Leukocytospermia and sperm preparation--a flow cytometric study.

BACKGROUND: Leukocytes 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. METHODS: Semen 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. RESULTS: Both 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. CONCLUSION: Seminal 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.

Heterozygous nucleotide-binding oligomerization domain-2 mutations affect monocyte maturation in Crohn's disease.

AIM: To investigate the function of monocytes in Crohn's disease (CD) patients and to correlate this with disease-associated nucleotide-binding oligomerization domain-2 (NOD2) gene variants. METHODS: Monocytes from 47 consecutively referred CD patients and 9 healthy blood donors were cultured with interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF), and stimulated with lipopolysaccharide (LPS) or muramyldipeptide (MDP), the putative ligand of NOD2. RESULTS: We found that monocytes from CD patients differentiated in vitro to mature dendritic cells (DCs), as determined by immunophenotype and morphology. NOD2 genotype was assessed in all subjects, and we observed high CD86 expression on immature and LPS-stimulated DCs in NOD2 mutated CD patients, as compared with wtNOD2 CD patients and controls. By contrast, CD86 expression levels of DCs induced to maturity with MDP derived from NOD2-mutated subjects were comparable to those of normal subjects. The amount of IL-12p70 in patient-cell cultures was larger than in controls after LPS treatment, but not after treatment with MDP. CONCLUSION: Our results suggest that DCs obtained from patients with mutations in the NOD2 gene display an activated phenotype characterized by high CD86 expression, but have a diminished response to MDP when compared to the terminal differentiation phase. We speculate that the altered differentiation of monocytes might lead to an imbalance between inflammation and the killing ability of monocytes, and may be relevant to the pathogenesis of CD.

Interaction of leptospires with murine microglial cells.

Leptospires, the agents of leptospirosis, exert tropism for the central nervous system, in the course of mammal infection. We evaluated the interaction between murine microglial cells and strains of pathogenic L. interrogans leptospires and non-pathogenic L. biflexa leptospires, mainly by flow cytometric assays. In the absence of opsonic conditions microglia are capable of ingesting--even quite slowly--the spirochetes and killing the non-pathogenic strain. The adhesion to microglia, which is quick and relevant for all the strains, does not involve the CR3 integrin receptor. These findings suggest that the murine microglia--in non opsonic conditions in vitro--do not effectively clear the pathogenic leptospires.

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.

Leptospira interrogans binds to the CR3 receptor on mammalian cells.

Pathogenic leptospires adhere to phagocytes even in the absence of specific antibodies; we demonstrate that the CR3 integrin (known as Mac-1 or CD11b/CD18 receptor), expressed on neutrophils and CHO Mac-1 transfected cells, recognizes and binds leptospires. The I-domain of the molecule seems to be involved in the recognition and data suggest that this occurs via fibronectin absorbed at the Leptospira surface. On the other hand, since N-acetyl-D-glucosamine does not behave as antagonist of the binding, the lectin-like domain of the receptor is not involved in the recognition.

Lymphocyte apoptosis co-cultured with Borrelia burgdorferi.

Borrelia burgdorferi is the causative agent of Lyme disease. We investigated whether the in vitro co-cultivation of lymphocytes with spirochetes would induce apoptosis in human lymphocytes. Peripheral blood mononuclear cell were mixed with various ratio of cell/spirochetes (1:10, 1:20, 1:50, 1:100) and incubated in a humified atmosphere of 5% CO(2) at 37 degrees C. Apoptosis was determined at 0, 4, 24 h by Annexin V binding assay and propidium iodide staining, and by CD95 Apo-1 expression. Analysis was performed by multiparametric flow cytometry on CD3, CD4, CD8, CD19 subset of lymphocytes. The binding of Annexin V increased at 24 h in T lymphocytes infected by living spirochetes at ratio 1:50; similar results were obtained with inactivated or sonicated spirochetes and lipidated OspC. The rate of Annexin V binding and pattern of CD95 over-expression were different in CD3, CD4, CD8 and CD19 subset; interleukine-10 (IL-10) was measured in supernatants of cultures after treatment with Borrelia preparations and with OspA and OspC, lipidated or not. Our data suggest that spirochetes were able to induce apoptosis on lymphocytes; the phenomenon appears associated with number of spirochetes, incubation time and the release of IL-10 in co-cultures. Moreover apoptosis was probably Fas-mediated and the cells involved were prevalently CD4.

Measurement of phagosomal pH of normal and CGD-like human neutrophils by dual fluorescence flow cytometry.

BACKGROUND: Phagosomal pH is thought to play an important role in the antimicrobial activity of polymorphonuclear leukocytes (PMNs). In this study, we set up a method for a rapid and accurate measurement of phagosomal pH in PMNs with the use of Candida albicans doubly labeled with a pH-insensitive and a pH-sensitive probe and flow cytometry. METHODS: Heat-killed, serum-opsonized C. albicans were doubly labeled with fluorescein, a pH-sensitive probe, and rhodamine, a pH-insensitive probe, and incubated with human PMNs. Flow cytometric readings of PMN-associated Candida were then taken, and the intraphagosomal pH was calculated on the basis of the ratio of fluorescein:rhodamine fluorescence by using a calibration curve obtained after equilibration of phagosomal pH with different external pH values after addition of digitonin. RESULTS: A rapid rise in phagosomal pH, which reached pH 7.8, was observed 2 min after initiation of phagocytosis and progressively declined to pH 6.9 after 15 min. Such a rise was not observed in PMNs with defective microbicidal activity (deficient in nicotinamide adenine dinucleotide phosphate oxidase), where phagosomal pH dropped to pH 6.6, 2 min after phagocytosis. The abnormal initial acidification in PMNs deficient in nicotinamide adenine dinucleotide phosphate oxidase was prevented by using lysosomotropic weak bases or the vacuolar-type H(+) pump inhibitor concanamycin A. CONCLUSIONS: Phagosomal pH of PMNs can be easily and accurately measured by dual fluorescence flow cytometry. The method can be applied to assess phagosomal pH in PMNs with defective microbicidal activity and to monitor the outcome of pharmacologic interventions aimed at correcting its abnormalities.

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 C8 beta.

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

UNLABELLED: 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.