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 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.

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.

Apoptosis in human sperm: its correlation with semen quality and the presence of leukocytes.

BACKGROUND: Apoptosis plays an important role in regulating spermatogenesis. However, the biological significance of apoptosis in ejaculated sperm is not yet clear. This study set out to investigate how apoptosis correlates with semen quality and the presence of seminal leukocytes. METHODS: Fifty-seven semen samples from the male partners of infertile couples were classified as normal or abnormal according to World Health Organization guidelines. Flow cytometry was used to evaluate sperm populations and seminal leukocytes. Preliminary flow cytometry analysis using 6-carboxyfluoresceindiacetate (6-CFDA), which identifies live cells, and propidium iodide (PI), which stains only dead cells, was performed in order to pinpoint the sperm region accurately. Having thus gated the sperm population, bivariate Annexin V/PI analysis was then carried out in order to measure the percentage of apoptotic and necrotic sperm and the apoptotic index (the ratio between apoptotic:live sperm). Leukocytes were counted by the standard peroxidase test and by flow cytometry using monoclonal antibody (mAb) anti-CD45 or anti-CD53. RESULTS: No significant differences in the apoptotic index and the percentage of live and apoptotic sperm were detected between the subjects with normal and abnormal semen. A significant inverse correlation between sperm concentration and the apoptotic index was observed only in the normal sperm group. There was no correlation between the concentration of leukocytes, detected either by peroxidase or by mAb anti-CD45 or anti-CD53, either with the percentage of apoptotic sperm or with the apoptotic index. In contrast, the ratio between CD45 positive leukocytes and sperm showed a significant correlation with the apoptotic index. A weaker correlation was found when leukocytes were counted by peroxidase, while no correlation was observed using mAb anti-CD53. CONCLUSIONS: Sperm apoptosis did not seem to be correlated with semen quality. In the absence of genito-urinary infection, one of the main functions of seminal leukocytes is probably to provide for the removal of apoptotic sperm.

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.

Evidence of involvement of the mannose receptor in adhesion of Borrelia burgdorferi to monocyte/macrophages.

The mannose receptor (MR) plays an important role in the recognition of some pathogens in nonopsonic phagocytosis and in antigen presentation to T cells. We found that Borrelia burgdorferi, the agent of Lyme borreliosis, adheres to monocyte-derived macrophages and to rat MR-transfected cells but not to untransfected cells. Antibodies to MR and sugars such as mannose, mannan, fucose, and some lectins significantly lowered the adhesion, confirming participation of the MR in the binding.

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.

TNF-Induced shedding of TNF receptors in human polymorphonuclear leukocytes: role of the 55-kDa TNF receptor and involvement of a membrane-bound and non-matrix metalloproteinase.

A down-modulation of both the 55-kDa (TNF-R55) and the 75-kDa (TNF-R75) TNF receptors is observed in neutrophils exposed to a variety of stimuli. Proteolytic cleavage of the extracellular region of both receptors (shedding) and, with TNF, internalization of TNF-R55 and shedding of TNF-R75 are the proposed mechanisms. We have characterized the TNF-induced shedding of TNF receptors in neutrophils and determined the nature of the involved proteinase. Neutrophils exposed to TNF release both TNF receptors. A release of TNF receptors comparable to that observed with TNF was induced with TNF-R55-specific reagents (mAbs and a mutant of TNF) but not with the corresponding TNF-R75-specific reagents. A hydroxamic acid compound (KB8301) almost completely inhibited shedding of TNF-R55 and to a lesser degree shedding of TNF-R75. KB8301 also inhibited FMLP-induced shedding to a similar extent. Shedding was also inhibited by 1,10-phenanthroline, but this effect was considered nonspecific as the compound, at variance with KB8301, almost completely inhibited TNF and FMLP-induced PMN activation. Diisopropylfluorophosphate partially inhibited shedding of TNF-R75, suggesting the contribution of a serine proteinase to the release of this receptor. Shedding activity was not affected by matrix metalloproteinases inhibitors nor was it released in the supernatants of FMLP-stimulated neutrophils. These results suggest that TNF induces release of its receptors, that such a release is mediated via TNF-R55, and that a membrane-bound and non-matrix metalloproteinase is involved in the process. The possibility that ADAM-17, which we show to be expressed in neutrophils, might be the involved proteinase is discussed.

Leukocyte detection in human semen using flow cytometry.

This study set out to establish a new method, using flow cytometry, to evaluate leukocytes in semen. Ejaculates of 59 males, asymptomatic for genitourinary infections, were examined. Routine semen analyses were carried out as well as peroxidase and polymorphonuclear granulocyte-elastase detection. Leukocytes were detected combining flow cytometry and monoclonal antibodies (anti-CD45, anti-CD53). This technique reliably assessed the total number of leukocytes and differentiated subpopulations even at low concentrations. The peroxidase test and elastase determination showed good specificity, but only moderate sensitivity versus flow cytometry combined with monoclonal antibodies. No significant association was observed between semen parameters and leukocytospermia whether evaluated by conventional methods or flow cytometry except for a moderate correlation between spermatozoa and CD53-positive cell concentrations. A first comparison of data from patients grouped on the basis of leukocytospermia (>10(6) white blood cells, WBC/ml) or non-leukocytospermia revealed no significant differences in semen parameters; lowering the threshold value for leukocytospermia to 2x10(5) WBC/ml, sperm concentration was reduced in the group with a low number of WBC identified by monoclonal antibodies. Flow cytometry using monoclonal antibodies was seen to be a simple, reproducible method that enables leukocytes in semen to be accurately detected and to identify WBC subpopulations without preliminary purification procedures.

In vitro incubation of bone marrow and peripheral stem cells with vincristine and methylprednisolone: functional T-cell depletion for haploidentical and autologous transplants.

A mismatched bone marrow transplantation is feasible only if the donor's marrow lymphocytes are eliminated from the graft. This can be achieved by several methods, but all have the disadvantage of inducing a long-lasting immune deficiency while the risk of graft rejection and leukemic relapse increase. We use a sort of functional T-cell depletion by treating the cells with vincristine and methylprednisolone. This method is surely the cheapest and has allowed us to perform 60 transplants with a tolerable risk of GVHD. The treatment of the donor's lymphocytes has already been demonstrated to be able to block the mixed lymphocyte culture reaction in vitro. In this experiment Th1 and Th2 activities were almost completely blocked without reduction of lymphocyte viability and apoptosis induction.

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.

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.

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.

Prevalence of celiac disease in patients with juvenile chronic arthritis.

We estimated the prevalence of celiac disease in children with juvenile chronic arthritis (JCA), using antiendomysium antibodies as the screening test to select patients for intestinal biopsy. We studied 119 children with JCA and found four patients with antiendomysium antibodies. In three of these patients (2.5%), intestinal biopsy revealed villous atrophy; in the fourth the intestinal mucosa was normal. We conclude that the prevalence of celiac disease is increased in patients with JCA.

Flow cytometric assay for the evaluation of phagocytosis and oxidative burst of polymorphonuclear leukocytes and monocytes in myelodysplastic disorders.

BACKGROUND: Myelodysplastic syndromes are clonal diseases characterized by pancytopenia of variable degree. Neutropenia is common and several morphologic and functional abnormalities of polymorphonuclear neutrophilic granulocytes (PMNs) and/or monocytes have been described. On the basis of these observations, the phagocytic and oxygen intermediates production of PMNs and monocytes was determined in a group of forty-seven patients affected by myelodysplastic syndromes of varying severity. METHODS: A rapid, simple and reliable flow cytometric method was developed to evaluate, in a one-step procedure, the phagocytosis rate and the oxidative burst in PMNs and monocytes using a small amount of whole blood. RESULTS: Phagocytosis of PMNs and monocytes was not significantly reduced in refractory anemia (RA), while in refractory anemia with excess of blasts (RAEB) and in chronic myelomonocytic leukemia (CMML) a clear decrease (p < 0.05) of this function was found in both PMNs and monocytes. The production of oxygen intermediates by PMNs and monocytes was significantly (p < 0.01) reduced in RA as well as in RAEB and in CMML. CONCLUSIONS: This study indicates the presence in myelodysplastic syndromes of a severe reduction in phagocytosis and oxygen intermediates production (two crucial functions to protect the host against pyogenic agents) in both PMNs and monocytes. This observation could explain the severe morbidity and mortality from infections in patients affected by these hematological malignancies.

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.

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.

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.

Biological activity of a peptidoglycan extracted from Leptospira interrogans: in vitro studies.

Peptidoglycan (PG) has been isolated from some species of spirochaetes, including Leptospira interrogans. Although leptospiral PG has been chemically characterized, no study has been carried out on its potential biological activity. Since PG of Treponema and Borrelia is biologically active both in vivo and in vitro, we investigated the capacity of a leptospiral PG preparation to induce relevant biological effects. PG extracted from L. interrogans strain Teramo was mitogenic at 0.1 microgram ml-1 for human peripheral blood mononuclear cells (PBMC) since it increased the PBMC fraction positive for Ki-67, an antigen expressed by human proliferating cells; at 4 micrograms ml-1, PG was able to induce complement consumption and to stimulate leucocyte phagocytosis and the metabolic burst of resting as well as phagocytosing leucocytes. These findings indicate that Leptospira PG may play a role in modulating the immunocompetent cell functions and suggest that PG can contribute to the host response during Leptospira infection.