulin on the surfaces of enterocytes. In contrast, HLA-DR positive cells were increased in the sepsis compared with the control group. The plasma cells present showed cytological changes suggestive of apoptosis. CONCLUSIONS: Stress associated with sepsis and its immediate causes might result in increased plasma glucocorticoid levels that bring about apoptosis of mucosal plasma cells (or their precursors). The consequent reduction in expression of IgA and IgM may favour bacterial adhesion to the enterocytes and facilitate bacterial translocation into the tissues.

Absence of lysozyme (muramidase) in the intestinal Paneth cells of newborn infants with necrotising enterocolitis.

AIM: To determine immunocytochemically whether preterm and newborn infants with necrotising enterocolitis (NEC) show differences in numbers of lysozyme positive Paneth cells compared with normal controls, and to relate the findings to the possibility that lysozyme deficiency may facilitate the bacterial infections thought to be associated with this condition. METHODS: Tissues from 10 infants with NEC and from 11 matched controls were sectioned and stained immunocytochemically for lysozyme. Differences in the numbers of Paneth cells and degree of lysozyme positivity in the tissues were assessed. RESULTS: Tissues from NEC patients showed no, or very few, lysozyme positive Paneth cells, whereas controls showed strong positive staining. CONCLUSIONS: A deficiency or developmental defect in Paneth cells, resulting in an absence of lysozyme, may render the intestine more susceptible to bacterial infection, allowing organisms to adhere and translocate across the mucosa. Such enhancement of infection may contribute to the pathogenesis of NEC.

Immunocytochemistry of mucosal changes in patients infected with the intestinal nematode Strongyloides stercoralis.

AIM: To investigate the immunopathological changes in duodenal tissues induced by strongyloidiasis and to relate these to degrees of clinical severity. METHODS: Tissues taken from 21 patients showing mild, moderate or severe symptoms of strongyloidiasis, and from non-infected controls, were sectioned and stained immunocytochemically for IgA, secretory component (SC) and HLA-DR. Immunopathology was assessed by changes in numbers, intensity and distribution of stained cells. RESULTS: Parasitised individuals showed villous atrophy and crypt hyperplasia. There was notable infiltration of the lamina propria by IgA positive plasma cells and of the epithelium by intraepithelial lymphocytes. Infection was also associated with increased expression of SC and decreased expression of HLA-DR in epithelial cells. Changes in all parameters correlated with degree of clinical severity. CONCLUSIONS: Profound mucosal changes are induced by strongyloidiasis. Some are analogous to those seen in coeliac disease, but others seem quite unusual. It is likely that these changes are functionally related to the immunopathophysiological consequences of infection seen in patients with severe disease.

Epstein-Barr virus--a possible missing link in the initiation of cervical carcinogenesis?

Salient epidemiological and molecular biological features of Epstein-Barr virus (EBV) infection correlate well with the natural history of carcinoma of the cervix. It is therefore hypothesised that the incorporation of EBV into the genome of cervical epithelial cells at an early age (teens) could be an important early event in cervical carcinogenesis.