The two membrane isoforms of human IgE assemble into functionally distinct B cell antigen receptors.

The human C epsilon gene expresses two membrane IgE heavy chain mRNAs which differ in the sequence that encodes their extracellular membrane-proximal domain. In the long IgE isoform (mLIgE), this domain contains a stretch of 52 amino acids which are absent in the short variant (mSIgE). We have now generated B cell transfectoma cell lines that express these two isoforms and show that both types of mIgE form functional B cell antigen receptors (BCR). Both receptors associate with the Ig-alpha/Ig-beta heterodimer, as well as with protein kinases that are capable of phosphorylating this complex. Upon their cross-linking, both receptors can activate protein tyrosine kinases that phosphorylate the same substrate proteins. Both IgE receptors also associate with two novel proteins that do not bind to mIgM. Apart from these similarities, the two IgE-BCRs show several differences of which some are analogous to the differences between the IgM- and IgD-BCRs. First, the mSIgE is transported to the cell surface at a higher rate than the mLIgE. Second, the two IgE-BCRs associate with differently glycosylated Ig-alpha proteins, the mLIgE associates with the completely glycosylated form, whereas the mSIgE associates with an Ig-alpha glycoform that is partially sensitive to endoglycosidase H. Third, the kinetics of protein tyrosine phosphorylation induced by receptor cross-linking is significantly different for the two IgE-BCRs. Finally, cross-linking of the mSIgE-BCR leads to growth inhibition of the B cell transfectoma, whereas signaling through the mLIgE-BCR does not affect the cellular proliferation. These data show that the two human membrane IgE isoforms assemble into functionally distinct antigen receptors which can induce different cellular responses.

Fas receptor is not present on ejaculated human sperm.

BACKGROUND: Apoptosis appears to have an essential role in the control of testis germ cell number and Fas expression has been reported in apoptotic spermatocytes and spermatids. We investigated if Fas (CD95) was present on ejaculated human sperm and any relationship between Fas on sperm and the apoptotic marker Syto16. METHODS: Semen samples from 77 male partners of infertile couples were evaluated. Each sample was analysed both before and after semen preparation by conventional microscopical procedures and by flow cytometry (FC). A multiparameter FC analysis to assess simultaneously sperm concentration, sperm viability, sperm apoptosis, CD45 positive (leukocyte) and CD95 (Fas) positive cell concentration was carried out. A further 10 samples were studied by indirect immunofluorescence to confirm results. RESULTS: The mean concentration of CD95 positive cells was very low (<1%), with no significant difference between normozoospermic and non-normozoospermic men. There was no correlation between apoptotic sperm and CD95 positive cell concentration. A linear correlation was found between CD95 positive cell and leukocyte (CD45 positive) concentration (r = 0.9946, P < 0.0001). CD95 mean fluorescence intensity of leukocytes was 10-fold greater than that of sperm and of isotypic control. Both incubation with activating anti-Fas antibody and betulinic acid induced apoptosis in leukocytes. Incubation with betulinic acid, but not with activating anti-Fas antibody, induced apoptosis in sperm. Pre-incubation with neutralizing anti-Fas antibody suppressed CD95 expression on leukocytes, whereas it did not change sperm CD95 peak fluorescence. CONCLUSIONS: There is no detectable quantity of Fas on human ejaculated sperm.

A new flow cytometric assay for the evaluation of phagocytosis and the oxidative burst in whole blood.

Phagocytes play an important role in host defence against microorganisms and different techniques are needed to evaluate their functional activities. Here we describe a rapid, simple and reliable one step procedure to measure the phagocytosis rate and oxidative burst activation of both polymorphonuclear leukocytes (PMN) and monocytes, by means of flow cytometry using only small quantities of whole blood. The two species of bacteria employed as test microorganisms, Staphylococcus aureus and Candida albicans showed a somewhat different behaviour. We found that the present method could be used as an alternative test in the diagnosis of chronic granulomatous disease (CGD). Moreover we were able to analyse, in a one step procedure, defective phagocyte functions in a group of paediatric patients suffering from recurrent microbial infections.

Flow cytometric detection of anti-gliadin antibodies.

A very sensitive solid-phase fluorescent immunoassay to detect anti-alpha-gliadin IgA class antibodies is described. The solid phase consisted of polystyrene carboxylated microspheres, of 5 microns diameter, coated with alpha-gliadin. Serum-specific antibodies bound to the alpha-gliadin were measured by flow cytometry using fluorescein-conjugated anti-human IgA. 41 samples were tested and the results compared with those obtained by a standard method: an enzyme-linked immunosorbent assay (ELISA). A good correlation was found between the two techniques (r = 0.96). The sera of untreated coeliac children showed significantly higher antibody values than the sera of children on a gluten-free diet or healthy control groups. The flow cytometric method was more sensitive when the Kolgomorov/Smirnov test was used to analyse the histograms. This method provides an alternative screening test for coeliac disease and may also be used to confirm borderline results obtained in the ELISA test.

Simultaneous measurement by flow cytometry of phagocytosis and metabolic burst induced in phagocytic cells in whole blood by Borrelia burgdorferi.

This paper describes the interactions between a strain of Borrelia burgdorferi and phagocytic cells, measured in whole blood, by a two-color flow cytometric method, which allowed the simultaneous quantification of both the phagocytosis rate and the oxidative burst activation. The data obtained indicated that: a) phagocytosis and metabolic activation increased as a function of spirochete concentration; b) the number of ingesting cells peaked within 10 min but activation followed later, and did not involve all the phagocytosing cells; c) opsonization of borreliae with a patient's serum enhanced the two cellular activities, mostly phagocytosis. The intensity of such functions was lower than those found for Staphylococcus aureus. The flow cytometric assay of phagocytes interactions with Borrelia burgdorferi assessed in whole blood represents an experimental approach which simulates the physiological conditions in nature.

Study of IL-2, IL-6, TNF alpha, IFN gamma and beta in the serum and synovial fluid of patients with juvenile chronic arthritis.

In the last few years the important role played by various cytokines in the pathogenesis of chronic inflammatory diseases has emerged. In the present study, serum and synovial fluid levels of IL-2, IL-6, TNF alpha, IFN beta and IFN gamma were evaluated in a group of 66 patients with juvenile chronic arthritis (JCA). At the same time the ESR, CRP, hemoglobin, immunoglobulins, platelet count and Ritchie index were measured. In the serum of pauciarticular patients, IL-6 and TNF alpha levels were only slightly elevated compared with controls, but there was no correlation between these cytokines and clinical and other laboratory parameters. Serum IL-2 and IFN gamma were undetectable. In contrast, in the synovial fluid IL-6 levels were very high in all of the patients examined and there was a significant correlation between synovial fluid IL-6 levels and Ritchie's articular index. TNF alpha tended to be elevated but to a lesser extent, while synovial fluid IL-2 and IFN gamma were undetectable or very low, as in the serum. In polyarticular and systemic patients, on the other hand, serum IL-6 was elevated and statistically correlated with the majority of the laboratory parameters and with the Ritchie articular index. TNF alpha levels were only slightly elevated; on the other hand, IL-2 and IFN gamma were undetectable. There was an inverse correlation between IFN beta levels and the Ritchie articular index and a significant correlation with hemoglobin levels. In conclusion, our study demonstrates that not only IL-1 (as shown in other studies), but also IL-6 and to a lesser extent TNF alpha play a central role in the pathogenesis of JCA. IFN beta on the other hand, would seem to play an anti-inflammatory role.

Treatment of juvenile idiopathic arthritis with intra-articular triamcinolone hexacetonide: evaluation of clinical effectiveness correlated with circulating ANA and T gamma/delta + and B CD5+ lymphocyte populations of synovial fluid.

OBJECTIVE: The aims of the study were to assess the effect of intra-articular treatment with triamcinolone hexacetonide (TH) in juvenile idiopathic arthritis (JIA) and to investigate whether treatment response correlates with the presence of antinuclear antibodies (ANA) in the serum and/or B CD5+ and T gamma/delta + lymphocytes in the synovial fluid. METHODS: A total of 37 patients (81% females, 56% ANA+) with oligoarticular JIA involving knees were treated with intra-articular injections of TH after failing to respond to NSAIDs for two months. Eighteen patients were treated within 6 months of onset, 19 were treated more than 6 months after onset. RESULT: Mean duration of remission was 13.9 months. Twelve patients (7 ANA+) had stable remission after a single injection; 13 patients (3 ANA+) experienced more than 6 months' remission but subsequently had a relapse; 12 patients (11 ANA+) had a relapse within six months of injection. Of 20 patients treated within 6 months of onset, 17 had stable remission whereas only 8 out of 17 who were treated during relapse attained stable remission (p = 0.03). The mean percentage of T gamma/delta + and of B CD5+ lymphocytes in synovial fluid was the same as in peripheral blood of normal subjects. CONCLUSION: Our data indicate that local treatment with slow-release steroids is very effective in oligoarticular JIA. Prolonged remission was less likely in the presence of ANA positivity, probably because the disease is immunologically more active. Finally, our data suggest that the earlier the treatment, the easier it is to obtain a protracted, and possibly permanent, response.

Flow cytometric analysis of DNA content in cervical lesions.

Flow cytometry is used to screen gynaecologic specimens for Cervical Intraepithelial Neoplasia (CIN) and its precancerous lesions, possibly associated with human papilloma virus (HPV), in order to investigate the role of aneuploidy as a biological marker in HPV and CIN lesions. A total of 299 cervical samples was collected by scraping and the cellular DNA content was measured using the propidium iodide staining procedure. Two groups of patients were studied; a group of 142 negative controls for cytology and groups of patients assigned to mild, moderate or severe dysplasia, carcinoma in situ diagnosed by histologic classification according to the Papanicolaou staining technique. Pathological samples showing an alteration of the DNA index or perturbation of the cellular phase S compartment ranged from 6.4% to 41.9%. Our results confirm those obtained by other authors and suggest the hypothesis that aneuploidy is present with permanence of viral DNA in early stages of carcinogenesis, which can be used as a marker in the transition from benign to malignant cells. This work is of potential interest for the possible follow-up of patients having condyloma and could aid the early diagnosis of cervical carcinoma.

CD69 expression on alpha-gliadin-specific T cells in coeliac disease.

Coeliac disease (CD) is a T-cell mediated immunological disease of the small intestine which is triggered in susceptible individuals by ingestion of gluten. The pathogenic mechanism of coeliac disease, and the role that alpha-gliadin specific T cells play in mucosal lesions and their involvement in peripheral blood is not yet explained at all. Previous studies have reported proliferative response to alpha-gliadin measured with the classic assay of 3HTdR incorporation. We analysed the activation antigen CD69 on T cells from CD patients and normal individuals following stimulation with alpha-gliadin and different antigens (tetanus toxoid, peptides unrelated to gliadin and PHA). CD69 coexpression with T cell CD3+ and proliferation marker Ki67 was evaluated with time. CD69 coexpression with T cell CD3+, CD4+ and CD8+ was also evaluated. It was found that peripheral blood mononuclear cells (PBMC) of coeliac patients increased their percentage of CD69 positive T cells when stimulated with alpha-gliadin, in comparison with cells from controls. Significant T cell activation was found only in subjects not treated with the gluten free diet; a positive response was found also in two coeliac patients with selective IgA deficiency, anti-endomisium negative, without circulating IgA anti alpha-gliadin or anti-tissue transglutaminase antibodies. The CD69 expression after stimulation was compared with the standard method of 3HTdR incorporation. Our data show that CD69 expression is useful to asses a specific T cell response to alpha-gliadin in coeliac disease. in a very short time. Moreover, the method allows to investigate T cell response at the lymphocyte subsets level, which represents a useful tool in the diagnosis of coeliac disease.

Evaluation of the serum T cell population variations induced by PUVA therapy.

In the present study we evaluated the effects of UVA irradiation on the serum T cell subpopulations (CD3, CD4, CD8, CD4/CD8, NK, T lymphocytes/mm3) by means of a Fluorescence Activate Cell Sorter (F.A.C.S. 420, Becton Dikinson). Twenty-eight psoriatic patients, never previously treated with PUVA, received UVA radiations (cabin Waldman 8001 K). Blood samples were taken before therapy and after 60 J/cm2 and 120 J/cm2 irradiations. Results were compared with those of our healthy control group of volunteers. A decrease of CD4 positive cells was observed in the psoriatic group before therapy. PUVA did not induce any statistically significant modification of the populations studied, except for a progressively increasing trend of CD4 positive cells. These data seem to stress that a medium-term PUVA therapy does not significantly modify the serum level of the T cell populations.

Surface receptors of neutrophils towards B. burgdorferi.

The spirochetal agent of Lyme borreliosis, Borrelia burgdorferi, is able to induce an infection which develops in three stages: an early, localized infection, disseminated infection and a third stage, chronic infection, which probably indicates that a protected niche has been established in one or more tissues, where the spirochetes persist even if a specific immune response has been initiated. During the first stage, immediately after their entry into the host tissue, B. burgdorferi meet the motile phagocytic cells, neutrophils and monocytes; this is followed by consequent phagocytosis and killing. Although the rate and mechanism of this killing is not entirely clear, there is evidence that phagocytosis by both neutrophils and monocytes proceeds even in the absence of specific antibodies. We have demonstrated in both neutrophils and CHO Mac-1 (CR3 integrin) transfected cells, that one phagocyte receptor which is involved in B. burgdorferi adhesion in non osponic phagocytosis is the CR3 complement receptor known as integrin alpha m beta 2. Both recognition domains of the integrin, the iC3b site and the COOH terminal lectin site, bind to B. burgdorferi. Data presented here show that inhibition of adhesion on CR3 Mac-1 transfected cells and neutrophils is induced by mannose as well as by N-acetyl-D-glucosamine, sugars known to be specific inhibitors of the COOH terminal lectin-site of the integrin CR3. The inhibitory effect was serum complement independent. On the contrary, monoclonal antibody VIM12 directed towards the lectin domain not only failed to inhibit but improved adhesion, suggesting that, as a consequence of the binding, the integrin becomes more receptive to B. burgdorferi attachment at the I domain. Pretreatment of the borrelias with NalO4 eliminated adhesion, suggesting that the sugar residue/s recognized by CR3 is located on the bacteria.

A new multiparameter flow cytometric method for human semen analysis.

BACKGROUND: The objectives of this study were (i) to evaluate whether the combined use of Syto 16 and 7-amino-actinomycin-D (7-AAD) allows the detection of sperm apoptosis and (ii) to describe a new multiparameter flow cytometric method to assess simultaneously sperm concentration (SC), viability and apoptosis as well as leukocyte concentration. METHODS: Semen samples from 68 patients were evaluated according to World Health Organization (WHO) criteria (normal, n=26; abnormal, n=42). The detection of activated caspases before and after betulinic acid (BA) incubation was carried out in 13 semen samples by flow cytometry using fluorescein-labelled inhibitors of caspases (FLICA). A multiparameter flow cytometric analysis was performed in 55 semen samples. Fluorescent microspheres were used to assess SC. Sperm apoptosis was detected by staining sperm with Syto 16 and 7-AAD. Leukocytes were counted using monoclonal anti-CD45. RESULTS: A significant correlation between the percentage of the spermatozoa with low Syto 16 fluorescence and the percentage of spermatozoa containing activated caspases was found (r=0.68, P=0.0106; n=13). After incubation with BA, an increase of the percentage of apoptotic cells was observed in all samples, using both the Syto 16/7-AAD and the caspase activation methods. There was a good correlation between flow cytometry and optical microscopy for sperm (r=0.98, P < 0.0001) and leukocyte counting (r=0.64, P <0.0001). The percentage of apoptotic sperm was inversely correlated with both SC (r=-0.303, P=0.0246) and morphology (r=-0.384, P=0.0050) but not with motility. CONCLUSIONS: The combination of Syto 16/7-AAD provides a sensitive assay to detect sperm apoptosis. The multiparameter flow cytometric method described offers the possibility of a simultaneous, simple, rapid and accurate assessment of several semen parameters.

Simultaneous flow cytometric method to measure phagocytosis and oxidative products by neutrophils.

We developed a rapid and sensitive two-color flow cytometric method which allows the simultaneous quantification of both the phagocytosis rate and the oxidative burst activation of polymorphonuclear leukocytes (PMNLs). The oxidation of hydroethidine (HE) to ethidium bromide (EB) was performed by the oxidative neutrophil products within the cells during the respiratory burst, which was stimulated by phagocytized fluorescein-labeled Staphylococcus aureus. By means of flow cytometry we measured red EB fluorescence emission together with green fluorescence, which was emitted by the ingested fluoresceinated bacteria. The fluorescence intensity was proportional to the number of bacteria ingested. Adherent bacteria were distinguished from the ingested ones. This two-color cellular staining permits measurement of two different functions of neutrophils in one step. This method could be of interest for the determination of the interactions between neutrophils and bacteria and for the investigations on infectious diseases in chronic granulomatous disease patients.

An analysis of T-lymphocyte subpopulations in psoriasis using monoclonal antibodies.

We evaluated subpopulations of T-lymphocytes by using the monoclonal antibodies OKT3, OKT4 and OKT8 in two groups of psoriatic patients, with active and stable psoriasis respectively. Whereas data from the patients with stable psoriasis were similar to those obtained from the control group, the patients in an acute flare condition revealed a relative decrease in lymphocytes reactive with OKT8, and a slight increase in the proportion of lymphocytes reactive with OKT4. The results of this study are analysed.

Integrin CR3 mediates the binding of nonspecifically opsonized Borrelia burgdorferi to human phagocytes and mammalian cells.

Like other pathogens, the spirochete Borrelia burgdorferi, the agent of Lyme disease, possesses multiple pathways for cell binding; adhesion to phagocytic cells is of particular interest since it reportedly occurs even in the absence of specific antibodies. This study sets out to investigate how B. burgdorferi binds to human polymorphonuclear leukocytes (PMNs) when an exogenous complement is added and how the CR3 complement receptor, known as Mac-1 or alpha(m)beta2 integrin, is involved in the binding process. Experiments performed on PMNs and CHO Mac-1-expressing cells demonstrate that binding is inhibited by monoclonal anti-iC3b site antibodies, fibrinogen, and N-acetyl-D-glucosamine. These findings, which are not present with non-Mac-transfected CHO cells, indicate that the integrin alpha(m)beta2 acts as a receptor for spirochetes in nonimmune phagocytosis; furthermore, binding occurs on different domains of the CD11b subunit, involving the iC3b site and the lectin domain. The interaction of B. burgdorferi with alpha(m)beta2 integrin adds a novel pathway to Borrelia-phagocyte binding; not only does this binding affect the early stages of phagocytosis, but also it can influence the effector intracellular mechanisms which are activated by the beta2 integrin, as are the cytotoxic mechanisms.

Rapid flow cytometric studies of Borrelia burgdorferi phagocytosis by human polymorphonuclear leukocytes.

The interactions between a strain of Borrelia burgdorferi and human polymorphonuclear leukocytes were studied by flow cytometry in the presence of specific or non-specific opsonizing factors. The capacity of the borrelias to stimulate leukocyte metabolism was also investigated. The results indicated that a low phagocytosis by isolated purified polymorphonuclear leukocytes did occur in the presence or absence of specific antibodies. Within whole blood the percentages of phagocytosting leukocytes increased in the presence of non-specific opsonizing factors. No stimulation of the oxidative metabolism stimulated by Borrelia was observed and PMA or zymosan stimulation of leukocytes was inhibited by the spirochaetes.

Interaction with Borrelia burgdorferi causes increased expression of the CR3 integrin and increased binding affinity to fibronectin via CR3.

We have previously demonstrated that the alphaMbeta2 integrin (known as CR3 or Mac-1) expressed on neutrophils (PMNs) and/or on CHO Mac-1 transfected cells,in the presence of serum complement binds B. burgdorferi and promotes an increased non -opsonic adhesion, in the presence of serum complement. In this study we demonstrate that: 1) living motile B. burgdorferiand recombinant lipidated OspA and OspC, up-regulate CR3 expression on PMNs; 2) in the absence of serum, B. burgdorferi induces increased adhesion of CHO cells expressing CR3 to fibronectin, an extracellular matrix protein. Both the I-domain and the lectin-like domain of CR3 are involved in the binding recognition and activation because mAb anti I-domain and N-acetyl-glucosamine inhibit cell adhesion to fibronectin. These data indicate that B. burgdorferi whole cells, but not Osps, activate CR3 integrin; since this receptor plays a key role in priming neutrophils to important inflammatory events, the interaction of B. burgdorferi with neutrophils via the CR3 may enhance their role both in defence and in disease.