Sandra Perticarari

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.

Serum-Resistant Strains of Borrelia burgdorferi Evade Complement-Mediated Killing by Expressing a CD59-Like Complement Inhibitory Molecule

Borrelia burgdorferi, the etiological agent of Lyme disease, comprises three genospecies, Borrelia garinii, afzelii, and burgdorferi sensu strictu, that exhibit different pathogenicity and differ in the susceptibility to C-mediated killing. We examined C-sensitive and C-resistant strains of B. burgdorferi for deposition of C3 and late C components by fluorescence microscope and flow cytometry. Despite comparable deposition of C3 on the two strains, the resistant strain exhibited reduced staining for C6 and C7, barely detectable C9, and undetectable poly C9. Based on these findings, we searched for a protein that inhibits assembly of C membrane attack complex and documented an anti-human CD59-reactive molecule on the surface of C-resistant spirochetes by flow cytometry and electron microscopy. A molecule of 80 kDa recognized by polyclonal and monoclonal anti-CD59 Abs was identified in the membrane extract of C-resistant strains by SDS-PAGE and Western blot analysis. The molecule was released from the bacterial wall using deoxycholate and trypsin, suggesting its insertion into the bacterial membrane. The CD59-like molecule acts as C inhibitor on Borrelia because incubation with F(ab′)2 anti-CD59 renders the serum-resistant strain exquisitely susceptible to C-mediated killing and guinea pig erythrocytes bearing C5b-8, unlike the RBC coated with C5b-7, are protected from reactive lysis by the bacterial extract. Western blot analysis revealed preferential binding of the C inhibitory molecule to C9 and weak interaction with C8β.

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.

Semen preparation methods and sperm apoptosis: swim-up versus gradient-density centrifugation technique.

OBJECTIVE To compare the effects of density-gradient centrifugation and swim-up on sperm apoptosis by using a multiparameter flow cytometric method. DESIGN Autocontrolled split-sample study. SETTING Tertiary infertility center. PATIENT(S) Sixty-two male partners of couples undergoing infertility investigations. INTERVENTION(S) Each sample was analyzed both before and after semen preparation by optical microscopy and by flow cytometry. MAIN OUTCOME MEASURE(S) Percentage of viable, apoptotic, and necrotic sperm and recovery rate of total motile, progressive motile, and viable sperm before and after the two sperm preparation methods. RESULT(S) Compared with the original semen, the mean percentages of apoptotic and necrotic sperm were significantly lower after both sperm preparation methods. The mean percentage of viable sperm was significantly higher after swim-up compared with gradient centrifugation. The recovery rates of total motile, progressive motile, and viable sperm were significantly higher using gradient centrifugation compared with swim-up. The viable sperm percentage and the progressive sperm motility were significant predictors for negative difference between the two methods in terms of viable sperm percentage after preparation. CONCLUSION(S) Both sperm preparation methods allow obtaining a sperm population with a low percentage of apoptotic sperm. Therefore, the risk of using apoptotic sperm for clinical treatment seems to be rather low. The choice of method will depend on whether IVF/ICSI or intrauterine insemination is to be performed.

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

ABSTRACT 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.

Leukocytospermia and sperm preparation - a flow cytometric study

BackgroundLeukocytes represent the predominant source of reactive oxygen species both in seminal plasma and in sperm suspensions and have been demonstrated to negatively influence sperm function and fertilization rate in assisted reproduction procedures. Peroxidase test is the standard method recommended by WHO to detect semen leukocytes but it may be inaccurate. The aims of this study were (i) to compare the efficiency of swim-up and density-gradient centrifugation techniques in removing seminal leukocytes, (ii) to examine the effect of leukocytes on sperm preparation, and (iii) to compare flow cytometry and peroxidase test in determining leukocyte concentration in semen using a multiparameter flow cytometric method.MethodsSemen samples from 126 male partners of couples undergoing infertility investigations were analyzed for leukocytospermia using standard optical microscopy and flow cytometry. Sixty-nine out of 126 samples were also processed using simultaneously the swim-up and density-gradient centrifugation techniques. A multiparameter flow cytometric analysis to assess simultaneously sperm concentration, sperm viability, sperm apoptosis, and leukocyte concentration was carried out on neat and prepared sperm.ResultsBoth sperm preparation methods removed most seminal leukocytes. However, the concentration of leukocytes was significantly lower after swim-up compared to that after density-gradient centrifugation preparation. Leukocytes concentration, either initial or in prepared fractions, was not correlated with sperm parameters (optical microscopy and flow cytometry parameters) after semen processing. There was no correlation between leukocyte concentration in the ejaculate and sperm recovery rate, whereas a significant correlation was found between the concentration of the residual leukocytes in prepared fractions and viable sperm recovery rate. Although the overall concordance between the flow cytometry and the optical microscopy was satisfactory, the sensitivity of peroxidase test for the detection of leukocytospermia resulted low.ConclusionSeminal leukocytes do not seem to influence sperm preparation results. However, for assisted conception, semen samples containing leukocytes should be processed using swim-up method. Although peroxidase-test is recommended by WHO as the standard method for determining semen leukocytes, it should not be used in clinical research study.

Chronic infantile neurological cutaneous articular syndrome: CD10 over-expression in neutrophils is a possible key to the pathogenesis of the disease

Chronic, Infantile, Neurological, Cutaneous and Articular Syndrome (CINCA) or Neonatal/Infantile Onset Multisystem Inflammatory Disease (NOMID/IOMID) is a rare, multisystem inflammatory disease characterised by neonatal onset of urticarial symptoms, persistent rash, ocular inflammatory lesions, progressive articular and neurological involvement and associated with characteristic overgrowth of the ossification nucleus of the patella. The tissues involved are extensively infiltrated by inflammatory cells, mostly neutrophils. This paper describes the clinical features of three new cases as well as a study of activation markers in neutrophils and search for mutations of the CIAS1gene in these patients. Clinical records of three cases of CINCA are reported. For genetic analysis, exon 3 of the CIAS1gene was amplified and sequenced. Immunophenotype, oxidative burst and phagocytosis were analysed in neutrophils obtained from all the three CINCA patients as well as from eight juvenile idiopathic arthritis (JIA) patients and eight healthy controls. Functional assays in neutrophils were normal in all three patients with CINCA syndrome and did not differ from those of JIA patients and healthy controls. The surface density of CD10 was significantly higher on neutrophils from CINCA patients as compared to those of JIA and controls ( P <0.0005). In one subject a new missense mutation in the CIAS1gene was identified. Conclusion: the hyper expression of the activation antigen CD10/NEP in neutrophils from these three cases of CINCA, as compared to JIA patients and healthy controls, irrespective of the presence of mutations in CIAS1, could be a marker of the inflammatory disorder typical of some patients with CINCA syndrome.

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.

ELISA method for quantitative measurement of IgA and IgG specific anti-gliadin antibodies

Summary In this study we describe the application of an enzyme-linked immunosorbent assay (ELISA) for the measurement of anti-α-gliadin antibodies (AGA) in absolute units (μmlg protein/ml). Enriched samples of IgA and IgG AGA were obtained by means of protein A chromatography after immunoaffinity purification of pooled sera from untreated celiac patients. No cross-reactivity with other food antigens (β-lactalbumin, soya proteins, ovalbumin) was detected. The quantitative evaluation of protein content in IgA and IgG AGA samples obtained by immunoaffinity chromatography, was performed by means of ELISA sandwich method using a reference curve obtained with pure standard human immunoglobulins. Scalar concentrations of purified IgA and IgG were then used to obtain a reference standard curve by means of an ELISA method. Such standard curve was utilized for titrating AGA in 214 sample sera. The minimal detectable concentration of IgA and IgG AGA was 0.02 μmlg/ml. The reproducibility of within- and between-assay resulted very good for IgA-AGA and acceptable for IgG-AGA. The method here described seems to be satisfactory not only for quantitative diagnostic purposes in routine screenings but also in epidemiological studies. Moreover, it can constitute a suitable way to solve practical problems of quality control of AGA ELISA assay.

A dot immunobinding assay to detect anti-α-gliadin antibodies in celiac disease

A dot immunobinding assay to detect anti-α-gliadin-specific antibodies in the sera or whole blood of enteropathic patients is described here. The method is based on the adsorption of α-gliadin as a spot onto nitrocellulose sheets. After incubation with the patient sample, the detection of specific antibodies is performed with alkaline phosphatase-conjugated goat anti-human (IgA or IgG) antibodies. Twenty-one celiac serum samples together with 18 enteropathic or disease controls and 44 healthy controls were analyzed. The classical ELISA test and the dot test gave comparable results. The dot test gave reliable result even when whole blood was tested. The method proved to be simple and sensitive.